Development of BMP type I receptor kinase inhibitors for the
treatment of fibrodysplasia ossificans progressiva and the
study of the BMP signaling pathway.
by
Agustin Humberto Mohedas
B.S. Biomedical Engineering
Texas A&M University, 2007
SUBMITTED TO THE HARVARD-MIT DIVISION OF HEALTH SCIENCES AND
TECHNOLOGY IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF
DOCTOR OF PHILOSOPHY IN MEDICAL ENGINEERING AND MEDICAL PHYSICS
AT THE
MASSACHUSETTS INSTITUTE OF TECHNOLOGY
JUNE 2014
C 2014 Massachusetts Institute of Technology. All rights reserve
A4C
T
TS INS
OF TECHOLOGY
UN 18 2014
Signature redacted
S u
Certified by:
Harvard-MIT Division of Health Sciences and Technology
May 19, 2014
Signature redacted
Paul B. Yu, MD, PhD
Assistant Professor of Medicine
Thesis Supervisor
I
6-'
Accepted by:
BRARIEo
Signature red acted
Emery N. Brown, MD, PhD
Director Harvard-MIT Program in Health Sciences and Technology
Professor of Computational Neuroscience and Health Sciences and Technology
I
E
2
Development of BMP type I receptor kinase inhibitors for the
treatment of fibrodysplasia ossificans progressiva and the
study of the BMP signaling pathway.
by
Agustin Humberto Mohedas
Submitted to the Harvard-MIT Division of Health Sciences and Technology
on May 19, 2014 in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy in
Medical Engineering and Medical Physics
Abstract
The BMP signaling pathway is essential for embryonic development and the maintenance
of tissue homeostasis. Dysregulated BMP signaling, both loss and gain-of-function, has been
demonstrated in the pathogenesis of diseases including cancer, atherosclerosis, anemia and
particularly hereditary disorders such as pulmonary arterial hypertension, hereditary hemorrhagic
telangiectasia, and fibrodysplasia ossificans progressiva (FOP). FOP is a rare and disabling
condition caused by a highly recurrent mutation in the ACVR1 gene encoding the BMP type I
receptor activin-like kinase 2 (ALK2), characterized by the progressive heterotopic ossification
(HO) of skeletal muscle and connective tissue leading to widespread joint immobilization, with
significant morbidity and premature mortality. There are currently no effective treatments for
FOP. The goal of this thesis is to develop and characterize highly selective BMP type I receptor
inhibitors targeting ALK2 for the treatment of FOP.
Despite the high degree of structural homology between all the BMP and TGF-p type I
receptors, I hypothesized that potent and selective inhibitors targeting a single BMP type I
receptor, ALK2, could be developed based on a previously identified pyrazolo[1,5-a]pyrimidine
core scaffold. I screened a library of pyrazolo[1,5-a]pyrimidine derivatives in a high throughout
sensitive radiometric assay of BMP and TGF-P type I receptor kinase activities. I identified a
derivative with a unique chemical moiety (5-quinoline) that demonstrated high selectivity for
ALK2, but with lower potency than the parent molecule. We synthesized a new 5-quinoline
derivative with increased potency and selectivity for ALK2 over the other BMP type I receptors
and greatly improved selectivity against the TGF--P type I receptors. I used this highly selective
compound to examine ALK2-mediated BMP signaling in vitro and demonstrated in vivo efficacy
in two mouse models of HO.
In a complementary approach, we generated a library of novel BMP type I receptor
inhibitors based on the 2-aminopyridine core scaffold. I developed a structure activity
relationship to determine the key structural elements responsible for potency and selectivity. We
3
identified a several novel derivative compounds with improved potency and selectivity for ALK2
over the parent. We successfully used this set of derivatives to address a specific question in
FOP biology, of whether ATP-competitive kinase inhibitors exert differential activity against
wild-type or diverse FOP-causing ALK2 mutants.
Finally, in our SAR of pyrazolopyrimidine compounds, we identified a highly potent
inhibitor of both BMP and TGF-$ type I receptor activity. I characterized the ability of this
compound to inhibit ligand-induced BMP and TGF- signaling in a variety of cell culture
models, as well as inhibit the activity of individual type I receptors. We then used this
compound to examine the contribution of individual BMP and TGF-P receptors to signal
transduction. We used the broad activity of this inhibitor to limit signaling of all endogenous
BMP and TGF-P type I receptors in cells, while reconstituting the activity of specific type I
receptors using engineered, inhibitor-resistant mutant receptor kinases which we developed by
modifying gatekeeper residues critical for interactions with inhibitor. These mutant receptor
kinases demonstrated preserved basal and ligand-mediated signaling functions which were
unaffected by inhibitor. These results demonstrate proof-of-principle of a system for examining
the function of individual receptors of this pathway in isolation.
The work presented in this thesis advances the development of novel BMP type I receptor
kinase inhibitors of high selectivity and potency which could serve as important tools for the
study of BMP signaling and as therapies for diseases of excessive BMP signaling such as FOP.
Development of highly potent and selective inhibitors of ALK2 offers the hope of rational
disease modifying therapy for the treatment of FOP.
Thesis Supervisor: Paul B. Yu
Title: Assistant Professor of Medicine
4
Acknowledgments
I dedicate this thesis to my grandfather, Umberto Panza, who has inspired me throughout
my life. He has served as a standard to which I hold myself and hope to one day match.
This thesis would not have been possible without the love, commitment, and patience of
my fiance Megan Baugh. I thank you most of all.
I would like to thank my parents Teresa and Sergio Mohedas, who from an early age
instilled in me the importance of education and the power of hard work. I would not be where I
am today without the unconditional support they have given me throughout my life. Thank you
to my brother Ibrahim and sister Jimena for the fond memories and insults we've shared.
I thank my mentor and thesis advisor Dr. Paul Yu who throughout these years has been
nothing but supportive of my research and goals. To my lab colleagues Kelli Armstrong, Dr.
Jana Bagarova, Patrick McManus, Dr. Ivana Nikolic, Sam Paskin-Flerlage, Ashley Vonner, and
Dr. Lai-Ming Yung thank you for putting up with me all of these years!
Thank you to my thesis committee members Dr. Collin Stultz, Dr. Thomas Michel, and
Dr. Greg Cuny who listened carefully and provided crucial guidance. The work presented in this
thesis is the result of multiple fruitful collaborations and I am forever grateful to Dr. Alex
Bullock, Dr. Greg Cuny, and Dr. Joerg Ermann. I want to also thank the other scientists that
contributed to this work; specifically Dr. Xuechao Xing and Dr. You Wang for synthesizing
compounds and Dr. Caroline Sanvitale for solving crystal structures and assaying compounds.
I am also thankful to have had the opportunity to work with Dr. Arthur Lee and all of the
great people at the Therapeutics for Rare and Neglected Diseases program at the National Center
for Advancing Translational Sciences (NCATS) at the National Institutes of Health (NIH). I
hope my contributions to this program have in some small way helped advance the development
of treatments for FOP.
Finally, I would like to thank all of my friends and the unique people that I've had the
pleasure of knowing during my time at MIT. It has been quite a journey.
5
Table of Contents
Chapter 1
Introduction.............................................................................................................................17
Chapter 2
Background and M otivation.................................................................................................
The TGF-p Fam ily ......................................................................................................................
2.1
20
20
2.1.1
H istorical perspective.....................................................................................................
21
2.1.2
Canonical TGF-p and BM P signaling............................................................................
21
2.1.3
Role of TGF-P and BMP signaling in development and homeostasis .............................
23
D iseases of TGF-3 and BM P Signaling .................................................................................
24
2.2.1
TGF-P and BM P signaling in disease ............................................................................
24
2.2.2
Fibrodysplasia ossificans progressiva...........................................................................
26
2.2
2.3
K inase Inhibitors .........................................................................................................................
28
2.3.1
K inase inhibitor developm ent and clinical use...............................................................
28
2.3.2
TGF-p type 1 receptor kinase inhibitors .........................................................................
30
2.3.3
BM P type 1 receptor kinase inhibitors ............................................................................
30
Sum m ary .....................................................................................
2.4
Error! Bookm ark not defined.
Characterization of dorsom orphin derivatives ...................................................................
Chapter 3
33
3.1
Background and M otivation...................................................................................................
33
3.2
Experim ental M ethods ................................................................................................................
34
3.2.1
Traditional radiom etric kinase assay...............................................................................
34
3.2.2
H igh throughput radiom etric kinase assay .....................................................................
36
3.3
Results and D iscussion................................................................................................................37
3.3.1
Km determ ination for A LK 1-5 ..........................................................................................
37
3.3.2
Screening of dorsom orphin deriviatives against ALK 1-5...............................................
38
3.4
Chapter 4
Conclusion ..................................................................................................................................
Developm ent of potent and selective ALK2 inhibitors.....................................................
44
45
4.1
Background and M otivation...................................................................................................
45
4.2
Experim ental M ethods ................................................................................................................
46
4.2.1
Cell culture ..........................................................................................................................
46
4.2.2
Luciferase reporter assay.................................................................................................
46
4.2.3
Ligand induced SM A D phosphorylation .......................................................................
47
4.3
4.3.1
Results and D iscussion................................................................................................................48
Characterization of BM P inhibitors ................................................................................
6
48
4.3.2
LDN-212854 binding mode..............................................................................................55
4.3.3
Kinase profiling of LDN-193189 and LDN-212854...........................57
4.4Concluson.........................................................5
4.4
Conclusion .................................................................................................................................. 62
Chapter 5
Study of BM P signaling using an ALK2 selective inhibitor...............................................
5.1
Background and Motivation........................................................................................................64
5.2
Experimental M ethods................................................................................................................65
64
5.2.1
BM P-Induced ALP activity............................................................................................
65
5.2.2
IL-6 induced hepcidin expression ..................................................................................
65
5.2.3
caALK2 (Q207D) M ouse M odel of FOP.......................................................................
66
5.3
Results and Discussion................................................................................................................66
5.3.1
LDN-212854 preferentially inhibits ALK2.....................................................................
5.3.2
IL-6 induced hepcidin expression is predominantly mediated by ALK3........................67
5.3.3
LDN -212854 inhibits ALK2Q207D-induced heterotopic ossification ...............................
69
5.3.4
LDN-212854 inhibits heterotopic ossification in Bmall-'- m ice .....................................
71
5 .4
C on c lu sio n ..................................................................................................................................
Chapter 6
Development of 2-aminopyridine BMP kinase inhibitors ..................................................
66
76
77
6.1
Background and M otivation.....................................................................................................
77
6.2
Experimental M ethods ................................................................................................................
78
6.2.1
Therm al shift kinase assay .............................................................................................
78
6.2.2
Cell viability assay ..............................................................................................................
78
6.3
Results and Discussion................................................................................................................79
6.3.1
Structure activity relationship (SAR) of solvent exposed group.....................................
79
6.3.2
Structure activity relationship (SAR) of hydrophobic pocket position ...........................
83
6.3.3
Structure activity relationship (SAR) of hinge binding position.....................................
85
6.3.4
Structure activity relationship (SAR) of K02288 and LDN-193189 hybrid molecules...86
6.3.5
Kinome selectivity of LDN-212838 and LDN-214117 ..................................................
6.3.6
FOP mutations, inhibitor binding affinity, and implications for therapeutics.................96
6.3.7
Cytotoxicity of kinase inhibitors....................................................................................
98
6.3.8
Structural basis of inhibitor binding................................................................................
99
6.4
Chapter 7
Conclusion ................................................................................................................................
Development of a potent dual BM P/TGF-p inhibitor ...........................................................
7.1
Background and M otivation......................................................................................................104
7.2
Experim ental M ethods ..............................................................................................................
7
88
102
104
105
7.2.1
Kinase assay ......................................................................................................................
7.2.2
Luciferase reporter assay...................................................................................................105
7.2.3
Cell viability assay ............................................................................................................
105
106
Results and Discussion..............................................................................................................107
7.3
7.3.1
Lead optimization of LDN-193189 to improve metabolic stability.................................107
7.3.2
SAR of LDN-193189 derivatives reveals 2-methylquinoline reduces selectivity ........... 108
7.3.3
In vivo metabolism of TRND-343765 .............................................................................
7.3.4
Characterization of TRND-343765 as a potent dual BMP and TGF-P inhibitor .............. 111
7.3.5
M utant type I receptors and TRND-343765 as in vitro probes of signaling ..................... 113
C on clu sio n ................................................................................................................................
7 .4
Chapter 8
109
1 16
Conclusions...........................................................................................................................117
8.1
Summary of the thesis.................................................................................................117
8.2
Suggested future work...............................................................................................................118
8.2.1
Optimization of 5-quinoline position ................................................................................
118
8.2.2
Role of BM P signaling in heterotopic ossification in Bmall-.- .........................................
119
8.2.3
Improved selectivity for TRND-343765 ...........................................................................
119
8.2.4
Resistant BM P and TGF-P type I receptors to study signaling .........................................
120
B iblio grap hy ..............................................................................................................................................
8
12 1
9
List of Figures
Figure 2.1. Phylogenetic tree of the TGF-p super family of signaling ligands, receptors, and intracellular
20
transduction proteins (SM ADs). Adapted from [2]. ..............................................................................
Figure 2.2. A schematic representation of the canonical BMP and TGF- signaling pathways
demonstrating ligand binding to tetrameric complexes of type I and type II receptors that phosphorylate
22
R-SMADs, which translocate to the nucleus and effect specific transcriptional programs. ..................
Figure 2.3. (a) A color coded grid with the percent sequence identity for each BMP and TGF-0 type I
receptor was determined using the alignment tool (http://www.uniprot.org/blast/). (b) Superposition of
ALKI and ALK2 demonstrating the high degree of similarity between these receptors despite their unique
3
fu n ctio n s......................................................................................................................................................2
Figure 2.4. (a) FOP-causing mutations in both the GS loop and kinase domain. (b) Soft tissue nodules
and HO lesions on the back of a 4-year-old child affected by FOP. (c) The same child at 8 years of age
27
showing extensive HO of the back. Adapted from [73] and [74]. ..........................................................
Figure 2.5. (a) Structural overview of ALK2 demonstrating the bilobular structure of kinases including
the kinase domain, hinge, GS domain, ATP binding pocket, and interacting regulatory protein FKBP 12.
(b) The highly conserved ATP binding region of kinases showing hydrogen bonding interactions between
adenine and the hinge as well as hydrophobic pockets that are amenable to inhibitor binding. Adapted
29
fro m [8 8 ]. ....................................................................................................................................................
Figure 3.1. (a) Structure of the pyrazolo[1,5-a]pyrimidine core with R-groups at the 5-position of the
pyrimidine (RI) and the 2-(R2) and 3-(R3) positions of the pyrazolo. (b) The structure of dorsomorphin
with a piperidinyl ethoxy phenyl at R1, hydrogen at R 2, and 4-pyridyl at R3. (c) The structure of LDN33
193189 with a phenylpiperazine at Ri, hydrogen at R2, and 4-quinolyl at R3. ........................................
Figure 3.2. Schematic representation of a traditional kinase assay with the kinase bound radiolabeled
ATP (a) phosphorylating its protein substrate (b) which is then transferred to phosphocellulose paper (c)
and finally placed in a vial with scintillation fluid (d) for light output measurement.............................35
Figure 3.3. (a)Traditional kinase assay setup including 96 scintillation vials, caps, and racks. (b)
Equivalent high throughput radiometric kinase assay setup with 96-well plate. (c) Comparison metrics
between traditional kinase assay and our optimized high throughput assay..........................................36
Figure 3.4. Km determinations for (a) ALK1, (b) ALK2, (c) ALK3, (d) ALK4, and (e) ALK5. (f)
Summary of Km values for BMP and TGF-p type I receptor kinases.....................................................37
Figure 3.5. Structure-activity relationship of 4-methoxyphenyl pyrazolo[1,5-a]pyrimidine derivatives. (a)
In vitro kinase assay IC 50 measurements against BMP and TGF-P type I receptors show only the 4quinoline (LDN-193688) and 5-quinoline (LDN-193719) derivatives are active. (b) Structure of five
pyrazolo[1,5-a]pyrimidine derivatives with varying quinoline attachment sites. (c) Fold selectivity of
active inhibitors against ALK2 versus BMP and TGF-p type I receptors. Although slightly less potent
against ALK2 than LDN-193688, the 5-quinoline derivative LDN-193719 has much greater selectivity
43
for B M P vs. T G F-p type I receptors. ..........................................................................................................
10
Figure 4.1. Synthetic scheme of LDN-212854. (a) AcOH, MeOH, 80 C, (61%). (b) Pd(PPh 3) 4, 2.0 M
Na 2 CO 3 , dioxane, 101 0C, (82-95%). (c) NBS, DCM, (63%). (d) TFA, DCM, then sat. NaHCO 3, (55%).
.....................................................................................................................................................................
47
Figure 4.2. Potency and selectivity of BMP inhibitors based on in vitro kinase assay. a) Structure of
previously described BMP inhibitors and LDN-212854. b) In vitro kinase assay measurements of IC 50 for
BMP and TGF-p type I receptors show LDN-193189 is the most potent inhibitor of ALK2, followed
closely by K02288a and LDN-212854. Both DMH1 and dorsomorphin exhibited 10-fold lower potency
against BMP type I receptors. c) Fold selectivity of these inhibitors against ALK2 versus closely related
BMP and TGF-p type I receptors. d) Inhibition of ALK2 (BMP) and ALK5 (TGF-p) kinase activity
demonstrates LDN-212854 exhibits increased selectivity for ALK2 versus ALK5. Data shown are
representative of 2-3 independent experim ents.......................................................................................
50
Figure 4.3. Selectivity of known BMP inhibitors and novel inhibitor LDN-212854 based on in vitro
kinase assay and cell-based BMP (BRe-Luc) and TGF-P (CAGA-Luc) transcriptional activity mediated
by constitutively active ALK 1-5. (a) Graphical representation of the fold selectivity of these inhibitors in
kinase assay against ALK2 versus closely related BMP and TGF-p type I receptors. (b) and (c) A
graphical representation of the fold selectivity of inhibitors for caALK2 over closely related BMP and
TGF-s type I receptors. LDN-212854 has 6-10 fold selectivity for caALK2 versus other BMP receptors
(caALK I and caALK3) and much greater selectivity (>100 fold) over the TGF-p receptors (caALK4 and
caALK5). Having much lower potency against caALK1, 2 and 3 in cells, both K02288a and DMH1
exhibit substantially lower selectivity. Data shown are calculated based on IC 50 generated from at least 3
independent experiments, with data plotted as mean ± S.E.M ................................................................
51
Figure 4.4. Potency Inhibition curves of BMP and TGF-3 transcriptional activity mediated by
constitutively activated ALK 1-5 in a cell-based luciferase reporter assays. Representative inhibition
curves for a) dorsomorphin, b) LDN-193189, c) LDN-212854, d) DMH1 and e) K02288a against
constitutively active BMP (ALKI, 2 and 3) and TGF-P (ALK4 and 5) type I receptors. Dorsomorphin
exhibits a similar selectivity profile to LDN-193189, but with approximately 10-fold decreased potency
against all receptors. Despite showing potency similar to LDN-193189 in kinase assay, K02288a is less
potent and selective in cells. DMH1 exhibits similar potency to dorsomorphin but with less selectivity.
In contrast to dorsomorphin and LDN-193189, LDN-212854 demonstrates improved selectivity for
ALK2 versus ALK4 and 5, as well as versus ALK 1 and ALK3. Data shown are representative of at least
3 independent experiments, with data plotted as mean ± S.E.M. (n=3 replicates per [c] point).............53
Figure 4.5. Potency and selectivity of BMP inhibitors based on on BMP (BRe-Luc) and TGF- (CAGALuc) transcriptional activity mediated by constitutively active ALK1-5. (a) In vitro cell-based assay IC50
measurements against constitutively-active BMP (caALK1, 2, and 3) and TGF-p (ALK4 and 5) type I
receptors demonstrates LDN-212854 to be more selective for BMP versus TGF-p receptors while
retaining low nanomolar potency against caALK2. (b-c) Inhibition of caALK1-5 by BMP inhibitors at
various concentrations demonstrates that LDN-212854 preferentially inhibits caALK2 at concentrations
near 100 nM, whereas other receptors are affected at higher concentrations. Data shown are calculated
based on IC 50 generated from at least 3 independent experiments, with data plotted as mean ± S.E.M.....54
Figure 4.6. Comparison of potency and selectivity of LDN-193189 and LDN-212854 in modulating BMP
and TGF-p ligand-mediated SMAD signaling. (a) Western blot analysis of BMP7 induced
phosphorylation of SMAD1/5/8 in BMPR2-/- PASMCs reveals low nanomolar inhibition by both LDN11
193189 and LDN-212854. (b) Western blot analysis of TGF-31 induced phosphorylation of SMAD2 in
wild-type PASMC revealed significant inhibition by LDN-193189 at concentrations greater than 1 gM,
and virtually no inhibition by LDN-212854 at concentrations up to 25 ptM. Data shown are representative
55
of 2 independent experim ents. ....................................................................................................................
Figure 4.7. (a) ATP pocket interactions of LDN- 193189 (magenta) co-crystallized with ALK2 (PDB
3Q4U). A single hydrogen bond to the hinge residue H286 is made by the central pyrazolo[1,5-a]
pyrimidine. The pendant 4-quinoline moiety forms a water-mediated hydrogen bond to the aC-helix
residue E248. Water is represented by a red sphere and labeled "Wat". Hydrogen bonds are shown as a
dashed line. (b) Model for the binding of LDN-193189 (magenta) to ALK5. The inhibitor was located by
the superposition of ALK2 (PDB 3Q4U) and ALK5 (SB-431542 complex; PDB 3TZM). ALK5 structures
show a conserved water position set further back in the ATP pocket between the aC-helix residues E245
and Y249. (c) Model for the binding of LDN-212854 (green) to ALK2. The pendant 5-quinoline group is
predicted to form an alternative water-mediated hydrogen bond to the catalytic lysine (K23 5). The new
water position was modeled from the ALK2-K02288 co-crystal structure (PDB 3MTF). (d) ATP pocket
interactions of LDN-212854 (yellow and blue) in the ALK2 co-crystal structure. The pendant 5-quinoline
moiety forms a water-mediated hydrogen bond with the catalytic lysine (K235) as predicted and also to
the aC -helix glutam ate residue (E248). ................................................................................................
56
Figure 4.8. Kinase inhibition profile of LDN-193189 and LDN-212854. Kinome dendrograms for (a)
LDN-193189 and (b) LDN-212854 showing both on-target hits from our kinase assay and the top offtarget hits from a screen of 198 human kinases. (c) IC 50 values for top off-target hits. RIPK2 was the most
potently inhibited off-target kinase followed by ABL 1 and PDGFR-p, while other kinases were inhibited
57
at much higher concentrations. ...................................................................................................................
Figure 5.1. LDN-212854 preferentially inhibits ALK2. (a) At low concentrations (34nM) LDN-212854
potently inhibits ALK2 (79%) whereas ALKI, ALK3, ALK4, and ALK5 remain largely active. In
contrast, at 34nM LDN-193189 inhibits both ALK2 and ALK3 equally. a) Representative inhibition
curves of caALK2 and b) caALK3 transcriptional activity (BRE-Luc) by LDN-193189 and LDN-212854
in C2C12 cells. Both compounds potently inhibit ALK2, whereas LDN-212854 is substantially weaker
against ALK3. Data shown are representative of at least 3 independent experiments, with data plotted as
67
m ean ± S.E.M . (n=3 replicates per [c] point).........................................................................................
Figure 5.2. LDN-212854 provides useful selectivity as a probe of signaling mediated by ALK2 versus
ALK3, and their respective ligands. (a) Alkaline phosphatase (ALP) activity induced in C2C 12 cells by
BMP6, which signals primarily through ALK2, was inhibited with comparable potency by LDN- 193189
and LDN-212854. (b) ALP activity induced in C2C12 cells by BMP4, which signals primarily through
ALK3, was more potently inhibited by LDN-193189 than LDN-212854. Data shown are representative of
at least 3 independent experiments, with data plotted as mean ± S.E.M. (n=3 replicates per [c] point).....68
Figure 5.3. IL-6 induced hepcidin expression in HepG2 cells was potently inhibited by LDN- 193189 and
less potently by LDN-212854, consistent with a primarily ALK3-dependent mechanism of IL-6 induced
hepcidin expression. Data shown are representative of at least 2 independent experiments, with data
69
plotted as mean ± S.E.M . (n=4 replicates per [c] point) .........................................................................
Figure 5.4. In vivo efficacy of LDN-212854 in a mouse model of fibrodysplasia ossificans progressiva
(FOP). Mice expressing an inducible constitutively-active ALK2Q2 07 D (CAG-Z-EGFP-caALK2)
transgene were treated with (a) vehicle, (b) LDN-193189 or (c) LDN-212854 at 6 mg/kg IP BID.
12
Heterotopic ossification following injection of Ad.Cre was observed by X-ray (top panels) and staining
for alizarin red (calcium) and alcian blue (glycosaminoglycans) (bottom panels). Heterotopic ossification
following Ad.Cre injection was observed 100% of vehicle-treated mice, whereas ossification was
essentially absent in mice treated with LDN-193189 or LDN-212854. Transgene-mediated expression of
GFP (middle panel) was observed at the site of Ad.Cre injection to confirm recombination and ALK2Q2 7 D
expression. (d) Passive range-of-motion was progressively impaired in vehicle-treated mice starting on
day 10, whereas mobility was almost entirely preserved in mice treated with LDN- 193189 and LDN-
2 12 8 5 4 ...............................................................
. ......................................................................................
70
Figure 5.5. LDN-212854 was significantly better tolerated than LDN-193189. (a) Weight change of
mouse pups throughout the treatment period from P7 to P35. (b) LDN-193189 resulted in a 32%
reduction in the rate of growth, while LDN-212854 resulted in a significantly lower reduction of 14%...71
Figure 5.6. Voluntary wheel-running behavior in mice. Dark bars represent periods of wheel-running.
Wild-type mice (a) and (b) Bmallknock-out mice were subjected to day-night cycles (day 0 - 21)
followed by complete darkness (day 22-70). Wild-type mice maintained circadian rhythms with a period
of 23.6 hours, while Bmal-/- mice did not. Figure adapted from [126]. ...............................................
71
Figure 5.7. Progressive arthropathy and joint ankylosing seen in Bmal' mice both in the intervertebral
joints and at the Achilles anthesis. Adapted from [136]. ........................................................................
72
Figure 5.8. Relative expression of Bmallin the liver and the Achilles enthesis of Bmallf-Prx1-cremice
compared to BmaIf mice. Prx]-cre mice demonstrated disrupted and abnormal cycling of Bmall over 24
hours in the peripheral tissue of the Achilles enthesis, while maintaining normal circadian rhythm in the
liv e r. ............................................................................................................................................................
73
Figure 5.9. Heterotopic ossification (HO) at the Achilles enthesis. (a) Bmall 1 Prx]-Cre mice develop
Achilles HO which is first visible by X-ray at 8 weeks of age. The HO continues to develop increasing in
severity until 27 weeks of age. (b) Penetrance is of the Achilles enthesis HO is 100% but the severity of
H O at 8 weeks is highly variable. ...............................................................................................................
74
Figure 5.10. Quantification of heterotopic ossification in Bmall 1 Prx1-Cre mice. (a) A region of interest
(ROI) is selected just above the calcaneus and lateral to the tibia in the area of the Achilles tendon. Using
ImageJ a threshold of 42 is set and the number of pixels above this threshold is counted. (b)
Quantification of HO in Bmall] PrxJ-Cremice treated with vehicle, LDN-212854, or ALK3-Fc shows
significant inhibition of H O by LDN -212854.........................................................................................
75
Figure 6.1. Superposition of the ALK2 and ALK5 co-crystal structures with K02288 and SB431542,
respectively, showing selected interactions of ALK2 with K02288. The ATP pocket in many ALK5 cocrystal structures shows a more open conformation with a subtle movement of the N-lobe away from Clobe. Such conformational differences, which change the shape, volume and dynamics of the ATP
pocket, are likely to im pact inhibitor selectivity....................................................................................
80
Figure 6.2. Potency and selectivity of K02288 derivatives based on thermal shift, biochemical kinase
activity, and ligand induced transcriptional activity assays. (a) The 2-aminopyridine scaffold of K02288
(b) Modifications to the solvent exposed domain (R1 ) of K02288. (c) Thermal shift (ATm), biochemical
enzymatic inhibition (IC 50 ) for ALK2 and ALK5 kinase proteins, and inhibition of cell-based BMP6 and
TGF-3 1-induced transcriptional activity (IC 5o) by K02288 derivative compounds (nd = not determined).
(d) Correlation of thermal shift and cell-based BMP/TGF-p inhibition assays. ....................................
81
13
Figure 6.3. A strong negative log-linear correlation is seen between thermal shift and biochemical IC50
for both (a) BMP (ALK2) and (b) TGF-p (ALK5) type 1 receptors. A strong negative log-linear
correlation is seen between thermal shift of BMP type I receptors and ligand induced cell-based IC 50 for
both (c) BM P6 (ALK2) and (d) TGFbI (ALK5). .................................................................................
82
Figure 6.4. Potency and selectivity of compound 15 derivatives based on thermal shift, biochemical
kinase activity, and ligand induced transcriptional activity assays. (a) The 2-aminopyridine scaffold of 15
(b) Modifications to the ATP-binding pocket hydrophobic domain (R2) of compound 15. (c) Thermal shift
(ATm), biochemical enzymatic inhibition (IC 5o) for ALK2 and ALK5 kinase proteins, and inhibition of
cell-based BMP6 and TGF-1 -induced transcriptional activity (IC 5o) by compound 15 derivatives (nd =
not determined). (d) Correlation of thermal shift and cell-based BMP/TGF-p inhibition assays...........84
Figure 6.5. Potency and selectivity of K02288 derivatives based on thermal shift, biochemical kinase
activity, and ligand induced transcriptional activity assays. a) The 2-aminopyridine scaffold of 15 b)
Modifications to the primary amine kinase hinge binding domain (R3) of compound 15. c) Thermal shift
(ATm), biochemical enzymatic inhibition (IC 5o) for ALK2 and ALK5 kinase proteins, and inhibition of
cell-based BMP6 and TGF-p 1-induced transcriptional activity (IC 5o) by compound 15 derivatives (nd =
not determined). d) Correlation of thermal shift and cell-based BMP/TGF- inhibition assays. ........... 86
Figure 6.6. Potency and selectivity of K02288 derivatives based on thermal shift, biochemical kinase
activity, and ligand induced transcriptional activity assays. (a) Structure of hybrid derivatives. (b)
Thermal shift (ATm), biochemical enzymatic inhibition (IC 50) for ALK2 and ALK5 kinase proteins, and
inhibition of cell-based BMP6 and TGF-3 1-induced transcriptional activity (IC 5o) by hybrid molecules
(nd = not determined). (c) Correlation of thermal shift and cell-based BMP/TGF-p inhibition assays......88
Figure 6.7. Kinome dendrogram plot for (a) LDN-212838 (15) and (b) LDN-214117 (10) showing an
improved selectivity profile for LDN-214117, albeit with reduced potency for BMP type I receptor
k in a se s.........................................................................................................................................................8
9
Figure 6.8. FOP causing ALK2 mutations do not affect inhibitor binding. a) Strong correlation of
thermal shift data for ATP competitive kinase inhibitors binding to wild-type ALK2 versus known FOP
causing GS domain mutations of ALK2 and b) known FOP causing kinase domain mutations suggests the
potency of ATP competitive inhibitors are not affected by these disease causing mutations. m = slope, R2
= correlation coefficient..............................................................................................................................97
Figure 6.9. Cell viability. HepG2 cells were exposed to 1, 10, and 100 tM of compounds for 4 or 24
hours. The average cell viability of three experiments is shown with green indicating >75%, orange
indicating 25-75% , and red <25% ...............................................................................................................
99
Figure 6.10. Plots of cell-based BMP (a) and TGF-p (b) IC 50 versus cell viability show no correlation
100
betw een potency and toxicity ....................................................................................................................
Figure 6.11. Binding mode of 26. (a) The inhibitor (yellow) forms a single hydrogen bond to the hinge
amide of H286 as well as a water-mediated bond to the catalytic lysine K235. (b) Plot of the interactions
of the inhibitor (purple) in the binding pocket of ALK2. The plot was generated by LigPlot+.[154]......101
Figure 6.12. Docking model for 10. Docking was performed using the ICM-Pro software package
(Molsoft) and the ALK2-26 structure as a template. Compound 10 (cyan) is predicted to bind similarly to
the parent molecule K02288 (PDB 3MTF) as well as the close derivative 26 (PDB 4BGG). The hinge
14
binding orientation of this 2-aminopyridine series differs compared to the pyrazolo[1,5-a]pyrimidine
scaffold of LDN-193189 (dark blue thin sticks; PDB 3Q4U). .................................................................
101
Figure 7.1. (a) In vitro studies of metabolism for LDN-193189 revealed major metabolism via oxidation
of the quinoline (NIH-Q55) mediated by aldehyde oxidase. Analine formation at the phenylpiperazine
position was also observed. (b) In vivo studies confirmed the in vitro findings and found that LDN193189 is quickly metabolized by the liver into a low potency metabolite, NIH-Q55. Furthermore, the
metabolite accumulates in tissues (e.g. muscles) at concentrations far exceeding those of LDN-193189.
...................................................................................................................................................................
108
Figure 7.2. (a) Methylation at the 2-position of the quinoline in LDN-193189 and derivatives was used
as a strategy to block aldehyde oxidase metabolism at this position. (b) In each case there was a dramatic
decrease in B M P versus TG F-p selectivity...............................................................................................109
Figure 7.3. Significant improvements to in vivo metabolic stability seen with TRND-343765. (a) TRND-
343765 had improved half-life, AUC, and oral bioavailability (F) as compared with LDN-193189. (b)
TRND-343765 demonstrated superior pharmacokinetics to LDN-193189 with high plasma concentrations
at 7 hours after administration, while LDN-193189 was almost completely cleared. .............................. 110
Figure 7.4. Characterization of the inhibitory profile of TRND-343765 compared with LDN-193189. (a)
Kinase assay inhibition curves for ALKI-6 showing that at 200 nM TRND-343765 completely inhibits all
BMP and TGF-p type I receptor kinases, while LDN-193189 only inhibits BMP type I receptor kinases.
(b) IC 5 o and 1C 9o values for LDN-193189 and TRND-343765 showing potent dual BMP and TGFinhibition. (c) Cell-based ligand induced transcriptional activity inhibition curves. ................................ 111
Figure 7.5. Cell-based inhibition of BMP and TGF-3 transcriptional activity. (a-c) LDN-193189 and
TRND-343765 both potently inhibited all BMP ligand and constitutively active type I receptor
transcriptional activity (d-f) Only TRND-343765 potently inhibited all TGF-p ligand and constitutively
active type I receptor transcriptional activity............................................................................................112
Figure 7.6. (a) Abstract representation TRND-343765 used in conjunction with inhibitor resistant type I
receptors. (b) Deep hydrophobic pocket of ALK2 showing threonine gatekeeper residue that was mutated
to isoleucine to generate inhibitor resistant mutants. (c) Baseline and ligand-induced signaling of wild
type ALK2 and caALK2 is inhibited by TRND-343765 while T2831 mutants show inhibitor resistance in
b oth c a se s..................................................................................................................................................1
15
15
List of Tables
Table 2.1. Members of the TGF-p and BMP signaling family including receptors, ligands, co-receptors,
and endogenou s inhibitors...........................................................................................................................24
Table 3.1. Dorsomorphin derivatives and their respective IC 50 (nM) values for BMP4 induced
pSMAD1/5/8 in cell western and biochemical kinase assay for ALK1-5. * n.a. = no activity...............42
Table 4.1. Kinome profiling for LDN-193189 and LDN-212854 at 100 nM and 1IM ranked by kinases
with the greatest inhibition of enzym atic activity. .................................................................................
62
Table 6.1. Comparison of compounds 15, 26, and 10 across multiple assays including thermal shift kinase
assay, ligand induced transcriptional assay, and constitutively active ALK 1-5 transcriptional activity
demonstrates increased selectivity for ALK2 for compound 10 albeit with a reduction in potency. ......... 87
Table 6.2. Kinome profiling for LDN-212838 and LDN-214117 at 100 nM and I pM for >200 kinases
representing a wide sampling of the human kinome ranked by kinases with the greatest inhibition of
enzy m atic activ ity . ......................................................................................................................................
95
Table 6.3. Km values for wild type ALK2 and various constitutively active mutant versions of ALK2 seen
in F O P .........................................................................................................................................................
16
96
Chapter 1 Introduction
Bone morphogenetic proteins (BMP), which belong to the transforming growth factor beta
(TGF-p) family of over 30 structurally diverse ligand molecules, are critical for development and
homeostasis. BMPs, like TGF-p ligands, signal through binding to signaling complexes of type I
and type II receptors, whose intracellular kinase domains phosphorylate and activate SMAD
effector proteins. Activated SMADs serve as broadly-acting transcriptional
regulators,
modulating a wide variety of cellular mechanisms including differentiation, proliferation, and
migration.
Novel reagents such as selective inhibitors of the type I receptor kinases, have
become critical tools for studying this complex signaling pathway.
BMP and TGF-p signaling have been implicated in a wide variety of diseases, including
fibrosis, cancer, and anemia. In particular, an activating mutation of the BMP type I receptor
kinase, activin-like kinase 2 (ALK2), is the known cause of an extremely rare genetic disorder
known as fibrodysplasia ossificans progressiva (FOP). FOP-causing mutations in ALK2 lead to
increased BMP signaling and the development of heterotopic ossification (HO) lesions in
skeletal muscle and connective tissue.
This pathological bone formation begins in the first
decade of life, progresses inexorably, and causes disability through ankylosis or fusion of joints.
FOP is a life-shortening and highly morbid disease with no effective treatments.
This thesis was motivated by the need for novel, highly refined inhibitors of the BMP type I
receptor kinases. These compounds could serve as important scientific tools for the study of
BMP signaling in vitro and in vivo. Additionally, the development of highly selective ALK2
inhibitors would offer a rational therapeutic approach to treating FOP. This thesis builds on
previous work in which a low potency compound was modified to yield a potent, albeit, less
selective BMP type I receptor kinase inhibitor. This thesis describes the work done to further
expand the understanding of the chemical structural features that modulate BMP type I receptor
kinase potency and selectivity. The thesis is divided into several chapters as outlined below.
Chapter 2 provides a detailed overview of the TGF-p and BMP signaling pathways
including a historical perspective on their discovery, the mechanisms by which a variety of
ligands signal and exert their pleiotropic effects, the role of these pathways in development and
tissue homeostasis, and finally the role of these pathways in a variety of diseases. We focus on
17
the example of FOP and how this work could contribute to the development of therapy for this
devastating disease.
We close the chapter by describing the field of kinase inhibitors, their
development as therapeutics, and the work to date on TGF-p and BMP kinase inhibitors.
In Chapter 3 we describe the characterization of a previously synthesized but incompletely
characterized library of dorsomorphin derivatives. Dorsomorphin is a low potency BMP type I
receptor kinase inhibitor discovered previously by our laboratory. By analyzing this derivative
library using a highly sensitive radio-kinase assay for the BMP and TGF-P type I receptors
ALKi, ALK2, ALK3, ALK4, and ALK5 we were able to discover a unique structural insight
that greatly improved selectivity for BMP versus TGF- signaling.
In Chapter 4 we describe the synthesis and characterization of a novel potent and selective
inhibitor of ALK2 based on the structural findings in Chapter 3.
We compare this novel
derivative with all of the previously described BMP type I receptor kinase inhibitors in
biochemical and cell-based assays of BMP signaling.
We demonstrate this novel inhibitor
provides superior potency and selectivity as compared to other inhibitors. We also identified key
off-targets across the human kinome.
Finally, we describe an inhibitor-ALK2 co-crystal
structure that highlights aspects of the binding mode that impart a high level of selectivity in this
compound series.
In Chapter 5 we build on the characterization of the compound developed in Chapter 4 and
show its utility as a scientific tool for the study of BMP signaling both in vitro and in vivo. In
particular, we demonstrate this compound has sufficient selectivity for ALK2 versus other BMP
type I receptors, such as ALK3, and that it can be used to ascertain the relative contribution of
these type I receptors in signaling phenomena. We also use this compound in a mouse model of
FOP and demonstrate complete prevention of pathological bone formation as well as improved
tolerability as compared to a previously developed less selective inhibitor. Finally we use this
compound to demonstrate that BMP signaling plays a role in the development of heterotopic
ossification in a novel model of arthropathy suggesting a potential for using BMP type I receptor
kinase inhibitors in other diseases such as ankylosing spondylitis and trauma induced heterotopic
ossification.
Chapter 6 describes the development of a unique set of kinase inhibitors based on a 2aminopyridine scaffold discovered by our collaborator. We developed a large set of derivatives
and explored the structure-activity relationships that modulate potency and selectivity. We were
18
able to discover a potent and selective BMP type I receptor kinase inhibitor that demonstrated
very low cytotoxicity and improved kinome-wide selectivity. Finally, we used this derivative
library to answer a particular question in the field of FOP concerning the ability of BMP type I
receptor kinase inhibitors developed against wild type ALK2 protein to inhibit the many
different FOP-causing ALK2 mutants.
In Chapter 7 we describe a novel compound that was discovered as part of our collaboration
with the NIH that is a potent inhibitor of both BMP and TGF-$ type I receptor kinase inhibitors.
In addition this compound demonstrated superior pharmacokinetic properties as well as
significantly less cytotoxicity. We fully characterized the ability of this compound to inhibit
both BMP and TGF-
signaling in cells in response to a wide-variety of ligands. Finally, we
demonstrate a proof of concept that we can use this dual BMP and TGF-p inhibitor as a way of
studying this complex signaling pathway. By engineering BMP and TGF-p type I receptors with
mutations at the conserved gatekeeper residues we can create mutant receptors that are resistant
to the inhibitor. We can then apply the inhibitor to cells effectively shutting down all BMP and
TGF-p type I receptor kinase activity and then selectively express resistant mutant kinases to
assess the function of that receptor or combinations of resistant mutant receptors for particular
biological processes.
Chapter 8 provides a summary of the main conclusions of the thesis and their implications.
I also discuss future work based on the results presented.
19
Chapter 2 Background and Motivation
The purpose of this chapter is to provide a high level background and context for the work
described in this thesis. We begin with an overview of the BMP and TGF-3 signalingpathways
and their role in development, homeostasis, and disease. We pay particular attention to
fibrodysplasia ossificans progressiva, a very rare disease of BMP signaling. We then discuss
kinase inhibitordevelopment and clinical use. Finally we look specifically at the development of
both TGF-3 and BMP kinase inhibitorsandpresent the motivationfor this thesis work
2.1
The TGF-P Family
The transforming growth factor beta (TGF-P) superfamily (Figure 2.1) of signaling ligands,
transmembrane receptors, and intracellular transduction proteins is essential for embryonic
development and the homeostatic regulation of a multitude of cellular processes, including cell
proliferation, differentiation, migration, and apoptosis[1, 2]. The TGF- superfamily also
regulates many processes of disease pathophysiology and is implicated in cancer, fibrosis,
atherosclerosis as well as a multitude of hereditary disorders including familial pulmonary
arterial hypertension (PAH), Hereditary hemorrhagic telangiectasia (HHT), juvenile polyposis
syndrome (JPS), and fibrodysplasia ossificans progressiva (FOP) [3, 4].
UgaNdS__
Receptors
BMP2
lypt I mmcpwr
ACVRXc (ALK7M
DPP (Decapentaplegic)
(
GBAB
(BA )
TKV (Thickveins)
GD2AVRLI0I
BMP5
SX(aophone)
Uecps
MPAType
r
BMP8B
TGFRW (rGFORwI
GBB (Glass bottom boat)
SCW (Screw)
AMHR2 (MISRJ)
Tful?
thinking)
n
GD
ACVR2 ACM Q
AcvR2(AcC1u)
BMP3
PUT (Punt)
GDF
..
-
MsTN
SMADs
ynG
Myoglianin
SMADI
SMAO5
SMAD9 (SMAD8)
MAD (Mothers against dpp)
SMAD2
=7TU12
T (Maverick)
GDF
5
AMH
Alp23B (Dawdle)
INHB (activin
NHBE (acfdWActivin
BIMP1S
)
3
RMADs
SMAD3 SMOX (Smad on X)
)
I4MADs
SMAD6
ShAD7
GDF9DAD
(Daughters against dpp)
Figure 2.1. Phylogenetic tree of the TGF-p super family of signaling ligands, receptors, and intracellular
transduction proteins (SMADs). Adapted from [2].
20
2.1.1
Historicalperspective
TGF-Ps were first described in 1978 by Joseph de Larco and George Todaro at the National
Cancer Institute [5].
They describe the secretion by sarcoma virus-transformed mouse
fibroblasts of polypeptide growth factors with the ability impart transformed-like properties, such
as cell proliferation and anchorage independent growth onto normal fibroblasts. Further work in
the early 1980s led to the purification of TGF-Ps from many non-neoplastic tissues, including
human placenta and their characterization as 25-kD dimers active at 2-3 pM and critical to
wound healing [6, 7]. As work in the TGF-$ progressed throughout the 1980s, the complexity of
this signaling pathway was just beginning to be uncovered with the discovery of high affinity
type I, type II, and type III receptors as well as the seemingly paradoxical effects of TGF-s [8,
9]. For example, in normal epithelial cells TGF-Ps inhibit growth and suppress tumor formation
while in cancer cells they can promote tumor progression and metastasis [10, 11]. Despite the
identification of many TGF-
signaling family members (Figure 2.1) and almost 60,000
publications to date, there remains a significant need to further understand the intricacies of this
complex signaling family in development, homoeostasis, and particularly in disease.
2.1.2
CanonicalTGF-3 and BMP signaling
Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-
beta (TGF-)
signaling family, which includes over 30 different ligands including TGF-Ps,
growth and differentiation factors (GDFs), and Activins [2, 12, 13]. BMP signaling is essential
for numerous processes including cell fate determination, embryonic patterning, and iron
homeostasis [13, 14]. The BMP signaling cascade parallels that of TGF-$ signaling (Figure 2.2).
BMP ligand dimers facilitate the assembly of tetrameric receptor complexes consisting of two
constitutively-active type II receptor kinases (BMPRII, ACTRIIA, or ACTRIIB), which
transphosphorylate and activate two type I receptor kinases (ALKI, ALK2, ALK3, or ALK6)
[15]. Activated type I receptors phosphorylate effector proteins (SMAD1/5/8) that complex with
SMAD4, translocate to the nucleus, and activate BMP responsive genes, such as the inhibitor of
differentiation (Id) gene family. Activin and TGF-$ ligands similarly recruit TGF-s or Activin
type II receptors (TGFpR2, ACTRIIA, or ACTRIIB) with a set of type I receptors (ALK4,
ALK5, or ALK7) to activate SMADs 2 and 3, which translocate with SMAD4 to the nucleus to
regulate distinct transcriptional programs.
21
T
I
)1
I
W!
C D4To
1) AD1IL5
BMP Regulated Genes
4
GF-p Regulated Genes
Nucleus
SNA
Figure 2.2. A schematic representation of the canonical BMP and TGF-P signaling pathways
demonstrating ligand binding to tetrameric complexes of type I and type II receptors that phosphorylate
R-SMADs, which translocate to the nucleus and effect specific transcriptional programs.
Biological specificity in BMP and TGF-P signaling is conferred in part by preferential
binding of ligands with specific combinations of type I and type II receptors, although there is
considerable functional redundancy among ligands and receptors [16]. Functional and anatomic
specificity of BMP signaling is also regulated by the spatio-temporal expression of ligands and
their cognate receptors, as well as the expression of endogenous BMP antagonists such as noggin
(Table 2.1) [2, 16]. The diversity of upstream ligand and receptors signals, and their pleiotropic
downstream effects raises questions of how specificity is recognized and translated into
biological outcome in this pathway [17].
The various permutations of type 1 and type 2
tetrameric receptor complexes allows for context dependent and selective responses to the
various permutations of BMP and TGF-P ligand dimers.
However, there is considerable
functional redundancy in this pathway at the level of ligands and receptors, posing some
challenges for understanding the specific roles of its individual components. Despite serving
diverse biological functions, there is a tremendous degree of structural homology between the
receptors of the BMP and TGF-P signaling pathways (Figure 2.3).
Kinase domain sequence
identity is particularly high for ALK1 and ALK2 (79%), ALK3 and ALK6 (86%), and ALK4
and ALK5 (90%) [18].
22
(a)
Kinase Domain Sequence Identity
ALK1
ALK2
ALK3
ALK2
ALK3
ALK4
ALK5
ALK6
64.95
66.32
64.26
"4.91
W54
ALK7
ACVRLi (ALKi)
ACVRi (ALK2)
Hinge
63.86
67.37
AMK
(b)
'
68.04
AMK
65.26
ALK6
ALK7
Figure 2.3. (a) A color coded grid with the percent sequence identity for each BMP and TGF-P type I
receptor was determined using the alignment tool (http://www.uniprot.org/blast/). (b) Superposition of
ALK 1 and ALK2 demonstrating the high degree of similarity between these receptors despite their unique
functions.
2.1.3
Role of TGF-6 and BMP signalingin development and homeostasis
During embryogenesis BMP, nodal and activin signaling play major roles in tissue
patterning and axis determination [19]. Concentration gradients of ligands (e.g. BMP4 and
BMP7) and endogenous antagonists (e.g. noggin) lead to specific downstream signaling in the
relevant cells that determines the ventral to dorsal axis [20, 21]. Additionally, nodal signaling
through type I receptors ALK4 and ALK7 plays a role in the formation of the three germ layers:
endoderm, mesoderm, and ectoderm [22, 23]. In addition to embryonal patterning members of
the TGF-P superfamily of signaling ligands are involved in the development of many organs.
For example, TGF-p ligands are known to induce the epithelial-mesenchymal transition (EMT)
of endocardial cells leading to invasion of the heart cushion and eventually heart valve formation
[24]. BMPs and GDFs are also known to play critical roles in limb and digit formation [25, 26].
Long after development, the TGF-P superfamily continues to be important for the maintenance
of tissue homeostasis.
For example, signaling by BMP9/10 through ALKI and co-receptor
endoglin serves as an important endothelial quiescence factor in the maintenance of the
vasculature [27, 28]. TGF-p signaling has been shown to be important in wound healing and
maintenance of extracellular matrix (ECM) [29, 30].
As can be appreciated from this brief
review, signaling from the TGF-p superfamily of ligands is absolutely essential for normal
23
development and tissue homeostasis.
Many diseases have been identified that involve
disruptions in TGF-3 signaling and are described in 2.2.1.
TGF-P/Nodal Family
BMP Family
Type I Receptors
TGFPRII
ActRila, ActRilb, BMPRII
Ligands
TGFP1-3, Activins, Nodal
BMPs
Table 2.1. Members of the TGF-p and BMP signaling family including receptors, ligands, co-receptors,
and endogenous inhibitors.
2.2
Diseases of TGF-p and BMP Signaling
TGF-3 and BMP signaling have been implicated in many common diseases such as
atherosclerosis to extremely rare conditions such as fibrodysplasia ossificans progressiva. In
many cases TGF-P and BMP signals mediate certain aspects of disease pathophysiology but are
not in and of themselves disease causing. In other cases, however, gain or loss of function in
TGF-p and BMP signaling is directly responsible for the disease phenotype. In this section we
provide a brief overview of the role of TGF-P and BMP signaling in disease with a particular
focus on a rare genetic disorder caused by excessive BMP signaling.
2.2.1
TGF-3 and BMP signalingin disease
It has become increasingly appreciated that disordered BMP signaling contributes to
developmental and postnatal disease [31, 32]. There are at least six known heritable disorders
caused by germline mutations in members of the TGF- superfamily including heritable forms of
pulmonary arterial hypertension (PAH), hereditary haemorrhagic telangiectasia (HHT1/2),
juvenile polyposis syndrome (JPS), Loeys-Dietz syndrome, and fibrodysplasia ossificans
progressiva (FOP). Familial PAH, caused by heterozygous loss-of-function mutations in the
BMP type II receptor BMPR2 transmitted in an autosomal dominant fashion with incomplete
penetrance (-20%), is characterized by pathological remodeling of the small arterioles
(hypertrophy of medial smooth muscle and intimal thickening) in the lung leading to increased
24
resistance, elevated pressures (>25 mmHg), eventual right heart failure, and high mortality [27,
33-35]. HHT1 and HHT2, caused by loss-of-function mutations in the TGF-$/BMP co-receptor
endoglin and the BMP type I receptor ALKI, are characterized by dilated blood vessels at the
surface of the skin and mucous membranes known as telangiectasias that often cause epistaxis
and gastrointestinal bleeding and vascular abnormalities in other organ systems such as the lung
and brain, which can lead to significant morbidity and mortality [3, 36]. Up to 50% of JPS cases
are caused by loss-of-function mutations in the common TGF-$/BMP signaling mediator
SMAD4 or the BMP type I receptor ALK3 [37, 38]. JPS is characterized by the development of
hamartomatous gastrointestinal polyps and a up to 50% lifetime risk of developing cancer [13].
Loeys-Dietz syndrome, which shares many phenotypic similarities with Marfan syndrome
caused by deficiencies in extracellular matrix protein fibrillin-1, is caused by loss-of-function
mutations in TGF-
type I and type II receptors ALK5 and TGFBR2, respectively, and is
characterized by craniofacial abnormalities such as cleft palate, hypertelorism as well as
aneurysms of the aortic root and other vessels [39, 40].
FOP, caused by gain-of-function
mutations in BMP type I receptor ALK2, is a rare and debilitating disorder characterized by the
development of progressive heterotopic ossification of the skeletal muscle and connective tissue
leading to joint immobility, pain, and premature death [41, 42]. FOP is discussed in more detail
in section 2.2.2. As can be appreciated from this brief review of the currently known monogenic
diseases of TGF-p and BMP signaling, these pathways are critically important for homeostasis
and maintenance of many tissues and their disruption can incur a multitude of deleterious and
diverse effects.
In addition to monogenic diseases, the TGF-P and BMP signaling pathways
have been show to contribute to the complex pathogenesis of many diseases including fibrosis,
atherosclerosis, cancer and anemia of inflammation [31, 43-45] [46].
The role of TGF-P signaling as a tumor suppressor had long been suspected due to its ability
to inhibit cellular proliferation [47]. Two papers published in 1995 reported the first direct
evidence of TGF-P tumor suppression activity in both human disease, where mutations in
TGFpR2 were identified in 8 colon cancer cell lines, and in a mouse model of breast cancer,
where in vivo overexpression of TGFp1 significantly reduced tumor development [48, 49].
Since those early findings, a multitude of components of the TGF-P and BMP signaling pathway
have been implicated in cancer development and progression including SMAD4, ALK3, and
ALK2 [50, 51].
Despite serving a tumor suppression function, significant evidence has
25
demonstrated that after carcinogenesis TGF-
signaling promotes proliferation and metastasis
through effects on tumor microenvironment, cancer stem cells and epithelial to mesenchymal
transition (EMT) [11, 52, 53]. Recently, BMP signaling through ALKI has been shown to be
pro-angiogenic and could serve as a therapeutic target against tumor vascularization and
progression [54, 55]. Fibrosis is the accumulation of extracellular matrix connective tissue as a
result of injury repair mechanisms. When it occurs inappropriately or in excess, fibrosis leads to
a loss of normal function in affected tissues. Excess TGF- signaling has been demonstrated in
multiple models of fibrosis including pulmonary, hepatic, renal, cardiac, and scleroderma [44].
The TGF-s and BMP signaling pathways play critical roles in all stages of cancer and may
potentially serve as therapeutic targets.
Anemia of inflammation (AI) is typically a mild to moderate normocytic anemia
associated with chronic diseases including neoplastic disorders, rheumatoid arthritis, and
multiple myeloma [56, 57]. It is thought that inflammatory signals such as IL-6 stimulate the
synthesis of hepcidin in the liver to suppress iron absorption and bioavailability, leading to
impaired erythropoiesis [58, 59].
Recently the role of hepatic BMP signaling has been
implicated in the regulation of hepcidin synthesis in response to iron loading, IL-6 signaling, and
other types of inflammation [14, 60-63]. Use of BMP type I receptor kinase inhibitor LDN1931879 was shown to normalize iron levels and blood counts associated with chronic
inflammation [64]. These results suggest that therapeutic intervention with kinase inhibitors can
be an effective means of treating diseases where the pathogenesis results from excess or
potentially disregulated BMP signaling.
2.2.2 Fibrodysplasiaossificansprogressiva
One of the most striking examples of BMP signaling-related disease is seen in fibrodysplasia
ossificans progressiva (FOP), an extremely rare and disabling genetic disease with an estimated
prevalence of 1 in 2 million affecting an estimated 3,000 people worldwide and with only 800
diagnosed patients [42]. In familial cases, FOP is inherited as an autosomal dominant disorder.
Individuals with the classical form of FOP are nearly normal at birth except for cervical and
hallux valgus joint deformities.
However, during the first decade of life they develop
progressive formation of endochondral bone in muscles, fascia, and ligaments leading to severe
immobility, pain, and premature mortality a consequence of restrictive lung disease due to
26
thoracic involvement [41, 65].
A highly recurrent gain-of-function mutation in the glycine-
serine (GS) rich domain of the BMP type-I receptor ALK2 (c.617G>A; p.R206H) accounts for
more than 98% of cases of classic FOP [66].
Several other FOP-causing gain-of-function
mutations in both the GS and kinase domains of ALK2 have also been described in non-classic
or variant forms of FOP (Figure 2.4a) [67-70]. The arginine to histidine mutation within the GS
activation domain of ALK2 is non-conservative and abrogates the normal requirement for
phosphorylation for activation of the kinase. Structural modeling of the mutant and wild-type
receptor predicts that the R206H mutation disrupts alpha helical structure of the GS domain and
prevents intramolecular salt-bridging, exposing the active site and rendering the enzyme
constitutively-active [71, 72]. It is thought that enhanced ligand-induced, and potentially ligandindependent BMP signaling potentiate osteogenic differentiation in mesenchyme-derived
progenitors in these individuals. Constitutively-active mutant kinases, such as BCR-ABL have
been successfully targeted by small molecule inhibitors such as imatinib". ALK2R206 H, with a
constitutively-active intracellular kinase domain, is unlikely to be affected by endogenous
antagonists of BMP signaling such as chordin or noggin, which sequester BMP ligands, or by
receptor- or ligand-neutralizing antibodies. ALK2 R26 H thus represents an ideal therapeutic target
for a highly selective small molecule kinase inhibitor in the treatment of FOP.
(a)
anQ207E
(b)
~
(
Pi47jJ96ensL
G328E
375P
Figure 2.4. (a) FOP-causing mutations in both the GS loop and kinase domain. (b) Soft tissue nodules
and HO lesions on the back of a 4-year-old child affected by FOP. (c) The same child at 8 years of age
showing extensive HO of the back. Adapted from [73] and [74].
27
2.3
Kinase Inhibitors
In the last decade protein kinases have become an increasingly important class of drug
targets, accounting for 20% of preclinical research targets for pharmaceutical and biotechnology
companies. Strategies to block receptor kinase signaling include monoclonal antibodies, ligand
traps, and small molecule kinases inhibitors. The latter are particularly effective when targeting
constitutively-active kinases that function independently of activation or ligand-mediated
signaling, as is the case for many mutant kinases found in cancer. In fact, currently there are 22
FDA-approved small molecule kinase inhibitors, generating $30B in worldwide sales, 21 of
which have been approved for cancer indications, the majority targeting a few oncogenic kinase
such as BCR-AbL, VEGFR, EGFR, and RAF [75-77].
Just recently tofacitinib, which
selectively targets JAK3, was the first kinase inhibitor approved for a non-cancer indication,
rheumatoid arthritis. This development provides a proof of concept that kinase inhibitors can be
successfully developed to treat non-cancer diseases.
While certainly not all kinases will be
druggable, with only a small fraction (4%) of the human kinome currently targeted by FDAapproved drugs there is ample opportunity for further development of novel kinase inhibitor
therapeutics.
2.3.1
Kinase inhibitor development and clinical use
The human kinome consists of 518 protein kinases that share a bilobular structure and a
highly conserved ATP binding pocket (Figure 2.5a) [77]. The development of highly selective
kinase inhibitors is challenging, particularly given the conserved nature of the ATP binding site,
but is critical for reducing the potentially dose-limiting effects of off-target inhibition [78]. The
adenine ring of ATP forms hydrogen bonds at the kinase hinge that connects the amino and
carboxy-terminal domains of the kinase [79]. There are four classes of small molecule kinase
inhibitors: type I, type II, type III, and covalent.
Most protein kinases have a conserved
activation loop which regulates kinase activity, and is marked by a conserved DFG
(corresponding to its one-letter amino acid sequence) motif at the start of the loop.
Conformations of the activation loop include those that are catalytically active, and those that are
inactive in which the activation loop blocks the substrate binding site, a.k.a., the "DFG-out"
conformation. The vast majority of current kinase inhibitors mimic ATP by forming one to three
hydrogen bonds to the hinge and typically also interact with adjacent hydrophobic regions for
28
increased affinity [79, 80]. Type I kinase inhibitors reversibly bind the active conformation of
the kinase, compete with ATP, and generally are less selective given the highly conserved nature
of the ATP binding site [81].
Type II kinase inhibitors reversibly bind to the inactive
conformation of the kinase (DFG-out), do not compete with ATP, and are generally more
selective given they target an additional hydrophobic binding site [82]. Type III or allosteric
kinase inhibitors reversibly bind to sites outside of the ATP binding pocket, do not compete with
ATP, and are the most selective inhibitors because they bind highly unique pockets within their
target kinase and stabilize inactive kinase conformations [83] [84].
Finally, covalent kinase
inhibitors first bind non-covalently to kinases within ATP or allosteric pockets and then an
electrophilic moiety reacts with a nucleophilic thiol group of nearby cysteine residues that exist
in some kinases [85, 86]. Once bound, covalent kinase inhibitors permanently shut down their
target kinase activity until new protein is synthesized, resulting in unique pharmacokinetic
properties [86].
Recently the FDA approved the irreversible kinase inhibitors afatinib and
ibrutinib for the treatment of metastatic non-small-cell lung cancer and mantle cell lymphoma,
respectively, proving the value of this kinase inhibitor class [87].
(a)
(b)
residue
Hydrophobic
reion I
R . NH
0D
C
O
HN
R
0
H'. H Adenine
hinge
0
*
ATP
pocket
N H ,til'1
1
N
Phosphate-binding
R
-
HN
N
N
0.- P.~
region
1
Hydrophobic
region 1
\
HN'R
H
%R
0
HN
Figure 2.5. (a) Structural overview of ALK2 demonstrating the bilobular structure of kinases including
the kinase domain, hinge, GS domain, ATP binding pocket, and interacting regulatory protein FKBP12.
(b) The highly conserved ATP binding region of kinases showing hydrogen bonding interactions between
29
adenine and the hinge as well as hydrophobic pockets that are amenable to inhibitor binding. Adapted
from [88].
2.3.2
TGF-3 type ] receptor kinase inhibitors
TGF-
signaling inhibition has been investigated thoroughly by both academia and industry
as a potential therapeutic for renal, liver, and pulmonary fibrosis as well as for cancer [89, 90].
Previously, well-described small molecule inhibitors of the TGF-P type I receptor kinases, such
as A-83-01 and SB-505124, have both high potency and high (> 3 log) selectivity for TGF-p
versus BMP signaling [91, 92].
While TGF-P signaling inhibitors had potential utility as
therapeutic agents, preclinical animal studies have associated the administration of highly potent
ALK5 inhibitors with bone physeal abnormalities in immature animals, diffuse pulmonary
hemorrhages, and catastrophic hemorrhagic necrosis of heart valves in adult animals [90, 93-95].
The observed toxicity was class wide and not compound specific which resulted in several ALK5
inhibitor programs to be halted and abandoned [96]. Thus, a clinically viable BMP signaling
inhibitor, particular for long term indications such as FOP, should significantly reduce off-target
inhibition of Activin/TGF-3 type I receptor kinases (ALK4, ALK5, and possibly ALK7).
2.3.3
BMP type 1 receptorkinase inhibitors
Targeting individual ligands and receptors by genetic epistasis has yielded important
insights into function, but their interpretation is limited again by redundancy as well as
embryonic effects.
Pharmacologic strategies for modulating BMP and TGF-P signaling have
emerged as a promising strategy for elucidating function and specificity in these pathways.
These strategies include small molecule kinase inhibitors and recombinant protein ligand-traps
[32]. On the other hand, the high degree of homology between receptors of this pathway makes
the development of selective small molecule BMP type I receptor inhibitors particularly
challenging.
In a high-throughput phenotypic screen examining embryonic zebrafish development,
dorsomorphin was identified as a small molecule inhibitor of BMP-mediated signaling, based
upon its potent ability to induce dorsalization [63].
Subsequent medicinal chemistry efforts
resulted in a highly potent BMP inhibitor, LDN-193189, which demonstrated therapeutic benefit
in a mouse model of FOP [97].
In addition, K02288, based on a distinct 2-aminopyridine
scaffold, was recently identified by our collaborator Alex Bullock at the Oxford University
30
Structural Genomics Consortium [98].
These compounds demonstrate varying degrees of
selectivity for the BMP and TGF-P type I receptors. These molecules in some cases target an
array of other kinases, including several receptor tyrosine kinases (RTKs).
These off-target
effects within the TGF- signaling pathway and among RTKs may potentially limit their utility
as biologic probes of BMP function and therapies. Highly selective agents targeting ALK2 and
other individual receptors of this pathway would be valuable.
Selective BMP receptor inhibitors may have utility as pharmacologic probes of BMPmediated signaling, and therapies for BMP-mediated disease.
A common challenge to the
development of selective ATP competitive small molecule kinase inhibitors is the structural
homology of highly conserved ATP binding domains [99]. Structural homology is particularly
high between the type I receptors of the BMP and TGF-p signaling pathways. For example, the
ALK3 kinase domain possesses 66% sequence identity with that of ALK5 [18]. Even greater
kinase domain sequence identity is found between homologues within the BMP or TGF-$
families, such as ALKi and ALK2 (79%), ALK3 and ALK6 (86%), and ALK4 and ALK5
(90%). The high degree of structural homology between receptors poses serious challenges for
the development of highly selective small molecules that can discriminate between the individual
members of the TGF-P or BMP receptor families.
Highly selective inhibitors could be useful as therapeutics for diseases mediated by
inappropriate signaling of an individual type I receptor, exemplified best by FOP (2.2.2).
Clinically viable BMP receptor kinase inhibitors, particularly for indications requiring long term
treatment, will optimally have a high degree of selectivity within the BMP and TGF-P receptor
family, and in the kinome (2.3.2).
2.4
Summary
The overall goal of this thesis was to develop and characterize highly selective inhibitors of
the bone morphogenetic protein (BMP) signaling pathway for use as biological probes and as
potential therapeutic compounds. In preliminary work, small molecule inhibitors of the BMP
type I receptor serine-threonine kinases with varying degrees of potency and selectivity for
individual receptors within this family were developed. These reagents have proven to be useful
for investigating BMP signaling biology both in vitro and in vivo. We have refined the activities
of these small molecules, via medicinal chemistry, structural biochemistry, molecular modeling,
31
and using novel, highly sensitive assays for activity of individual BMP receptors. We confirmed
the efficacy of these compounds, and refined their properties further using cell-based and animal
models of disease mediated by excessive or inappropriate BMP signaling. A highly selective
and potent inhibitor of the BMP type I receptor, ALK2, would by clinically useful in the
treatment of a rare and severe genetic disorder, fibrodysplasia ossificans progressiva (FOP).
Such an inhibitor would also serve as an important scientific tool to identify functions of this
receptor and its cognate ligands in normal and disease physiology.
32
Chapter 3 Characterization of dorsomorphin derivatives
This chapter describes the characterizationof a previously synthesized but poorly described
set of dorsomorphin derivative. The goal of these studies was to establish a structure activity
relationship (SAR) based on this derivative libraryfor BMP and TGF-3 type I receptors (ALK]5). By using a highly sensitive high throughput radio-kinaseassay we developed, we were able to
accurately discern previously unknown activity of these molecules and uncover a novel and
highly selective BMP type I receptor kinase inhibitor. These observations have formed the basis
for novel compounds and hypotheses describedand tested in Chapter4.
3.1
Background and Motivation
During embryogenesis, BMP signaling gradients define the dorsoventral axis [100, 101].
These gradients are the result of highly specific interactions between ligands, receptors, and
antagonists [102, 103]. It has been shown that enhanced BMP signaling during embryogenesis
results in ventralization while reduced BMP signaling results in dorsalization [104, 105].
Previously, our lab exploited the embryonic patterning role of BMP signaling to develop an in
vivo phenotypic zebrafish screen. We tested approximately 7,500 compounds for BMP signaling
modulation, both antagonistic (dorsalization) and agonistic (ventralization), while also screening
against non-specific and undesirable biologic effects such as toxicity [63]. The result of this
screen was the discovery that compound C (Figure 3.1b), previously described as a weak AMPactivated protein kinase (AMPK) inhibitor [106], was a potent inhibitor of BMP signaling.
Compound C was subsequently renamed to dorsomorphin for its ability to dorsalize zebrafish
embryos. Dorsomorphin, based on a substituted pyrazolo[1,5-a]pyrimidine core (Figure 3.1a),
was also shown to inhibit BMP-4 induced phosphorylation of SMAD1/5/8 in cultured pulmonary
artery smooth muscle cells (PASMCs) with a half maximal inhibitory concentration (ICso) of
0.47tM, while showing no inhibition of TGF-P induced phosphorylation of SMAD2 [63].
(a)
(b)
(c)
HN
N
N-
N
R2
N
NN
N
R3
N
N
Figure 3.1 (a) Structure of the pyrazolo[l,5-a]pyrimidine core with R-groups at the 5-position of the
pyrimidine (R1) and the 2-(R 2) and 3-(R 3) positions of the pyrazolo. (b) The structure of dorsomorphin
with a piperidinyl ethoxy phenyl at R1,hydrogen at R2, and 4-pyridyl at R3. (c) The structure of LDN193189 with a phenylpiperazine at R1, hydrogen at R2, and 4-quinolyl at R3.
33
Derivatives of substituted pyrazolo[1,5-a]pyrimidine,
were synthesized as part of a
medicinal chemistry effort to improve upon dorsomorphin's moderate potency against BMP
signaling, selectivity, and metabolic stability [107].
Derivatives of dorsomorphin where
synthesized by varying the substituent groups at R1 , R2, and R3 (Figure 3.1a). An SAR focused
on potency of BMP signaling inhibition was developed using a cell-based assay measuring
BMP4-induced phosphorylation of SMADl/5/8, leading to the synthesis of a far more potent
BMP inhibitor LDN-193189 (Figure 3.1c).
The SAR also demonstrated that dorsomorphin's R3 substituted 4-pyridyl group was critical
for BMP inhibition as a 3-pyridyl, phenyl, or hydrogen R3 substitution resulted in an almost
complete abrogation of BMP signaling inhibition. R2 substitutions, apart from hydrogen, were
tolerated and detrimental to potency. A 4-methoxyphenyl Ri substitution resulted in a ten-fold
decrease in potency. Various RI aminoether substitutions at the 4-position of the pendent phenyl
ring did not significantly change the potency of BMP signaling inhibition. Finally, a ten-fold
increase in BMP signaling inhibition was observed with a 4-quinoline R3 substitution.
Furthermore the replacement of the aminoether on the 4-position of the pendent phenyl ring with
piperazine, as seen in LDN-193189, resulted in a further enhancement of BMP signaling
inhibition and an IC 50 of 5nM. LDN-193189 also demonstrated greater metabolic stability in
mouse liver microsomes over dorsomorphin with a half-life (tl/2) of 82 minutes versus 10.4
minutes for dorsomorphin.
Although potent, LDN-193189 retained activity against TGF-
signaling at higher concentrations (> 500 nM) and could not differentiate between the various
BMP type I receptors such as ALKI, ALK2, and ALK3[108]. Motivated by the in vivo toxicity
seen with TGF-p inhibitors, as described in 2.3.2, we sought to create a more thorough SAR of
this same library to develop highly selective inhibitors of BMP type I receptors.
3.2
Experimental Methods
3.2.1
Traditionalradiometrickinase assay
Purified recombinant ALK1 -5 and other kinase proteins (Invitrogen), ATP (Sigma), ATP
[y-32P] (Perkin Elmer), and dephosphorylated casein (Sigma) at final concentrations of 2.5nM,
6ptM, 0.05 pCi pL-,
and 0.5 mg mL-1 respectively were aliquoted in kinase buffer (Cell
Signaling) containing 0.2% bovine serum albumin supplemented with 10mM MnCl2 into 96-well
plates, in combination with inhibitor compounds diluted at varying concentrations in kinase
34
buffer (0.01nM to 10 pM) in triplicate. Positive control samples lacking inhibitor compounds,
and negative controls lacking recombinant kinase were also measured in triplicate. The mixture
was reacted at RT for 45 minutes, quenched with a final concentration of 2% phosphoric acid.
The reaction mixture in each well is individual transferred to 2.1 cm P81 phosphocellulose paper
discs (Whatman) and allowed to dry for 30 minutes. Once dry, discs were placed in a 4L
Erlenmeyer flask containing 2L of 1% phosphoric acid solution and vortexed by hand for 2 to 3
minutes. The wash was repeated 3 times. The discs were set out to dry for 30 minutes while
scintillation vials were labeled. Discs were then individually transferred to scintillation vials
which were then filled with scintillation fluid (Perkin Elmer), capped, and placed on racks.
Racks were transferred to a scintillation counter (Beckman) for measurement of the scintillation
(light output) per vial over the course of 5 minutes per vial and averaged (CPM). A schematic
representation of the traditional kinase assay can be seen in Figure 3.2.
(a)
(b)
(c)
(d)
Figure 3.2. Schematic representation of a traditional kinase assay with the kinase bound radiolabeled
ATP (a) phosphorylating its protein substrate (b) which is then transferred to phosphocellulose paper (c)
and finally placed in a vial with scintillation fluid (d) for light output measurement.
The amount of light output measured results from the effects of the ionizing radiation on the
scintillation fluid and is directly proportional to amount of radiation remaining on the washed
discs which is itself directly proportional to the amount of radiolabeled casein which is directly
proportional to kinase activity.
Data was normalized to positive controls at 100% enzyme
activity with negative controls being subtracted as background. GraphPad (Prism software) was
used for graphing and regression analysis by sigmoidal dose-response with variable Hill
coefficient.
35
3.2.2
High throughputradiometrickinase assay
To improve our efficiency in screening a library of diverse structural analogs, we developed
a high throughput radio-kinase assay. Figure 3.3 shows the size difference between to the assays
as well as quality control measures demonstrating that the high throughput assay, although
slightly more noisy, was very robust with a Z' factor of 0.71 and a signal to noise ratio of 19.
(b)
(C)
Traditional
High
Throughput
Coefficient
of variation
5.1%
9.8%
Z' Factor
0.79
0.71
Signa-onoise
42
19
Figure 3.3. (a)Traditional kinase assay setup including 96 scintillation vials, caps, and racks. (b)
Equivalent high throughput radiometric kinase assay setup with 96-well plate. (c) Comparison metrics
between traditional kinase assay and our optimized high throughput assay.
Purified recombinant ALK1-5 and other kinase proteins (Invitrogen), ATP (Sigma), ATP [y32 P] (Perkin
Elmer), and dephosphorylated casein (Sigma) at final concentrations of 2.5nM,
6pM, 0.05 pCi pL-1, and 0.5 mg mL-1 respectively were aliquoted in kinase buffer (Cell
Signaling) containing 0.2% bovine serum albumin supplemented with 1 OmM MnCl2 into 96-well
plates, in combination with inhibitor compounds diluted at varying concentrations in kinase
buffer (0.0lnM to 10 pM) in triplicate. Positive control samples lacking inhibitor compounds,
and negative controls lacking recombinant kinase were also measured in triplicate. The mixture
was reacted at RT for 45 minutes, quenched with a final concentration of 2% phosphoric acid.
The reaction mixture was transferred to 96-well P81 phosphocellulose filter plates (Millipore)
and bound for 5 minutes. The plates were washed twenty-times with 150 pL of 1% phosphoric
acid solution per well by vacuum manifold. Plates were dried at RT for 1 h, sealed, and assayed
with Microscint 20 scintillation fluid (Perkin Elmer) using a Spectramax L luminometer
(Molecular Devices) using the photon counting setting with an integration time of one second per
well. Data was normalized to positive controls at 100% enzyme activity with negative controls
36
being subtracted as background. GraphPad (Prism software) was used for graphing and
regression analysis by sigmoidal dose-response with variable Hill coefficient.
3.3
Results and Discussion
3.3.1
Km determination for ALK]-5
In order to screen our library of dorsomorphin derivatives against ALKi -5, we had to
first determine for each kinase the concentration of ATP necessary to achieve the half maximal
enzyme velocity (K). The half maximal inhibitory concentrations (IC 50 ) for each inhibitor
compound were at ATP concentrations equal to that particular kinase's Km and thus the IC 5o
values are comparable across the various BMP and TGF-P type I receptor kinases tested. Km
values for ALK1-5 were determined by reacting excess dephosphorylated protein substrate
(casein) with varying concentrations of ATP for 30 minutes (within the linear range of enzyme
activity).
The assay was then carried out as described in 3.2.2. The Km values for were
determined by non-linear regression analysis based on the Michaelis-Menten equation (Figure
3.4). Km values for BMP and TGF-3 type I receptors ranged from 2.7-16.7gM. These
corresponding ATP concentrations were used in subsequent compound screening (3.3.2).
(a)
(b'j
ALK Activity
1000-
Km = 2.7
800-
f
pM
ALK3 Activity
m=
5.8 pM
Km = 16.7 pM
1500,
2000
600
>
ALK2 Activity
2500-
1000
a 1500
40
1000
20
500
500.
0.
(d)
50
100
15C
[ATP] uM
50
(e)
ALK4 Activity
400&
0
Km= 3.9 pM
100
15(
[ATP) uM
50
(f
ALK5 Activity
Km = 6.8 pM
1500-1
0
[ATPJ uM
100
Kinase
Km (pM)
ALK2
5.8
150
i
X
3000]
1000.
C.
200&
500.
100
ALK4
6
200
400
60C
0
3.9
RP 1N m
n-
100
200
300
Figure 3.4. Km determinations for (a) ALKI, (b) ALK2, (c) ALK3, (d) ALK4, and (e) ALK5. (f)
Summary of Km values for BMP and TGF-0 type I receptor kinases.
37
.i1Mii!
3.3.2
Screening of dorsomorphinderiviatives againstALKJ-5
With appropriate Km values for each kinase determined, we conducted a high throughput
screen of 34 dorsomorphin derivatives generating IC 50 (nM) values for the BMP type I receptors
ALKl-3 and the TGF-P type I receptors ALK4 and ALK5. These findings as well as the results
from a previous cell-based BMP4-induced pSMAD assay are summarized below (Table 3.1.).
Compound
Structure
BMP4 ALKI
ALK2 ALK3 ALK4 ALK5
HO
N
193189
5
N
6
2
63
952
197
9,904
n.a.
n.a.
n.a.
n.a.
n.a.
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...
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.....
.....................................
.............
..
....
.... /.
N
... ...
193599
1,000 11,516
999
0
CO2Me
N
193649
n.a.
N
N
38
18,515 11,778
0
N
193651
15,000
N
N
801
130
600
7,889
7,080
n.a.
n.a.
n.a.
n.a.
N
193689
n.a.
18,954 18,724 17,892
..
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...
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41
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zt,
193720
N
N
450
cl
39
371
354
1,424
n. a.
3,964
194731
W' N
ZPI
N
rl-HN
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
XJ N
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pS A 1// in cell western.....
=.noactvit
We preiusly asae-hsstofdrvtvsuineITensont
in cel wetr asa17.HwvrsneBPa
1/5/8 phshr1to
fAK 19.T
preferntiall
bidtML3 hsasywslkl isdtwrsihbtr
sa
sn
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ovecom
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high
creenagaint
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Inpriuaw in the..cofre tht heN nitgn ofth
confidence~~~~~~~~~~~
rsls
aans.hseknae.1969
pyrazloprmdn core wa.bsltlycitclfo.ctvt.
19360, 13651 and19555) nd sbstiutios .athe......... 7-poitinoth qu oiewre ot el
tolerated..
(19720 19592 and29857)
mroe
TGF-3 Llinhbto an.t.evlp
To a Hllevaetxct ocen soitdwt
SR
tools for understanding BMP signaling, we determndtesrcueatviyrltosi
42
elbsdBM4idcdSA
of pyrazolo[1,5-a]pyrimidine derivatives with varied quinoline attachment positions (Figure
3.5b). The resulting SAR demonstrated that 3-, 8-, and 6-quinoline substituted derivatives had
little activity against either BMP or TGF-P type I receptors (Figure 3.5a). However, the 5quinoline derivative LDN-193719 exhibited improved BMP versus TGF-p selectivity compared
to the 4-quinoline derivative LDN-193688 (550-fold versus 60-fold selectivity, Figure 3.5c).
LDN-193719 also demonstrated greater potency against ALK2 (IC 50 = 44 nM) than ALK3 (IC 5 o
= 1.5 gM), providing a potential explanation of the weak inhibition of BMP4-induced signaling
previously observed by in-cell western assay[ 107].
(a)
IC50O (PM)
Compound
Position
ALK1
ALK2
ALK3
ALK4
ALK5
LDN-193689
LDN-193718
6-quinoline
8-quinoline
>50
NA
>50
NA
>50
NA
NA
NA
NA
NA
LDN-193719
LDN-193721
5-quinoline
3-quinoline
.092
NA
.044
NA
1.5
NA
21.1
NA
24.2
NA
(b)
(c)
600
0>
Fold selectivity over ALK2
" LDN-1 93688
* LDN-1 93719
400
N
(0
200
N
0
0
ALK
ALK2
ALK3
ALK4
ALK5
Figure 3.5. Structure-activity relationship of 4-methoxyphenyl pyrazolo[1,5-a]pyrimidine derivatives. (a)
In vitro kinase assay IC50 measurements against BMP and TGF-P type I receptors show only the 4quinoline (LDN-193688) and 5-quinoline (LDN-193719) derivatives are active. (b) Structure of five
pyrazolo[1,5-a]pyrimidine derivatives with varying quinoline attachment sites. (c) Fold selectivity of
active inhibitors against ALK2 versus BMP and TGF-P type I receptors. Although slightly less potent
against ALK2 than LDN-193688, the 5-quinoline derivative LDN-193719 has much greater selectivity
for BMP vs. TGF-0 type I receptors.
43
3.4
Conclusion
By thoroughly screening a previously synthesized library of derivatives based on
dorsomorphin, a moderately potent BMP type I receptor kinase inhibitor, against ALK1, ALK2,
ALK3, ALK4, and ALK5 we were able to discover that the 5-quinoline moiety at the R3 position
resulted in far greater selectivity than the 4-quinoline moiety of LDN-193189. We additionally
hypothesized that this increased selectivity for BMP versus TGF-P and particularly for ALK2
might be generalizable to other 5-quinoline-substituted compounds.
Thus we synthesized a
novel derivative, LDN-212854, by combining the 5-quinoline moiety of LDN-193719 with the
phenyl-piperazine substituent of LDN-193189.
Characterizing this compound as well as all
known potent BMP signaling inhibitors is the focus of Chapter 4.
44
Chapter 4 Development of potent and selective ALK2 inhibitors
This chapter describes the development of a potent and selective inhibitor of the BMP type I
receptor kinase ALK2. Based on the results in Chapter 3 we hypothesized that the selectivity of
LDN-193189 could be significantly improved with a 5-quinoline group substituted at the R3
position of the pyrazolo[1,5-a]pyrimidine core. We synthesized this novel derivative, LDN212854, and characterizedits activity in comparison to all known potent BMP type I receptor
inhibitors including dorsomorphin, LDN-193189, DMH] and K02288. LDN-212854,
demonstrateda significantimprovement in selectivity for the BMP versus TGF-3 type I receptors
and was biasedtowards ALK2 versus the other BMP type I receptors ALK] and ALK3.
4.1
Background and Motivation
Pharmacologic strategies for modulating BMP and TGF-p signaling have emerged as a
promising strategy for elucidating function and specificity in these pathways. These strategies
include small molecule kinase inhibitors and recombinant protein ligand-traps [32]. A common
challenge to the development of selective ATP competitive small molecule kinase inhibitors is
the structural homology of highly conserved ATP binding domains [99]. Structural homology is
particularly high between the type I receptors of the BMP and TGF-$ signaling pathways. For
example, the ALK3 kinase domain possesses 66% sequence identity with that of ALK5 [18].
Even greater kinase domain sequence identity is found between homologues within the BMP or
TGF-3 families, such as ALKI and ALK2 (79%), ALK3 and ALK6 (86%), and ALK4 and
ALK5 (90%).
The high degree of structural homology between receptors poses serious
challenges for the development of highly selective small molecules that can discriminate
between the individual members of the TGF-p or BMP receptor families.
Highly selective inhibitors could be useful as therapeutics for diseases mediated by
inappropriate signaling of an individual type I receptor, exemplified best by fibrodysplasia
ossificans progressiva (FOP). FOP is caused by a constitutively-active intracellular kinase,
ALK2P206 H and is unlikely to be affected by endogenous antagonists of BMP signaling such as
chordin or noggin, which sequester BMP ligands, or similar ligand-traps.
ALK2R20 6 H thus
represents an ideal therapeutic target for a highly selective small molecule kinase inhibitor as a
treatment for FOP.
45
4.2
Experimental Methods
4.2.1
Cell culture
C2C12 Bre-Luc and 293T CAGA-Luc cells were were cultured in DMEM supplemented
with 10% FBS, L-glutamine, penicillin, and streptomycin (Life Technologies) at 37 0 C and 10%
CO 2 . HepG2 human hepatoma cells (ATCC) were cultured in EMEM (Life Technologies)
supplemented with 10% FBS, L-glutamine, penicillin, and streptomycin at 37 0 C and 10% Co 2.
C2C12 myofibroblasts (ATCC) were cultured in DMEM supplemented with 10% FBS, Lglutamine, penicillin, and streptomycin at 37 0 C and 10% C02.
Pulmonary arterial smooth
muscle cells (PASMCs) were isolated from both wild type and BMPR2fl"jflo mice and the latter
exposed to adenovirus specifying Cre recombinase (Ad. Cre) to generate BMP type II receptor
deficient (BMPR2del/del) cells as previously described [110]. PASMCs were cultured in RPMI
medium (Life Technologies) supplemented with 10% FBS, L-glutamine, penicillin, and
streptomycin at 37 0 C and 5% CO2.
4.2.2
Luciferase reporter assay
C2C12 myofibroblast cells stably transfected with BMP responsive element from the Idl
promoter fused to luciferase reporter gene (BRE-Luc) were generously provided by Dr. Peter ten
Dijke (Leiden University Medical Center, NL)[ 11]. Human embryonic kidney 293T cells stably
transfected with the TGF-P responsive element from the PAI-1 promoter fused to luciferase
reporter gene (CAGA-Luc) were generously provided by Dr. Howard Weiner (Brigham and
Women's Hospital, Boston, MA)[1 12]. C2C12 Bre-Luc and 293T CAGA-Luc cells were seeded
at 20,000 cells in DMEM supplemented with 2% FBS per well in tissue culture treated 96-well
plates (Costar@ 3610; Corning). The cells were incubated for 1 h (37 0 C and 10% C02) and
allowed to settle and attach. Compounds of interest or DMSO were diluted in DMEM and added
at final compound concentrations of 1 nM to 10 pM. Cells were then incubated for 30 min.
Adenovirus expressing constitutively active BMP and TGF- type I receptors (Ad.caALK1-5),
generously provided by Dr. Akiko Hata (University of California at San Francisco), were added
to achieve a multiplicity of infection (MOI) of 100. Plates were incubated overnight at 37 0 C.
Cell viability was assayed with an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium
bromide) colorimetric assay (Promega) per the manufacturer's instructions.
Media was
discarded, and firefly luciferase activity was measured (Promega) according to manufacturer's
46
protocol. Light output was measured using a Spectramax L luminometer (Molecular Devices)
with an integration time of one second per well. Data was normalized to 100% of incremental
BRE-Luc activity due to adenoviruses specifying caALK1, 2, or 3, or the incremental CAGALuc activity due to adenoviruses specifying caALK4 or 5. Graphing and regression analysis by
sigmoidal dose-response with variable Hill coefficient was performed using GraphPad Prism
software.
BocN
O
Br
H NH
a
/\
Br
H2N :
___O
1
N
B 1O
b*
b
_N
3
2
HO
-OH
B
BocN
BocN
N
N
N
c
N'
7
-N
b
N,-
5
N
6
N
b
Br
HN
BocN
NN
NN
N-
d
N
~
N
LDN-212854/
8
N
-
N
Figure 4.1. Synthetic scheme of LDN-212854. (a) AcOH, MeOH, 80 0C, (61%). (b) Pd(PPh 3)4, 2.0 M
Na 2CO3, dioxane, 101 IC, (82-95%). (c) NBS, DCM, (63%). (d) TFA, DCM, then sat. NaHCO 3, (55%).
4.2.3
Ligand induced SMAD phosphorylation
Both WT and BMPR2del/del PASMCs were seeded in 12-well plates (BD Biosciences) at
75% confluency (-375,000 cells). The cells were attached for 1 hour at 37 0 C. Compounds were
diluted in RPMI and added at final concentrations from 1 nM to 25.6 pAM, and incubated with
cells for 30 min.
Cells were then stimulated with BMP7 and TGF-p1 ligands at final
concentrations of 20 ng mL-1 and 5 ng mL-1 respectively. Positive controls were generated by
omitting compounds and negative controls were generated by omitting both compounds and
47
ligands. The phosphorylation state of downstream effector proteins (pSMAD1/5/8 and pSMAD2
for BMP and TGF-p respectively) was measured by western blotting performed 30 minutes after
ligand stimulation. Western blots were analyzed using ImageJ with positive controls at 100%
ligand induced phospho-SMAD and negative controls being subtracted as background. Graphing
and regression analysis by sigmoidal dose-response with variable Hill coefficient was performed
using GraphPad Prism.
4.3
Results and Discussion
4.3.1
Characterizationof BMP inhibitors
We profiled the selectivity of the known inhibitors of BMP signaling dorsomorphin,
LDN-193189, DMH1, the 2-aminopyridine inhibitor K02288a, and our novel pyrazolo[1,5a]pyrimidine 5-quinoline derivative LDN-212854 (Figure 4.2a) [113, 114]. Based on the above
SAR data, we hypothesized that the 5-quinoline derivative might improve selectivity for the
BMP pathway, while maintaining potency. All inhibitors tested primarily targeted the BMP type
I receptors, particularly the highly homologous receptors ALKI and ALK2 (Figure 4.2b).
Importantly, whereas other investigators have assayed inhibition of
BMP receptor kinase
activity using > 100 nM enzyme [113], resulting in the lowest measurement of IC 5 o being
bounded by half the active enzyme concentration (e.g. ~ 50 nM), we used 2.5 nM of purified
kinase in this radiometric assay, permitting the measurement ICso values substantially lower than
those previously reported. For example, Vogt et al. reported an in vitro IC 50 of 45 nM for LDN193189 against ALK2, as compared to an IC50 of approximately 0.7 nM for ALK2 in our assay
[115].
Among compounds tested, LDN-193189 was the most potent inhibitor of ALK2,
followed closely by both K02288a and LDN-212854 (both with IC 5o ~ 1.2 nM). Compared to
these potent compounds, dorsomorphin and DMH1 were weaker by approximately 10-fold.
While LDN-193189 and K02288a inhibited the TGF-$ type I receptor ALK5 with approximate
IC 5 o measurements of 110 nM and 230 nM, respectively, whereas LDN-212854 inhibited ALK5
in the micromolar range, and thus had substantially greater selectivity for ALK2 versus ALK5
(Figure 4.2c,d). The selectivity for BMP versus TGF- signaling, based on the ratio of the IC 50
for the inhibition of ALK5 to ALK2, was approximately 7000-fold for LDN-212854, as
compared to 800-fold, 175-fold, 470-fold, and 200-fold for dorsomorphin, LDN-193189, DMH1,
and K02288, respectively (Figure 4.3). Thus, in a kinase inhibition assay using lower kinase
48
concentrations than previously described, LDN-212854 demonstrated much greater selectivity
for BMP versus TGF-
type I receptors than other compounds while retaining nanomolar
potency.
To confirm kinase assay results in cellular assays of BMP- and TGF-P-induced
transcriptional activity, we used C2C12 cells stably expressing BMP responsive (BRE-Luc) and
293T cells stably expressing TGF-s responsive (CAGA-Luc) luciferase reporter transgenes
[111].
Cells were transfected with adenoviruses expressing constitutively-active BMP type I
receptors (caALK1, caALK2, and caALK3) and constitutively active TGF-P type I receptors
(caALK4 and caALK5) in low serum conditions and in the absence of exogenous ligand, with
varying concentrations of inhibitors.
LDN-193189 was the most potent inhibitor of BMP
signaling (IC 50 ~ 11 nM for caALK2). LDN-193189 inhibited caALK5 with an IC50 ~ 213 nM,
demonstrating ~20-fold selectivity for caALK2 versus caALK5 in cells (Figure 4.2a).
Dorsomorphin was less potent than LDN-193189, and exhibited approximately 9-fold selectivity
for caALK2 vs. caALK5. While the cellular assay results for these two compounds aligned
closely with those obtained using the kinase assay, the activity of DMH1 and K02288a differed
significantly between kinase and cell-based assays. DMH1 was a relatively weak inhibitor of
caALK2 in cells (IC 5 o~ 230 nM) and had greater activity against caALK5 signaling (IC 50
700
nM) than might be expected based on kinase assay results. Thus, the selectivity of DMH1 for
caALK2 vs. caALK5 was only 2.5-fold in cells versus 470-fold in kinase assays. These cellbased IC 5 o data confirmed that DMH1 is a less potent inhibitor of BMP signaling than LDN193189, however, in our hands DMH1 did not significantly improve selectivity for BMP versus
TGF-P signaling as previously reported [113].[113] Despite data suggesting similar potency to
LDN-193189 in kinase assays, K02288a was significantly less potent in cells, inhibiting both
caALK2 (IC 50 - 225 nM) and caALK5 (IC 50
700 nM) modestly and without the same degree of
selectivity observed in kinase assays (- 3-fold vs. 200-fold).
49
(a)
HN3
HN
O
N
NNN
N
N
Dorsomorphin
LDN-19 3189
\
/
N
LDN-212854
\ N/
N\
>YO
N-N
.
HO
N
N
N
DMH1
(b)
NH 2
K02288a
N
'C50
(nM)
Compound
ALK
ALK2
ALK3
ALK4
ALK5
Dorsomorphin
LDN-193189
LDN-212854
DMH1
K02288a
19.5
1.48
2.40
77.9
3.65
9.76
0.67
1.30
12.62
1.20
222
14.3
85.8
241
25.8
3,080
108
2,133
11,023
232
7,829
117
9,276
5,971
236
(C)
Fold Selectivity over ALK2
Compound
ALK
ALK2
ALK3
ALK4
ALK5
Dorsomorphin
LDN-193189
LDN-212854
DMH1
K02288a
2
2
2
6
3
1
1
1
1
1
23
21
66
19
22
316
802
175
7,135
473
197
(d)
161
1,641
873
193
ALK2 Activity
ALK5 Activity
100
10
10
0
0
osoopi
50
* Dorsomorphin
0LDN-193189
* LDN-212854
k
DosMH1
>
hi
*DMH1
0 * KO2288a
-2
-1
0
0
log([])
nM
nM
2
-1
*KO2288a
0
1
2
log([inhibitor)
3
4
nM
Figure 4.2. Potency and selectivity of BMP inhibitors based on in vitro kinase assay. a) Structure of
previously described BMP inhibitors and LDN-212854. b) In vitro kinase assay measurements of IC for
50
BMP and TGF-P type I receptors show LDN-193189 is the most potent inhibitor of ALK2, followed
closely by K02288a and LDN-212854. Both DMH1 and dorsomorphin exhibited 10-fold lower potency
against BMP type I receptors. c) Fold selectivity of these inhibitors against ALK2 versus closely related
BMP and TGF-p type I receptors. d) Inhibition of ALK2 (BMP) and ALK5 (TGF-)
kinase activity
demonstrates LDN-212854 exhibits increased selectivity for ALK2 versus ALK5. Data shown are
representative of 2-3 independent experiments.
50
(a )
Fold Selectivity over ALK2 in Kinase Assay
8,000
m Dorsomorphin
7,135
mLDN-1 93189
m LDN-212854
*DMH1
4,000 -
* K02288a
1,641
0
ALK
(b)
15
1
66
ALK2
ALK3
2
(C)
BMP Receptors: Ratio of
IC50 to caALK2
T
ALK4
150
ALK5
TGF-p Receptors: Ratio of
IC50 to caALK2
*caALK1
mcaALK2
10
1%-100
10
5
131
mcaALK3
* caALK2
*caALK4
*caALK5
10s
6
,a
5.
.
O
[0.
1a
35
2
2
2
50
22
LL
2220
9~
V
1
1
132 3
nd1
6W
n
V
IV
Figure 4.3. Selectivity of known BMP inhibitors and novel inhibitor LDN-212854 based on in vitro
kinase assay and cell-based BMP (BRe-Luc) and TGF-p (CAGA-Luc) transcriptional activity mediated
by constitutively active ALKI-5. (a) Graphical representation of the fold selectivity of these inhibitors in
kinase assay against ALK2 versus closely related BMP and TGF-P type I receptors. (b) and (c) A
graphical representation of the fold selectivity of inhibitors for caALK2 over closely related BMP and
TGF- type I receptors. LDN-212854 has 6-10 fold selectivity for caALK2 versus other BMP receptors
(caALK1 and caALK3) and much greater selectivity (>100 fold) over the TGF- receptors (caALK4 and
caALK5). Having much lower potency against caALKl, 2 and 3 in cells, both K02288a and DMH1
exhibit substantially lower selectivity. Data shown are calculated based on IC 50 generated from at least 3
independent experiments, with data plotted as mean ± S.E.M.
In contrast, the BMP selectivity of LDN-212854 demonstrated in the kinase assay was
confirmed in cells with an IC50 for caALK2 of 16 nM and an IC5o for caALK5 of approximately
2 pM, resulting in more than 130-fold selectivity for caALK2 vs. caALK5 (Figure 4.2a and
Figure 4.5).
Representative inhibition curves for dorsomorphin, LDN-193189, LDN-212854,
51
K02288a, and DMH1 against constitutively active BMP and TGF-P type I receptors also
demonstrated the improved selectivity of LDN-212854. At 100 nM, LDN-212854 inhibited 98%
of caALK2-mediated signaling while exerting minimal effect on caALK4 or caALK5, whereas
all other compounds inhibited caALK4 and caALK5 significantly at concentrations required to
suppress caALK2 signaling to the same degree (Figure 4.3 and Figure 4.5b,c). In addition to
having improved selectivity for caALK2 versus caALK5, LDN-212854 demonstrated a bias
towards caALK2 within the BMP type I receptor family. LDN-212854 inhibited caALK2 with
6- and 10-fold more potency than caALK1 or caALK3, respectively (Figure 4.3).
To confirm results obtained using constitutively-active type I receptors, we tested the
selectivity of LDN-193189 and LDN-212854 against specific BMP and TGF-P ligands.
We
examined the impact of inhibitors upon BMP7-induced (20 ng mL-, 30 min.) activation of
SMAD1/5/8 using BMPR2-deficient pulmonary vascular smooth muscle cells, which we have
previously found to use ALK2 almost exclusively for the transduction of BMP7 signaling [110].
We used TGF-P ligand (5 ng mL-1, 30 min) to induce TGF- f signaling and phosphorylation of
SMAD2.
In this ligand-based cellular assay, LDN-212854 and LDN-193189 exhibited
comparable, low nanomolar potency in blocking the phosphorylation of SMAD1/5/8 induced by
BMP7 in BMPR2-/- cells (Figure 4.6a). Importantly, the selectivity of LDN-212854 for BMP
versus TGF-p signaling was even greater than that observed using constitutively active type I
receptors, with almost no inhibition of TGF-p1 signaling at the highest concentration tested (25
pM, Figure 4.6b), in contrast to significant effects observed above 1 pM for LDN-193189.
These ligand-mediated signaling assays confirmed that LDN-212854 inhibits BMP versus TGF-
p signaling with nearly 4-log selectivity, much greater than previously described BMP inhibitors
and comparable to the selectivity of A-83-01 for TGF-p versus BMP signaling [91].
52
(a)
(b)
Dorsomorphin
LDN-193189
100.
100-
0
(C
0
50
-j
0
I-
i
0
4
0
4
log([inhibitor]) nM
Iog(finhlbltor]) nM
(d)
LDN-212854
0
100-
2100-
4.
Y X
2
Oil
4
log([inhibitor])
100.
50-
. I
0
(e)
DMH1
i
0
4
Iog(inhibitorJ) nM
nM
K02288a
I
*
f - :5
caALK1
M caALK2
0
M,
caALK3
0j 50.
-x
T
*
'TIL
caALK4
J* caALK5
0
log([inhibitor]) nM
Figure 4.4. Potency Inhibition curves of BMP and TGF-p transcriptional activity mediated by
constitutively activated ALKI-5 in a cell-based luciferase reporter assays. Representative inhibition
curves for a) dorsomorphin, b) LDN-193189, c) LDN-212854, d) DMH1 and e) K02288a against
constitutively active BMP (ALKI, 2 and 3) and TGF-p (ALK4 and 5) type I receptors. Dorsomorphin
exhibits a similar selectivity profile to LDN-193189, but with approximately 10-fold decreased potency
against all receptors. Despite showing potency similar to LDN-193189 in kinase assay, K02288a is less
potent and selective in cells. DMH1 exhibits similar potency to dorsomorphin but with less selectivity.
In contrast to dorsomorphin and LDN-193189, LDN-212854 demonstrates improved selectivity for
ALK2 versus ALK4 and 5, as well as versus ALK 1 and ALK3. Data shown are representative of at least
3 independent experiments, with data plotted as mean ± S.E.M. (n=3 replicates per [c] point)
53
IC
(a)
50
(nM)
BRE-Luc
Compound
caALK1
Dorsomorphin
LDN-193189
LDN-212854
K02288a
DMH1
309±52
23±5
100±7
440±52
378±54
(b)
caALK2
110±13
11±2
162
225±9
230±29
CAGA-Luc
caALK3
caA LK4
caALK5
172±38
11±1
16619
237±59
317±87
2,412 ±365
238 ±57
1,684 ±165
812 ±42
503 ±41
980+285
213±31
I
2,103±99
693±28
658±23
Inhibition of Type I Receptors at 100nM [c]
00%
100%
98%
0
.0
50% iH-4r%
1/6
.19%
0%
Dorsomorphin
(C)
-- r
LDN-193189
LDN-212854
K02288a
DMH1
Inhibition of Type I Receptors at 300 nM [c]
100%
-
7noll
0
56%
50%
-
39%
0
15%
0%
Dorsomorphin
LDN-1 93189
LDN-212854
K02288a
DMH1
Figure 4.5. Potency and selectivity of BMP inhibitors based on on BMP (BRe-Luc) and TGF-p
(CAGALuc) transcriptional activity mediated by constitutively active ALK1-5. (a) In vitro cell-based
assay IC 50
measurements against constitutively-active BMP (caALK1, 2, and 3) and TGF-P (ALK4 and 5)
type I
receptors demonstrates LDN-212854 to be more selective for BMP versus TGF-P receptors while
retaining low nanomolar potency against caALK2. (b-c) Inhibition of caALK1-5 by BMP inhibitors
at
various concentrations demonstrates that LDN-212854 preferentially inhibits caALK2 at concentrations
near 100 nM, whereas other receptors are affected at higher concentrations. Data shown are calculated
based on IC 5o generated from at least 3 independent experiments, with data plotted as mean ± S.E.M.
54
(a)
193189 (nM)
BMP7
pSMAD1/5/8
SMAD1
212854 (nM)
BMP7
pSMAD1/5/8
SMAD1
o o
1
3
6
16
39
N
BMP7 Induced pSMADII1W8 Inhibition
2446 1 15303615
32
am
m
t 4
o 0
1
3
6
Am W
16 399
244
1
33815
10
0
Am
4
a
-
212854 (pM)
TGF-01
pSMAD2
SMAD1
109=10
-
(b)
193189 (pM)
TGF-01
pSMAD2
SMAD1
LDW1N9318
TGF-S Induced pSMAD2
0
0
0.O
0.1 0.2 0.4
- + + +
o
0
0.0O 0.1
0.2
0.A
1.6
m+ m
-+ + +
0.4
3.2 6.4 128 25.
0.6 1.6
A
Z
-
- +e m+
m esosm
-
Inhibition
32 6.4 12.6 25.6
M
m o+
smm m -
* LDN13169 1C
7.5 pEU
* LDN,212854 PCao
>
2
25 p
3
4
5
Figure 4.6. Comparison of potency and selectivity of LDN-193189 and LDN-212854 in modulating BMP
and TGF-3 ligand-mediated SMAD signaling. (a) Western blot analysis of BMP7 induced
phosphorylation of SMAD1/5/8 in BMPR2'- PASMCs reveals low nanomolar inhibition by both LDN193189 and LDN-212854. (b) Western blot analysis of TGF- 1 induced phosphorylation of SMAD2 in
wild-type PASMC revealed significant inhibition by LDN-193189 at concentrations greater than I pM,
and virtually no inhibition by LDN-212854 at concentrations up to 25 pLM. Data shown are representative
of 2 independent experiments.
4.3.2 LDN-212854 binding mode
To gain insight into its improved selectivity compared to LDN-193189, the binding mode
of LDN-212854 to ALK2 was investigated by a molecular modeling approach, using the
available co-crystal structure of LDN-193189 as a template [116]. In this complex, the pendant
4-quinoline moiety formed an important water-mediated hydrogen bond to the aC-helix residue
E248 (Figure 4.7a). Our modeling suggests this bond is lost upon substitution of the 5-quinoline
group in LDN-212854 (Figure 4.7c). Instead, a new water-mediated hydrogen bond to the
catalytic lysine residue K235 is predicted, as previously observed in the ALK2-K02288 structure
[114]. E248 and K235 are strictly conserved residues that form a necessary ion pair in all active
kinases (for example, ALK5 residues E245 and K232). Therefore, sequence alone cannot explain
55
the reduced binding of LDN-212854 to ALK5. However, inspection of the available ALK5
structures reveals a subtle shift in the orientation of the aC-helix and a conserved water molecule
that is shifted to a deeper location in the ATP pocket bound between ALK5 E245 and Y249
(Figure 4.7b). Subsequently, we solved the crystal structure of ALK2 co-crystalized with LDN212854 (Figure 4.7d) and found that the 5-quinoline nitrogen forms a water-mediate hydrogen
bond with the catalytic lysine (K235) as predicted by our model and also with the glutamate
(E248).
The water molecule involved in hydrogen bonding with LDN-212854 moves 1.6
angstroms closer to the 5-quinoline nitrogen. This water molecule appears favorable for
hydrogen bonding to the 4-quinoline of LDN- 193189, but not to the 5-quinoline moiety of LDN212854, potentially explaining the relatively poor inhibition of ALK5 by LDN-212854.
(a)
(b)
C helix
V234
Hinge region
V231
21C
Hinge region
Y282
K235
E287
Wa
LDN-1 93189
L3 A:353D354
E248
helix
K232
E28
atE245
0rftO354
40 A34
LDN-193189
D351
-
N341
342
(C)
ALK2 (PDB 3Q4U)
Y285
339
ALK5 (Model)
(IC helix
V234
Hinge region
(d)
Waterwith LDN-193189
K3
K235
T283
E287
LDN-212854
E248
354
3 353
Water with LDN-2128654
N34 1
342
ALK2 (Model)
Figure 4.7. (a) ATP pocket interactions of LDN-193189 (magenta) co-crystallized with ALK2 (PDB
3Q4U). A single hydrogen bond to the hinge residue H286 is made by the central pyrazolo[1,5-a]
pyrimidine. The pendant 4-quinoline moiety forms a water-mediated hydrogen bond to the aC-helix
56
residue E248. Water is represented by a red sphere and labeled "Wat". Hydrogen bonds are shown as a
dashed line. (b) Model for the binding of LDN-193 189 (magenta) to ALK5. The inhibitor was located by
the superposition of ALK2 (PDB 3Q4U) and ALK5 (SB-431542 complex; PDB 3TZM). ALK5 structures
show a conserved water position set further back in the ATP pocket between the aC-helix residues E245
and Y249. (c) Model for the binding of LDN-212854 (green) to ALK2. The pendant 5-quinoline group is
predicted to form an alternative water-mediated hydrogen bond to the catalytic
water position was modeled from the ALK2-K02288 co-crystal structure (PDB
interactions of LDN-212854 (yellow and blue) in the ALK2 co-crystal structure.
moiety forms a water-mediated hydrogen bond with the catalytic lysine (K235)
the aC-helix glutamate residue (E248).
4.3.3
lysine (K235). The new
3MTF). (d) ATP pocket
The pendant 5-quinoline
as predicted and also to
Kinase profiling ofLDN-193189 and LDN-212854
(a)
LDN-193189
TK
PDGFRO
RIPK2
LDN-212854
TK
PDGFRP
ALK3
RIPK2
ALK a.
ALK2
AL
(b)
ABL
ARG
ABL
CK1
CAMK
ALK3
ALKI
ALK2
KInase
193189
212854
ABLI
34t2
40±4
PDGFR-a
360 * 22
650 188
KIT
1,090 t 304
1,440 t 557
CK1
AGC
sIK2
(C)
IC50
AGC
0 < 1000 nM
0 < 100 nM
< 10 nM
CAMK
Figure 4.8. Kinase inhibition profile of LDN-193189 and LDN-212854. Kinome dendrograms for (a)
LDN-193189 and (b) LDN-212854 showing both on-target hits from our kinase assay and the top offtarget hits from a screen of 198 human kinases. (c) IC 5o values for top off-target hits. RIPK2 was the most
potently inhibited off-target kinase followed by ABLI and PDGFR-s, while other kinases were inhibited
at much higher concentrations.
Given the highly conserved nature of the ATP binding pocket across the human kinome,
many ATP-competitive kinase inhibitors exhibit significant off-target effects [81]. The off-target
effects of LDN-193189 and LDN-212854 were determined against a panel of 198 kinases
broadly representing the human kinome using an assay of enzymatic activity (Figure 4.8 and
Table 4.1).
Compounds were tested at 100 nM, a concentration chosen based on the high
potency of these compounds against ALK2, and at 1 pM, a standard concentration for profiling
off-target activities.
While the 5-quinoline moiety of LDN-212854 greatly increased BMP
versus TGF-P selectivity, it did not significantly improve kinome wide selectivity. At 100 nM,
57
LDN-193189 and LDN-212854 inhibited, respectively, 2 (1%) and 3 (1.5%) of the 198 kinases
tested by greater than 50%. At 1gM the number of off-targets increased to 20 (10.1%) for LDN193189 and 17 (8.6%) for LDN-212854 (Table 4.1). The IC 50 values of LDN-193189 and LDN212854 against several of these kinases were determined revealing significant activity against
RIPK2, ABL 1, and PDGFR-P (IC 50 < 100 nM) whereas the IC 5 o values for PDGFR-a, VEGFR2
and KIT were greater than 300 nM (Figure 4.8c). These data confirm a recent report that RIPK2,
in particular, appears to be a principal off-target kinase of LDN-193189 with an IC 5 o comparable
to that of ALK2 [81]. Thus LDN-212854 and LDN-193189, when used in the low nanomolar
range (<100 nM), would effectively inhibit BMP signaling while minimizing effects against
most kinases, except RIPK2. The use of LDN-193189 or LDN-212854 to inhibit BMP signaling
should consider these off-target effects and particularly RIPK2, known to have important roles in
modulating innate immunity and inflammation [117, 118].
% > 50% inhib.
1.0%
10.1%
1.5%
8.6%
# > 50% inhib.
2
20
3
17
% Inhibition
LDN-193189
LDN-212854
100 nM
1 M
100 nM
1 M
I
Kinase Name
__
K
3
4
5
MAPK4
__PDGFRf___
ARG (ABL2)
30.9
39.0
32.9
33.1
6
7
8
9
MINK
KDR
MAP4K2
TNK1
26.4
19.5
20.7
17.7
24.7
12.2
10
MARK3
11.0
15.8
11
MARK
13.1
7.0
12
MARK4
13.4
20.6
13
14
15
16
FYN
TYR3
BRK
PDGFRa
18.1
13.1
12.7
17.8
9.3
20.3
24.9
21.0
4.
17
18
19
20
21
22
CK1a
ARK5
DYRK1A
FGR
YES
LCK
14.4
16.0
10.4
13.3
11.9
11.7
-0.2
8.2
4.9
14.8
14.2
3.4
5.9
43.4
31.1
51.7
56.2
29.0
41.3
58
.3
5.0
56.7
56.
52.8
52.8
49.5
46.4
35.6
21.6
46.2
11.8
PAR-1 Ba
DYRK1B
6.9
7.1
KIT
LYNA
7.0
9.1
LRRK2-G2019S
10.0
LOK
MKNK1
CLK2
LYNB
HCK
SRC
8.3
7.1
2.3
7.4
9.7
8.0
MNK2
LRRK2
PHKy1
DDR2
3.7
8.7
5.2
5.5
FMS
MUSK
AURORA-C
3.7
5.8
4.2
Z. t
EPHB2
3.9
16.0
5.0
2.3
5.4
7.0
3.8
20.O
39.2
31.0
30.0
28.7
31.3
15.6
4.9
23.5
2.9
2.7
5.4
0.8
4.1
18.1
19.8
28.8
-2.8
31.9
25.3
8.0
-0.1
3.8
1.3
24.8
8.4
23.7
1.8
9.4
BLK
AURORA-A
AMP-A1B1G1
BRSK2
CDK6/cyclinD3
FGFRI
FGFR2
BRSK1
FLT-1
TBK1
FGFR3
TXK
3.9
4.2
5.2
4.4
7.2
3.4
5.0
3.0
0.0
4.5
5.0
4.1
23.6
23.1
22.6
22.4
21.9
18.8
17.6
17.2
16.7
16.5
16.4
16.4
3.2
10.0
1.9
0.2
2.2
11.3
14.8
2.0
0.6
2.3
0.7
7.9
26.2
4,
20.8
14.5
4.7
TIE2
RET
2.0
4.0
15.7
15.6
3.3
7.3
12.5
42-.$
PRKD1
AURORA-B
PTK5
4.6
2.3
4.1
15.1
14.8
14.3
4.0
2.5
2.7
28.5
12.4
CK1-y1
FLT-4
3.1
3.4
13.5
13.5
0.6
3.8
0.4
30.0
PRKD2
PRKD3
FLT-3
RON
4.5
3.2
3.4
-1.9
13.4
13.2
12.4
12.3
5.0
4.8
5.0
4.4
29.6
35.5
4M.
11.0
ALK
3.7
12.2
0.0
8.1
18.5
19.4
12.4
10.0
15.1
18.4
17.0
ERBB4
6.1
11.4
-0.3
12.3
TNK2
MER
EPH-A4
PRKG2
2.0
4.4
7.1
1.7
10.2
10.2
10.1
9.3
6.2
1.2
0.7
3.5
39.7
6.8
1.3
15.8
59
PYK2
SRMS
-0.9
9.0
8.9
CAMK26
3.1
PAK5
CDK5/p35
MET
6.2
1.4
6.2
78
CDK4/cyclinD
RSK1
79
80
81
82
83
71
72
73
74
75
76
77
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
101
102
103
104
4.6
-3.6
1.7
7.6
13.5
8.7
1.2
14.4
8.5
8.5
8.4
2.6
2.1
-0.3
3.9
2.7
1.8
0.8
0.6
8.3
8.0
1.7
3.0
5.1
10.1
MST1
4.0
7.9
0.4
1.9
TTK
IKK-E
4.5
7.5
7.6
7.4
-0.8
0.8
-5.7
10.8
TRKA
CDK3/cyclinE
MRCK-a
EPHB4
NEK9
EPHA3
CSK
MST2
P14-K-P
RSK3
1.9
1.6
0.8
-0.6
1.9
2.2
3.3
5.1
3.5
1.5
7.2
7.0
6.8
6.6
6.6
6.3
6.0
6.0
5.8
5.6
0.3
3.5
1.5
-4.8
0.8
0.1
-2.1
3.8
-1.4
0.2
3.3
3.9
2.0
-4.0
3.5
1.1
3.2
3.0
1.5
5.6
TEC
CK1-y3
IGF1R
RSK2
CDK2/cyclinE
TSSK1
BRAF
PAK6
1.8
2.3
4.1
1.2
2.8
3.5
3.5
8.8
5.5
5.5
5.4
5.4
5.3
5.2
5.2
5.1
16.8
1.4
0.3
1.8
3.0
0.1
1.4
1.0
15.5
1.1
-1.1
5.1
4.1
10.8
4.0
0.1
CDK1/cyclinB
HIPK4
0.5
-0.3
5.0
4.9
0.2
3.3
2.0
25.4
PAK1
4.3
4.9
0.1
-2.6
107
PHKy2
EGFR
INSR
HIPK1
EPHB3
3.5
1.5
3.8
0.6
5.6
4.9
4.8
4.5
4.4
4.3
-2.2
-2.1
-1.1
0.1
3.2
1.9
-4.6
-1.0
1.8
2.7
108
109
110
JAK1
CK2
PKC-y
2.7
0.7
2.1
4.2
4.2
4.1
2.6
3.0
-0.6
-0.1
2.0
-0.2
111
112
113
114
CDK2/cyclinA
1.2
4.1
0.3
6.9
LTK
3.6
4.0
-1.9
-0.1
BMX
TRKC
CK1-y2
RSK4
ROS
0.2
1.6
1.6
0.8
3.8
4.0
3.9
3.9
3.8
3.7
0.0
-1.6
1.5
1.1
-6.8
4.7
0.3
-1.4
1.8
-5.6
FER
2.8
3.7
-3.5
-4.4
105
106
115
116
117
118
60
119
120
121
122
123
124
125
126
127
128
129
130
131
132
133
134
135
136
137
138
139
140
141
142
143
144
145
146
147
148
149
150
151
152
153
154
155
156
157
158
159
160
161
162
163
ITK
1 .1
1.7
1.4
2.6
-5.3
0.4
9.8
4.8
0.8
2.1
-7.1
3.2
2.4
2.6
4.1
2.4
2.3
2.2
2.1
2.1
2.1
2.1
2.0
1.9
-2.6
0.9
-3.7
3.2
0.6
2.8
0.1
2.7
0.3
-4.4
2.7
-1.8
4.1
1.5
5.0
-1.5
6.5
2.3
0.7
1.9
-0.7
0.0
1.0
2.9
0.5
7.9
0.8
2.1
0.4
1.4
-3.3
0.2
1.4
2.6
0.3
1.9
0.8
1.1
1.2
1.4
3.9
0.2
1.0
2.8
1.0
3.2
-4.4
1.8
1.8
1.8
1.7
1.6
1.5
1.5
1.5
1.5
1.5
1.5
1.4
1.4
1.4
1.3
1.3
1.3
1.3
1.3
1.2
1.2
1.2
1.1
1.1
1.1
2.2
2.9
-4.9
2.0
-1.1
22.1
3.1
0.2
1.2
-0.6
0.0
2.1
-5.7
-3.0
-1.1
2.6
1.0
-1.0
2.2
-0.3
-0.1
6.3
0.2
-2.9
1.9
2.8
2.2
1.0
-1.9
-1.8
20.1
1.5
1.0
0.5
-1.6
1.9
1.2
-2.3
-3.0
6.7
2.0
0.2
-0.6
2.0
1.1
0.0
1.4
-0.6
3.1
2.1
DCAMKL2
NEK1
P38a
TRKB
CLK3
0.9
0.8
2.1
4.4
0.8
0.5
3.6
3.4
3.4
3.3
3.0
2.6
FES
2.2
FGFR4
SGK3
IRR
GSK3P
ERB-B2
PKA
MRCK-P
CHEK2
PDK1
0.3
1.6
1.9
2.7
2.1
1.4
0.0
1.5
1.1
IRAK4
AKT2
PAK3
BTK
NEK7
AKT1
P13-K-a
P38P
PKC-i
MSSK1
AKT3
ROCK2
MAPK1
IKK-P
PASK
PLK1
MSK1
P38y
PKC-G
JNK2
PRKX
PRKG1
NEK2
PKC-a
GSK3a
AXL
MEK1
1.4
1.0
1.6
0.9
164
165
ZAP70
CAMK16
MSK2
GRK7
4.7
0.8
0.9
1.8
1.0
0.9
0.8
0.8
0.8
1.5
-5.2
-15.3
-1.6
-3.1
-1.8
-12.5
166
SGK1
1.1
0.8
2.1
2.7
61
167
168
169
170
171
172
173
0.0
1.0
0.7
-1. 1
-0.1
P70S6KB1
0.6
-3.1
-3.4
P386
1.8
0.5
2.1
0.4
JAK2
MAPKAPK3
TSSK2
2.0
1.6
0.0
0.5
0.5
0.5
-3.3
2.3
0.8
-3.9
2.7
-2.8
ROCK1
DAPK1
1.5
0.4
1.2
1.4
174
175
CHEK1
2.9
0.3
-0.5
-0.6
DYRK2
2.3
0.2
-2.1
1.5
176
SGK2
PKC-P1
GRK6
CRAF
PRAK
JAK3
MAPKAPK2
PIM-2
2.3
-1.9
-0.1
0.6
0.8
-0.2
0.7
-0.3
0.2
0.2
0.2
0.1
0.1
0.1
-0.1
-0.3
-0.4
0.4
-16.0
-5.1
0.5
-0.2
2.5
-1.6
0.4
0.7
-9.0
2.1
1.2
3.0
-0.3
-0.1
PAK2
-0.2
-0.4
-0.9
-0.2
CAMK4
0.1
-0.4
7.3
3.7
PIM-1
NEK6
-1.0
2.3
-0.7
-0.8
-0.6
-0.1
0.6
0.7
-0.9
177
178
179
180
181
182
183
184
185
186
187
188
189
190
191
192
193
194
195
196
197
198
PIM3
2.0
-1.2
PKC-r
2.2
-1.9
4.6
5.3
SRPK1
MAPK3
IKK-a
TYK2
SPHK2
-0.5
1.9
3.0
-0.9
-1.2
-2.0
-2.2
-2.3
-2.3
-2.6
-0.9
2.6
3.9
-0.7
12.5
-0.3
1.2
2.0
3.2
7.0
EPH-A2
CAMK2a
SPHK1
-1.8
-0.2
-1.4
-3.3
-7.7
-10.8
1.3
0.9
8.3
2.0
1.0
3.7
SYK
-0.7
-11.9
-3.5
3.4
Table 4.1. Kinome profiling for LDN- 193189 and LDN-212854 at 100 nM and 1p.M ranked by kinases
with the greatest inhibition of enzymatic activity.
4.4
Conclusion
In Chapter 3 we hypothesized that the increased selectivity imparted by the 5-quinoline
moiety was generalizable to other pyrazolo[l,5-a]pyrimidine derivatives.
Thus, in this
chapter we described the synthesis and characterization of a novel derivative LDN-212854.
LDN-212854 maintained the phenylpiperazine, known to increase potency, of LDN-193189
but replaced the 4-quinoline with a 5-quinoline group, which we predicted would yield a
potent and highly selective BMP signaling inhibitor. We characterized LDN-212854 and
compared it against all known BMP type I receptor kinase inhibitors (dorsomorphin, LDN62
193189, DMH1, and K02288) by both highly sensitive radiometric kinase assay and cellbased assays of BMP and TGF-P signaling. We confirmed our hypothesis and found that
LDN-212854 was a highly potent and selective BMP type I receptor kinase inhibitor.
However, kinome-wide selectivity of LDN-212854 was not significantly different from that
of LDN-193189. This suggests that the 5-quinoline moiety specifically affects ALK4 and
ALK5 binding affinity but does not alter affinity to other off-target kinases. This suggests
that further modifications to LDN-212854 could yield even more selective derivatives with
greater specificity across the kinome.
The selectivity of LDN-212854 for BMP versus TGF-p signaling was significantly
improved versus the parent compound LDN-193189 (7,000-fold versus 175-fold). In fact,
LDN-21854 demonstrated a bias towards ALK2 over the other BMP type I receptors tested
(ALKI and ALK3) whereas LDN-193189 equally inhibited all BMP type I receptor kinases.
We hypothesized that the bias towards ALK2 of LDN-212854 would make it a useful probe
of BMP signaling. In Chapter 5 we tested this hypothesis and used LDN-212854 to study
the BMP signaling in vitro and in vivo.
63
Chapter 5 Study of BMP signaling using an ALK2 selective inhibitor
This chapter describes the use of LDN-212854 as a novel probe of BMP signaling in vitro
and in vivo. We use variousfunctional assays of ALK2 signalingto confirmed that LDN-212854
inhibits ALK2 preferentially as we first described in 4.3.1. We then use this bias to investigate
the role of ALK2 signaling in anemia of inflammation and heterotopic ossification seen in two
distinct mouse models of disease.
5.1
Background and Motivation
We previously described the identification of a small molecule BMP inhibitor,
dorsomorphin, and the development of a highly potent derivative, LDN-193189, based on the
same pyrazolo[1,5-a]pyrimidine core structure [63, 107].
LDN-193189 reduced heterotopic
ossification (HO) in a mouse model of FOP with an inducible constitutively active mutant
ALK2Q2 07 D(caALK2) transgene [108]. LDN-193189 is a potent inhibitor of BMP signaling, but
exhibits TGF- receptor inhibition at higher concentrations.
Previously, well-described small
molecule inhibitors of the TGF-p type I receptor kinases, such as A-83-01 and SB-505124, have
both high potency and high (> 3 log) selectivity for TGF-P versus BMP signaling [91, 92].
While TGF-P signaling inhibitors had potential utility as therapeutic agents, preclinical animal
studies have associated the administration of highly potent ALK5 inhibitors with bone and
connective tissue toxicities [96, 119].
Clinically viable BMP receptor kinase inhibitors,
particularly for indications requiring long term treatment, may need to minimize or eliminate offtarget inhibition of the TGF-p and Activin type I receptor kinases, ALK4, ALK5, and ALK7 in
light of these toxicities.
In Chapter 4 we described the development of LDN-212854, a novel BMP inhibitor that
exhibits substantially greater selectivity for BMP versus the TGF-p type I receptors. In addition,
LDN-212854 possesses a bias towards ALK2 versus ALKI and ALK3 compared to other
inhibitors.
LDN-212854 supports the concept that development of selective inhibitors of
individual type I receptors may be feasible despite the high degree of homology between these
receptors. We hypothesize that LDN-212854 provides useful selectivity in vitro for resolving
ALK2 versus ALK3 signaling and in addition LDN-212854 should provide comparable potency
in a mutant ALK2 transgenic mouse model of FOP to LDN-193189.
LDN-212854 or similar
compounds that selectively target individual BMP receptors could be used in vitro to ascertain
individual receptor functions, and may have favorable characteristics as clinical candidates.
64
5.2
Experimental Methods
5.2.1
BMP-InducedALP activity
C2C12 myofibroblasts cells were seeded in 96-well plates (Corning) at 2,000 cells per well
in DMEM supplemented with 2% FBS as previously described [120]. Compounds diluted in
DMEM and were added at final concentrations ranging from InM to 10 jIM, in quadruplicate.
BMP4 and BMP6 ligands diluted in DMEM were added to final concentrations of 20 ng mL-1.
Positive controls were generated by omitting compounds and negative controls were generated
by omitting both compounds and ligands. Cells were incubated for 6 days at 37 0 C and 5% C02
and subsequently harvested using 1% Triton X-100. Extracts from each well were incubated at
RT for 30 min with alkaline phosphatase (ALP) yellow (pNPP) liquid substrate for ELISA
(Sigma-Aldrich)
and ALP activity was measured by absorbance
at 405 nM per the
manufacturer's instructions. Absorbance data was analyzed with positive controls as 100% ALP
activity and negative controls being subtracted as background. Graphing and regression analysis
by sigmoidal dose-response with variable Hill coefficient was performed using GraphPad Prism.
5.2.2
IL-6 induced hepcidin expression
HepG2 cells were seeded in a 12-well plate at 75% confluence (~100,000 cells per well) in
EMEM supplemented with 0.1% FBS and starved for 6 hours at 37'C and 5% C02. Cells were
pretreated for 30 min by adding compounds diluted in EMEM to final concentrations ranging
from 1 nM to 125 nM in quadruplicate.
Human recombinant Interleukin-6 (IL-6) (R&D
Systems) was then added at a final concentration of 100 ng mL-1. Positive controls were
generated by omitting compounds and negative controls were generated by omitting both
compounds and IL-6. After 90 min, the media was removed and each well washed twice with
PBS. Both RNA isolation using TRIzol@ (Life Technologies) and cDNA synthesis using MMLV-reverse transcriptase (Promega) and the Mastercylcer® ep gradient S (Eppendorf) were
conducted per the manufacturer's instructions.
The expression of hepcidin transcripts was
measured using SYBR@ FAST real-time qPCR kit (Kapa Biosystems), human primers (Forward
5'-CTGACCAGTGGCTCTGTTTTC-3', Reverse 5'-GAAGTGGGTGTCTCGCCTC-3') and
Mastercylcer@ ep gradient S realplex 2 (Eppendorf) per the manufacturer's instructions. The
relative expression
of hepcidin was normalized to
GCTGGAATTACCGCGGCT-3',
18S human RNA (Forward 5'-
Reverse 5'- CGGCTACCACATCCAAGGAA
65
-3') with
negative controls as baseline expression and positive controls as maximal expression. Excel
(Microsoft) software was used for data analysis and graphing.
5.2.3
caALK2 (Q207D) Mouse Model of FOP
Heterotopic ossification was induced in mice containing a single allele of the gene encoding
a conditionally-expressed constitutively-active ALK2 (ALK2Q2
retropopliteal injection of Ad. Cre (1x10
8
7D
or caALK2) by postnatal (P7)
plaque-forming units) as previously described [108].
Mice (n=6 per group) were treated intraperitoneally twice daily for 4 weeks with LDN- 193189 or
LDN-212854 at 6 mg kg-' or vehicle control.
Impaired mobility, correlating with joint
involvement, was quantified by daily assessments of passive range of motion, via dorsiflexion of
the left ankle joint. A score was assigned based on dorsiflexion angle (O=normal flexion, 0 200, I=mildly impaired, 20 - 90', 2=moderately impaired, 90' - 135', and 3=severely impaired,
>1350). Mice were sacrificed, imaged by X-ray and GFP fluorescence (Carestream), and soft
tissues fixed and stained by the Alizarin red and Alcian blue method as previously described
[121].
5.3
Results and Discussion
5.3.1
LDN-212854 preferentially inhibitsALK2
In addition to inhibiting caALK2 more potently than caALK4 and caALK5 in cell based
assays (Figure 5.1a), LDN-212854 also exhibited approximately one log selectivity for caALK2
versus caALK3.
When C2C12 cells were transfected with caALK2, both LDN-193189 and
LDN-212854 inhibited BMP transcriptional activity (BRE-Luciferase) with comparable low
nanomolar efficacy (Figure 5.1a). When C2C12 cells were transfected with constitutively active
ALK3, LDN-193189 was nearly 14-fold more potent than LDN-212854 (11 nM versus 153 nM,
Figure 5.1c). We tested whether or not the selectivity of LDN-212854 for caALK2 versus
caALK3 could be used to discriminate between the utilization of these receptors in vitro using an
assay of known ALK2 versus ALK3 function. We examined the impact of compounds on the
BMP-induced osteogenic differentiation of myofibroblast C2C12 cells. When C2C12 cells were
stimulated with BMP4 or BMP6 (20 ng mL-1, 6 d), osteogenic differentiation assayed by alkaline
phosphatase (ALP) activity was observed. In this system, BMP6 has been shown to function
66
primarily via ALK2, whereas BMP4 interacts primarily with ALK3 to induce ALP [109, 122].
As predicted based on assays using caALK2 and caALK3, LDN-193189 inhibited both BMP6(b)
% Inhibition at 34nM [c]
(a)
I
*
.ALK1
100%
caALK2 Transfected BRE-Luc C2C12
* LDN-193189 ICso = 10.4 nM
LDN-212854 ICso = 15.4 nM
I*
*.000
caALK2
10%0%
mcaALK3
Um caALK4
76%
790
.
0so
mcaALK5
Iog([inhibitor]) nM
50%
(C)
C~01
29%
caALK3
Transfected BRE-Luc C2C12
- LDN-193189 lCso =11 nM
LDN-212854 1050 = 153 nM
10*
32%
08
0
0%
LDN-1 93189
LDN-21 2854
Iog([inhibitor]) nM
Figure 5.1. LDN-2 12854 preferentially inhibits ALK2. (a) At low concentrations (34nM) LDN-2 12854
potently inhibits ALK2 (79%) whereas ALKi, ALK3, ALK4, and ALK5 remain largely active. In
contrast, at 34nM LDN- 193189 inhibits both ALK2 and ALK3 equally, a) Representative inhibition
curves of caALK2 and b) caALK3 transcriptional activity (BRE-Luc) by LDN- 193189 and LDN-2 12854
in C2C12 cells. Both compounds potently inhibit ALK2, whereas LDN-2 12854 is substantially weaker
against ALK3. Data shown are representative of at least 3 independent experiments, with data plotted as
mean ±S.E.M. (n=3 replicates per [c] point)
and BMP4-induced ALP with comparable low-nanomolar potency (ICso ~5 nM, Figure 5.2). In
contrast, LDN-2 12854 inhibited BMP6-induced ALP expression more potently than BMP4 (IC50
~10 nM versus 40.5 nM). Thus, LDN-2 12854 exhibited greater selectivity for BMP6- versus
BMP4-induced osteogenic differentiation consistent with its preference for ALK2 versus ALK3.
5.3.2 IL-6 induced hepcidin expression is predominantly mediated by A LK3
Next, we used LDN-2 12854 to probe IL-6-induced expression of hepcidin in HepG2
hepatoma cells. The in vitro induction of hepcidin in this assay models the homeostatic pathway
governing serum iron levels mediated by the BMP- and IL-6-regulated expression of hepcidin in
the liver. In anemia of inflammation, IL-6 mediated signaling is thought to enhance hepcidin
regulation in a BMP6-dependent fashion, inactivating iron transporter ferroportin and thereby
decreasing circulating iron bioavailability for erythropoiesis [14, 59].
67
The dose-dependent
impact of LDN-193189 and LDN-212854 upon IL-6-induced (100 ng mL-, 90 min.) expression
of hepcidin mRNA was measured in cells by quantitative RT-PCR (Figure 5.3). We have
(a)
BMP6 Induced ALP Activity Inhibition
T
S100
S LDN-1 93189 IC50
=
(b)
BMP4 Induced ALP Activity Inhibition
5 nM
0 LDN-1 93189 IC50 = 6 nM
001
* LDN-212854 C50 = 10 nM
* LDN-212854 C50 = 41 nM
0
0
.$
50
50-
0
0.
01
2
log([inhibitor]) nM
3
4
0
1
2
log([inhibitor]) nM
3
4
Figure 5.2. LDN-212854 provides useful selectivity as a probe of signaling mediated by ALK2 versus
ALK3, and their respective ligands. (a) Alkaline phosphatase (ALP) activity induced in C2C12 cells by
BMP6, which signals primarily through ALK2, was inhibited with comparable potency by LDN-193189
and LDN-212854. (b) ALP activity induced in C2C12 cells by BMP4, which signals primarily through
ALK3, was more potently inhibited by LDN-193189 than LDN-212854. Data shown are representative of
at least 3 independent experiments, with data plotted as mean ± S.E.M. (n=3 replicates per [c] point)
previously shown that ALK3 is the dominant BMP type I receptor required for IL-6-induced
hepcidin expression in vitro and in vivo [123]. Stimulation with IL-6 alone resulted in an 18-fold
increase in hepcidin expression over control. Co-treatment with IL-6 and either LDN-193189 or
LDN-212854 resulted in the inhibition of hepcidin mRNA expression (with approximate IC 50
values of 5 nM and 125 nM, respectively, Figure 5.3). The relatively weaker inhibition of IL-6-
mediated hepcidin expression by LDN-212854 correlated with its weaker inhibition of ALK3
compared to LDN-193189 (Figure 5.1c), and supported the concept that ALK3 is the principal
receptor responsible for IL-6-regulated hepcidin expression. As previously observed, maximum
inhibition with either LDN-193189 or LDN-212854 did not reduce hepcidin expression to levels
of controls, suggesting BMP-independent signaling mechanisms may also contribute to IL-6
induced hepcidin expression [124]. Taken together these results demonstrate that LDN-212854
can provide some useful albeit limited resolution of signaling via ALK2 versus other BMP
receptors in vitro. It is unlikely that LDN-212854 would discriminate the activity of ALK2 in
vivo, in part due to wide ranging plasma concentrations during absorption and metabolism, but
subsequent derivatives with greater selectivity might be useful in vivo probes of ALK2 function.
68
IL-6 Induced Hepcidin mRNA
25
10
5
m LDN-1 93189
m LDN-212854
20
4015
Z10
E
CL
-IL-6
+IL-6
Compound[c] 0 nM
+IL-6
1 nM
+IL-6
5 nM
+IL-6
25 nM
+IL-6
125 nM
Figure 5.3. IL-6 induced hepcidin expression in HepG2 cells was potently inhibited by LDN-193189 and
less potently by LDN-212854, consistent with a primarily ALK3-dependent mechanism of IL-6 induced
hepcidin expression. Data shown are representative of at least 2 independent experiments, with data
plotted as mean ± S.E.M. (n=4 replicates per [c] point)
5.3.3
LDN-212854 inhibitsALK2Q2O7D-induced heterotopic ossification
To demonstrate the in vivo efficacy of LDN-212854, we employed a murine inducible
transgenic ALK2Q2 07 D model of heterotopic ossification [108]. Intramuscular injection of Ad.
Cre potently induces recombination of a floxed inducible ALK2Q207D and a GFP reporter, while
inducing myositis and necrosis. This combination of inflammation and mutant ALK2 expression
result in ectopic endochondral bone lesions reminiscent of FOP.
Following retropopliteal
injection of Ad. Cre (108 pfu on P7), mice were treated for 4 weeks with vehicle, LDN-193189
or LDN-212854 (6 mg kg', I.P., twice daily). Heterotopic bone was assessed by x-ray, and
alizarin red and alcian blue staining, while GFP expression was used to confirm recombination
of the ALK2Q2 07 D transgene at the injection sites (Figure 5.4a).
Bone formation and
corresponding hindlimb immobility was 100% penetrant in vehicle treated mice as measured by
passive range of motion scores (Figure 5.4d).
LDN-193189 and LDN-212854 treatment
prevented the formation of heterotopic bone and preserved limb range of motion with minimal or
no impairment in the majority of mice.
These results demonstrate that LDN-212854 can
effectively neutralize ALK2 signaling in vivo.
By virtue of increased BMP selectivity, 5-
quinoline substituted pyrazolo[1,5a-]pyrimidine compounds such as LDN-212854 could be
advantageous for the treatment of FOP by minimizing toxicity due to inhibition of TGF-p type I
69
receptors. In fact LDN-212854 was significantly (p < 0.05) better tolerated in the treated mouse
pups demonstrating a 14% reduction in the rate of growth as compared to a 32% reduction for
pups treated with LDN-193189 (Figure 5.5).
Further medicinal chemistry improvements to
LDN-212854 could yield a compound with an even greater therapeutic window.
(a)
(b)
AU
a.
U-
m
(d)*
p= 3.5 x 10-6
+Vehicle (n=6)
*
*ELDN-193189(n=6)
a
2- A LDN-212854 (n=6)
01
0-
CL
LL.
0
10
30
2D
Day post adCre injection
<20'
0-
90
go
0
1
>135'
135
2
3
Range of motion scoring guide
Figure 5.4. In vivo efficacy of LDN-212854 in a mouse model of fibrodysplasia ossificans progressiva
Mice expressing an inducible constitutively-active ALK2Q2 7 D (CAG-Z-EGFP-caALK2)
(FOP).
transgene were treated with (a) vehicle, (b) LDN-193189 or (c) LDN-212854 at 6 mg/kg IP BID.
Heterotopic ossification following injection of Ad.Cre was observed by X-ray (top panels) and staining
for alizarin red (calcium) and alcian blue (glycosaminoglycans) (bottom panels). Heterotopic ossification
following Ad.Cre injection was observed 100% of vehicle-treated mice, whereas ossification was
70
essentially absent in mice treated with LDN-193189 or LDN-212854. Transgene-mediated expression of
GFP (middle panel) was observed at the site of Ad.Cre injection to confirm recombination and ALK2Q2 07D
expression. (d) Passive range-of-motion was progressively impaired in vehicle-treated mice starting on
day 10, whereas mobility was almost entirely preserved in mice treated with LDN-193189 and LDN-
212854.
(a)
(b)
20
*Vehicle (n=6)
NLDN-193189 (n=6)
A
A
-5%
*
ALDN-212854 (n=6)
15
AMA A AAA
N< A
.
0>-15%
A
A
**
-25%
5
0
-35%
0
10
20
Day post adCre injection
*p <.05
30
*
p=.003
Figure 5.5. LDN-212854 was significantly better tolerated than LDN-193189. (a) Weight change of
mouse pups throughout the treatment period from P7 to P35. (b) LDN-193189 resulted in a 32%
reduction in the rate of growth, while LDN-212854 resulted in a significantly lower reduction of 14%.
5.3.4
LDN-212854 inhibits heterotopic ossification in Bmall-- mice
Circadian rhythms are physiological or behavioral processes that oscillate with a period of
approximately 24 hours (day-night cycle of Earth) [125]. These processes continue to occur with
predictable periodicity even under experimental conditions of complete darkness (Figure 5.6)
suggesting intrinsic regulators of circadian rhythms known as circadian clocks. Many circadian
== (b)
(a)
10
10-
-
-
20-
)20i
30
30
'40,'*6
40
50-
so,
-
-
60,
0
24
48
Tim. (hours)
0
24
Time (hours)
48
Figure 5.6. Voluntary wheel-running behavior in mice. Dark bars represent periods of wheel-running.
Wild-type mice (a) and (b) Bmallknock-out mice were subjected to day-night cycles (day 0 - 21)
followed by complete darkness (day 22-70). Wild-type mice maintained circadian rhythms with a period
of 23.6 hours, while Bmal-/- mice did not. Figure adapted from [126].
71
rhythms are controlled by the central clock located in the suprachiasmatic nucleus (SCN) of the
hypothalamus, but circadian clocks exist in nearly all tissues and even within individual cells
[127, 128]. In fact, it is estimated that up to 10% of the transcriptome is regulated by circadian
clocks [129-131]. Several master regulatory genes of the circadian clock in mammals have been
identified including Clock and Bmall [132]. CLOCK and BMAL 1 proteins form heterodimers
that act as transcription factors by bidning to E-box sequences (5'-CACGTG-3') and E'-box
sequences (5'-CACGTT-3') on the promoters of target genes such as Cry]/2 and Per1/2 which
are part of a negative feedback loop that then inhibit CLOCK-BMAL1 activity [133] [134].
When Bmall is disrupted it leads to a complete loss of circadian rhythms (Figure 5.6b)[135].
Bunger et al. described the generation and characteristics of a Bmall-~ knockout mouse that
demonstrated not only complete loss of both behavioral and molecular circadian rhythms, but
also premature mortality, reduced weight, and a progressive arthropathy [136]. This progressive
arthropathy ultimately resulted in ankylosing of the joints due to heterotopic ossification of
ligaments and tendons particularly in the intervertebral joints and the hind limb tarsocrural joint
(Figure 5.7b). We believe this phenotype closely resembles the ankylosing enthesitis seen in
26 weeks
35 weeks
26 weeks
35 weeks
(a)
IIB
(b)
E
Figure 5.7. Progressive arthropathy and joint ankylosing seen in Bmal-/ mice both in the intervertebral
joints and at the Achilles anthesis. Adapted from [136].
many human spondyloarthropathies [137, 138]. Previous reports have implicated BMP signaling
in endochondral bone formation and phenotypically similar joint ankylosis [139].
72
We hypothesized that the heterotopic ossification seen in Bmalt
mice was driven by
excessive BMP signaling that resulted from the disruption of the circadian clock. First we
sought to determine whether or not tissue specific deletion of BmallJ was sufficient to cause the
heterotopic ossification phenotype and that disruption of the central clock was not implicated.
We used a Bmal]ff mouse expressing cre recombinase driven by paired-related homeobox gene1 (Prxl-Cre)which is limited in expression to the mesenchymal limb bud cells. We confirmed
peripheral deletion of Bmall by RT-qPCR comparing the relative expression of Bmall in
Bmallf mice and Bmal1ff-Prx1-Cre mice in both the liver and the Achilles enthesis (Figure 5.8)
and found that Prxl-Cre did disrupt the normal circadian rhythm of Bmall only in peripheral
limb tissues. Next, we characterized the phenotype of this novel tissue specific Bmall knockout
mouse. Similar to the previously reported global Bmall knockout mouse, peripheral deletion of
Bmall in the limbs resulted in hind limb arthropathy and Achilles enthesis ossification that was
visible by X-ray at the earliest by week 8 (Figure 5.9a) and continued to increase in severity
throughout adulthood. The Achilles enthesis heterotopic ossification was 100% penetrant but
highly variable at 8 weeks of age (Figure 5.9b).
Expression of Bmal1 in Bmal1 4 and BmallfPrx1-cre
Liver
Achilles enthesis
1.51.
p 1.0
1.0
0.5
.2!0.5-
0.0
0.0
6
12
18
ZT (hours)
0
12
18
ZT (hours)
Figure 5.8. Relative expression of Bmallin the liver and the Achilles enthesis of BmalfILPrxJ-cre mice
compared to Bmaf mice. PrxJ-cremice demonstrated disrupted and abnormal cycling of Bmall over 24
hours in the peripheral tissue of the Achilles enthesis, while maintaining normal circadian rhythm in the
liver.
73
Figure 5.9. Heterotopic ossification (HO) at the Achilles enthesis. (a) Bmall" PrxJ-Cre mice develop
Achilles HO which is first visible by X-ray at 8 weeks of age. The HO continues to develop increasing in
severity until 27 weeks of age. (b) Penetrance is of the Achilles enthesis HO is 100% but the severity of
HO at 8 weeks is highly variable.
Having characterized the time course and phenotype of Bmal1f-Prx]-Cre mice we
hypothesized that the Achilles enthesis heterotopic ossification was the result of enhanced BMP
signaling resulting from the dysregulated circadian rythms in peripheral tissue. We previously
used LDN-212854, an ALK2-biased highly selective inhibitor of BMP signaling, to prevent
heterotopic ossification in a mouse model of FOP (5.3.3).
To test the hypothesis that the
heterotopic ossification seen in Bmall knockout mice was driven by enhanced BMP signaling
we treated mice once per day with 6 mg/kg of LDN-212854 IP for 4 weeks starting at week 4. A
vehicle-treated group served as a control. Data from our kinase and cell-based assays suggested
that LDN-212854 preferentially inhibits ALK2 in vitro, however, due to the pharmacokinetics of
bolus dosing in vivo it is likely that LDN-212854 also inhibits ALK3 signaling when plasma
concentrations are highest.
To help determine if the potential effects seen with LDN-212854
were due to ALK2 inhibition or ALK3 inhibition we treated a third group of
74
- -,
~
(C)
(a)
-1 1
m Vehicle
(e) mLDN-212854
a ALK3-Fc
125
100
p=.17
Select ROI
Select ROI
CL
(b)
75
(d)
50
p=.0002
25
0
Apply threshold
Apply threshold
Treatment Groups
Figure 5.10. Quantification of heterotopic ossification in BmallJ'Prx1-Cremice. (a) A region of interest
(ROI) is selected just above the calcaneus and lateral to the tibia in the area of the Achilles tendon. Using
ImageJ a threshold of 42 is set and the number of pixels above this threshold is counted. (b)
Quantification of HO in Bmalf' Prx]-Cre mice treated with vehicle, LDN-212854, or ALK3-Fc shows
significant inhibition of HO by LDN-212854.
mice every other day with 8mg/kg of ALK3-Fc (Acceleron) a recombinant ligand trap shown to
inhibit signaling of BMP ligands with high affinity for ALK3 such as BMP2 and BMP4 in vitro
and in vivo [45, 124].
(Carestream).
After 4 weeks of treatment mice were sacrificed and X-rayed
Images were analyzed using ImageJ by creating a region of interest (ROI) just
above the calcaneus in the area of the Achilles tendon (Figure 5.10a,c).
A threshold of >42
(approximate pixel value for bone) was applied to the ROI of the 8-bit greyscale images (0 =
black to 255 = white) and the number of pixels above this threshold were counted (Figure
5.10b,d). Five mice and a total of 10 limbs from each treatment group were analyzed and
quantified.
The results demonstrate that LDN-212854 significantly reduced Achilles tendon
heterotopic ossification by greater than 75% (p=0.000 2 ).
75
Interestingly, ALK3-Fc failed to
significantly reduce the amount of HO strongly suggesting that the effects of LDN-212854 are
the result of ALK2 signaling inhibition or that the ligands trapped by ALK3-Fc do not play a role
in the formation of Achilles enthesis HO. Further studies will be needed to determine the exact
mechanisms by which disruption of Bmall and circadian rhythms results in enhanced BMP
signaling.
5.4
Conclusion
In Chapter 4 we described the development and characterization of LDN-212854 a potent
and selective inhibitor of ALK2.
In this chapter we used LDN-212854 as a novel probe
compound to explore BMP signaling in vitro and in vivo. We demonstrated that the selectivity
of LDN-212854 for ALK2 versus ALK3 could be used to determine which type I receptor was
responsible for particular biological functions such as ligand induced alkaline phosphatase
expression. We used LDN-212854 to demonstrate that the expression of hepcidin by IL-6, a key
regulatory mechanism in anemia of inflammation, is likely mediated by ALK3 and not by ALK2.
This suggests that selective ALK3 inhibitors could serve as therapeutics for the treatment of
anemia of inflammation.
We also demonstrated that LDN-212854 was useful as an in vivo probe of BMP signaling.
We showed that LDN-212854 effectively prevented heterotopic ossification in a mouse model of
FOP and was better tolerated than its less selective parent compound LDN-193189.
We also
7
used LDN-212854 in a novel model, Bmal11E-Prx1-Cre,
of progressive arthropathy with
phenotypic changes reminiscent of those seen in spondyloarthropathies such as ankylosing
spondylitis.
LDN-212854 effectively prevented the Achilles enthesis heterotopic ossification
whereas ALK3-Fc was not effective.
These findings strongly suggest that ALK2-mediated
signaling is responsible for the heterotopic ossification seen in this model and suggests a
potential strategy for preventing ankylosing in spondyloarthropathies. Taken together we have
shown that LDN-212854 is a useful tool compound in vitro and holds promise as a potential
therapeutic drug for the prevention of heterotopic ossification.
76
Chapter 6 Development of 2-aminopyridine BMP kinase inhibitors
In this chapter we describe the development of a library of BMP type I receptor kinase
inhibitors based on a novel 2-aminopyridine core scaffold Previous BMP kinase inhibitors,
described by us and others, have been based on the pyrazolo[, 5-a]pyrimidine core. A novel
core allowsfor the development of structurallydistinct compounds with the potentialto elucidate
new structure-activityrelationships with the goal of developing inhibitors with higher potency,
improved selectivity, and other desirableproperties. Additionally, a novel core provides a new
class of compounds with the potentialfor therapeutic development to treat diseases mediated by
BMP signaling.
6.1
Background and Motivation
Inappropriate BMP signaling has been shown to contribute to the pathophysiology of
various disease processes.[32]
One of the most striking examples of BMP signaling-related
disease is seen in fibrodysplasia ossificans progressiva (FOP), a rare and disabling genetic
disease caused by a highly conserved gain-of-function mutation in the glycine-serine (GS) rich
domain of the BMP type-I receptor ALK2 (c.617G>A; p.R206H) [66, 67]. Several other FOPcausing gain-of-function mutations in both the GS and kinase domains of ALK2 have also been
described in non-classic or variant forms of FOP [68-70, 140].
We and others have previously reported the discovery and development of small
molecule inhibitors of BMP type-I receptors such as dorsomorphin, LDN-193189, LDN-212854,
and DMH1 all of which are based on the pyrazolo[1,5-a]pyrimidine scaffold [107, 113, 141].
These compounds have proven to be useful chemical reagents for the study of in vitro
phenomenon and several have demonstrated in vivo efficacy in a mouse model of FOP [108,
141]. More recently we described a structurally distinct BMP type-I receptor inhibitor, K02288,
which is based on a 2-aminopyridine scaffold and demonstrated greater kinome-wide selectivity
than LDN-193189 [98].
The 2-aminopyridine scaffold is also found in crizotinib, which was
recently approved by the FDA for the treatment of non-small cell lung cancer in patients with
activating mutations in the anaplastic lymphoma kinase [142].
Despite the high affinity and
selectivity of K02288 for BMP receptors in thermal shift and in vitro kinase assays, it has
comparatively weak potency in cell-based assays [141].
In this chapter we describe a structure-activity relationship (SAR) study of K02288 with
respect to ALK2 binding affinity, BMP and TGF-P signaling inhibition in biochemical and
cellular assays, selectivity, and cytotoxicity. These studies were pursued as part of an effort to
77
elucidate the BMP type I receptor inhibitor pharmacophore, while producing a set of compounds
with greater utility as physiologic probes. This SAR provides unique insights into features of 2aminopyridine derivative compounds that are required for potent and selective inhibition of BMP
versus TGF-
receptor signaling.
We found that substitution of the 3-phenol with 4-
phenylpiperazine greatly increased potency in cells, yielding a series of compounds more likely
to be useful as probes of BMP function. These included a 2-methylpyridine derivative that
exhibited potent inhibition of ALK2 activity in cell-based and in vitro kinase assays, high
selectivity for BMP versus TGF-b signaling, and low cytotoxicity. Additionally, we used this
novel set of derivatives to demonstrate for the first time that FOP-causing mutations do not affect
inhibitor binding affinity as compared to wild-type ALK2. This finding strongly suggests that
ATP-competitive kinase inhibitors identified on the basis of their activity against endogenous
BMP signaling, such as dorsomorphin and its derivatives, or by their affinity for wild-type
ALK2, as in the case of K02288, will inhibit with equal potency the mutant ALK2R206H found in
classical FOP as well as the other GS- and kinase-domain mutants of ALK2 that have been
described in non-classical or variant FOP. These results describe a novel series of specific and
potent probe compounds for the interrogation of BMP signaling that may have therapeutic
potential for FOP and other diseases of maladaptive or inappropriate BMP signaling.
6.2
Experimental Methods
6.2.1
Thermal shift kinase assay
Thermal melting experiments were performed using a Real Time PCR machine Mx3005p
(Stratagene) with a protein concentration of 1-2 [M and 10 pM inhibitor as described previously
[143]. Recombinant human kinases for thermal shift kinase assay screening were prepared by
Structural Genomics Consortium (SGC) using published methods [98].
6.2.2
Cell viability assay
HePG2 hepatocarcinoma cells were seeded in DMEM supplemented with 10% FBS at 25,000
cells per well in tissue culture treated 96-well plates (Costar® 3610; Corning). The cells were
incubated for 2 h (37'C and 5% CO 2) and allowed to settle and attach. Compounds of interest or
DMSO were diluted in DMEM and added at final compound concentrations of 1 pM, 10 piM,
and 100 pM. Cells were incubated for 4 hours and 24 hours after which the media was discarded.
78
Cells were lysed by adding 30 pL of passive lysis buffer (Promega) and shaken at RT for 15 min.
Cell viability was determined by quantifying the ATP present in each well by adding 10 pL of
CellTiter-Glo (Promega) per well and measuring the light output Spectramax L luminometer
(Molecular Devices) with an integration time of one second per well. Data was normalized to
100% viability for cells receiving only DMSO without any concurrent compound.
6.3
Results and Discussion
6.3.1
Structure-activityrelationship (SAR) of solvent exposed group
We previously demonstrated that K02288 exhibits similar potency to LDN-193189 in
biochemical kinase assays for inhibiting ALK2 and related BMP type I receptor kinases, but was
surprisingly less active in cell-based reporter assays using constitutively active BMP type I
receptors [98, 141]. We speculated the relatively weaker activity of K02288 in cells might be
due to poor solubility or impaired interactions with solvent water molecules that might be
addressed via modifications in the solvent-exposed domain.
We created six derivatives of
K02288 by modifying the 3-phenol substituent, which in the co-crystal structure of ALK2
occupied the solvent-exposed hydrophobic channel at the entrance of the ATP pocket (Figure
5.1). Here, several functional groups were used as replacements of the 3-phenol (Figure 5.2c),
chosen either to mimic hydrogen bonding of the phenol with Asp293 or to introduce an
electropositive charge (e.g. a protonated amine) to mediate an ionic interaction with Asp293,
thus maintaining a potentially important interaction. To gain insight into the potency and
selectivity for BMP vs. TGF-P signaling, derivatives were tested for their ability to bind BMP
type I receptor ALK2 and TGF-P type I receptor ALK5, using an in vitro thermal shift kinase
assay. This type of assay has been previously shown by us and others to be highly predictive of
biochemical kinase inhibition activity [144], which was also measured in a selected subset of the
derivatives. Tm shift data were found to correlate highly (r2 > 0.8) with biochemical inhibition
data
79
D281
H284
N-lobe
D980
83
Y282
Hinge Y285
IE287
E284
35
a
242
E24b
H285
G280
2
283
H286
01
MH5
GG286
D354,
ND351
Activ ation
lo :)P
D290N341
C-lobe
D293
Catalytic
loop
0
*
ALK2-K02288 (PDB 3MTF)
ALK5-SB431542 (PDB 3TZM)
Figure 6.1. Superposition of the ALK2 and ALK5 co-crystal structures with K02288
and SB431542,
respectively, showing selected interactions of ALK2 with K02288. The ATP pocket in many ALK5 cocrystal structures shows a more open conformation with a subtle movement of the N-lobe away from Clobe. Such conformational differences, which change the shape, volume and dynamics of the ATP
pocket, are likely to impact inhibitor selectivity.
(Figure 6.3).
To assess the potency and selectivity of these compounds in cells, inhibition of
BMP6-induced transcriptional activity (BRE-Luciferase) and TGF-01-induced transcriptional
activity (CAGA-Luciferase) was measured for each of the compounds (Fig. 2c), using cell lines
(C2C12 for BMP6 and HEK293T for TGF-l1) previously shown to express a complement of
BMP or TGF-P receptors required for ligand-mediated signaling.[145] In general, the magnitude
of ATm for ALK2 and ALK5 correlated inversely with the log-IC5o for inhibition of BMP and
TGF-p-induced transcriptional activity, but with some minor exceptions.
Notably, K02288
exhibited a large thermal shift for ALK2 kinase protein (ATm = 13.2 C), consistent with potent
inhibition of ALK2 activity by biochemical assay (IC 50 = 34 nM), but was substantially weaker
in the cell-based assay of BMP6 activity (IC 50 = 421 nM, Figure 6.3c). Of the variants at the 3phenol position, compound 13 exhibited the best in vitro inhibition of ALK2, whereas compound
15 demonstrated the best cell-based activity. The occasional discordance between biochemical
(ATm and enzymatic) and functional assays (ligand-induced transcription) highlighted the need
for multiple assays in an SAR aimed at identifying probes with useful potency and selectivity.
80
(a)
(d)
2-aminopyridine scaffold
Thermal Shift vs Cell-based IC
OMe
*
100
40
OMe
*ALK2 vs BMP6
10
OMe
N
U
100
I0
R,
50
10,000
0
0 0
*ALK5 vs TGFb1
10
NH 2
11
12
13
14
15
16
ATm C Thermal Shift
N
0 /
(b)
HO
K02288
(C)
NH
OMe
OH
12
ATM( 0C)
13
Diff.
I0
15
14
BMP6
ALK5
ALK2
AT
ALK5
ALK2
HN
N
11
ATM(OC)
N
HO
IC50 (nM)
cs
(nM)
(nM)
.............................
.....................
M...
TGFP1
Fold
IC50 (nM)
Select.
580±..50
28
IjIM
11
13.5
12.0
1.5
~~.
nd
nd
. ...............
....
20±..1
.
.
.
.
.
.
.
......
. . . .. .. ....
.. . .......... ...
13
14.4
13.4
0.9
15
151
139
12
6.2.
Figure
activity,
(b)
and
Potency
and
ligand
induced
to
Modifications
enzymatic
(d)
of
BMP6
the
K02288,
the
BMP6
(12)
but
3-phenol
a
TGF-$31
reduced
BMP6
with
selectivity.
3-
to
to
or
4-phenylpiperazine,
The
largest
with
as
81
previously
inhibition
of
while
=
K02288
and
BMP6
cell-based
not
determined).
3-phenol
(11)
with
potency
done
a
with
with
LDN-193
group
Replacing
the
to
compared
inhibition
occurred
of
degree
3-methoxy
selectivity.
BMP6
improved
potency
modest
a
Adding
similar
showed
increased
retaining
6.2c).
in
of
biochemical
(ATm),
(nd
4-phenol
(28-fold,Figure
increase
shift
solvent-exposed
K02288,
(13)
kinase
biochemical
scaffold
Thermal
compounds
the
modestly,
shift,
thermal
23
assays.
3-phenol
methylsulfonamide
bioisosteric
and
derivative
4
100 10
2-aminopyridine
(c)
inhibition
compared
inhibition
The
proteins,
BMP/TGF-3
signaling
on
K02288.
K02288
Replacing
~.2O-fold
versus
kinase
by
(a)
of
modifications
selectivity.
by
decreased
cell-based
)
1
260±220
4± 1
based
assays.
(R
ALK5
(ICso)
potency,
in
4-phenol
with
and
altering
signaling
for
3-phenol
of
to
and
ALK2
shift
alternations
selectivity
to
for
activity
thermal
addition
significant
against
)
0
transcriptional
Correlation
In
5
activity
domain
60±*10
186
derivatives
K02288
exposed
solvent
180
10
transcriptional
the
(IC
inhibition
TGF-p31-induced
of
selectivity
6
j
the
189,
replacement
likely
due
(a)
(C)
ATm(*C) ALK2 vs Biochemical ALK2 IC 50
10,000
R2
0
V 1,000
= 0.836
(A
-
100
Z
U
0 0
10
10
0
W
1
1
8
9
10
11
12
13
14
15
8
16
10
12
ATm( 0C) ALK2
ATm(*C) ALK2
(b)
(d)
ATm(*C) ALK5 vs Biochemical ALK5 ICSO
100,000
10,000
10,000
R = 0.800
0u
*
1E
R2=O0.6569
NIL
IC3E1
1,000
14
ATm(*C) ALK5 vs TGFbI IC 50
100,000
U
iC
00.7739
1,000
R2
100
E
ATm(*C) ALK2 vs BMP6 IC 50
10,000
N
1,000
07
100
100
0
10
10
1
1
4
6
8
10
12
14
16
4
ATm(*C) ALK2
6
8
10
12
14
16
ATm(*C) ALK2
Figure 6.3. A strong negative log-linear correlation is seen between thermal shift and biochemical
IC5 0
for both (a) BMP (ALK2) and (b) TGF-p (ALK5) type 1 receptors. A strong negative log-linear
correlation is seen between thermal shift of BMP type I receptors and ligand induced cell-based IC 50 for
both (c) BMP6 (ALK2) and (d) TGFbI (ALK5).
to the increased polarity of this substituent resulting in both improved inhibitor aqueous
solubility and increased enthalpic interactions with solvent water molecules.[107]
14 and 15 demonstrated 70-100 fold increases in BMP6 inhibition (ICso
=
Compounds
6 nM, and 4 nM)
compared to K02288, with modest improvements in selectivity. Compound 15 is structurally
similar to previously disclosed aminopyridine inhibitors of interleukin-2-inducible T-cell kinase
(ITK) and pyridine benzamide inhibitors of protein kinase D (PKD).[146]'[147]
To further
investigate the type I receptor selectivity of 15, cells were transfected with adenoviruses
expressing constitutively-active BMP type I receptors (caALKl, caALK2, and caALK3) and
constitutively-active Activin or TGF-P type I receptors (caALK4 and caALK5) in low serum
conditions and in the absence of exogenous ligand. Derivative 15 was most potent against BMP
receptors caALK2 and caALK3 with IC 5 o measurements of -3.5 nM, whereas caALK1, whereas
the Activin/TGF-P type I receptors and caALK1 were inhibited with with an IC 50 measurements
82
of ~20 nM, with nearly complete extinction of BMP and TGF-P type I receptor signaling
inhibition at approximately 250 nM. Taken together these data demonstrate that replacing the 3phenol in the solvent exposed region of K02288 with 4-phenylpiperazine dramatically improved
its potency in cells, but with relatively poor selectivity for BMP versus TGF-b signaling. These
results prompted us to explore structural variants at other positions that might refine selectivity
while retaining gains in potency afforded by modification of the solvent-exposed 3-phenol with
4-phenylpiperazine in 15.
6.3.2
Structure-activityrelationship(SAR) of hydrophobicpocket position
Further modifications of potent compound 15 were performed to develop an SAR for the
3,4,5-trimethoxyphenyl group (R2 ) (Figure 6.4a,b) to identify the role of each methoxy group on
potency and selectivity. The trimethoxyphenyl group has previously been shown to interact with
the hydrophobic back pocket in ALK2 where it forms water-mediated hydrogen bonds with the
catalytic lysine residue (K235) (Figure 6.1). Compounds were again profiled in thermal shift,
biochemical enzyme inhibition, and cell-based luciferase reporter assays (Figure 6.4c,d).
Removal of any of the methoxy groups resulted in a significant decrease in BMP inhibition.
However, particular methoxy groups were more crucial than others. For example, removing one
of the 3-methoxy groups (16) resulted in a 35-fold loss in potency compared to 15, although
selectivity for BMP6 inhibition versus TGF-P increased. Removing the 4-methoxy group (17)
decreased activity 10-fold, but did not improve selectivity.
Combining these changes (18)
demonstrated that the 4-methoxy group contributed less significantly to BMP6 inhibition as
compared to either meta-methoxy group. As expected, removal of both meta-methoxy groups,
while retaining the para-methoxy group (20) drastically reduced potency by almost 500-fold.
Incorporating the 3,4-dimethoxy groups into a benzo-1,4-dioxane (19) resulted in decreased
activity compared to 16, perhaps reflecting disruption of the hydrogen bond with ALK2 residue
K235. In addition, increasing the steric bulk of the 3-alkoxy group (21) or replacing the 4methoxy with a chlorine (22) or a methyl (23) were also not productive. In conclusion, of the
compounds studied the 3,4,5-trimethoxphenyl group resulted in the best balance between BMP6
inhibition and selectivity over TGF-P. This is likely due to its greater molecular volume for
occupying the hydrophobic pocket in ALK2, while retaining hydrogen bond acceptors in the
83
(a3)
2-aminopyridine
scaffold
HN
(d)
N
Thermal Shift vs Cell-based IC 5O
100,000
a
10,000
C.)
B.
1,000
100
R2
*ALK2 vs BMP6
*ALK5 vs TGFb1
10
N
NH2
2
OMe
(b)
OMe
OMe
0
15
(C)
N OMe
17
N
20
ALK2
ALK5
ATM( 0 C)
ATM( 0 C)
21 - N
ATM
Duff.
ALK2
ALK5
IC
(WM) 50
IC6
(nM)0
....... ... ...
11.9
14
..
......
OMe
CI
iiiiiiOMe
18
SMe
.aOMe
22
23
BMVP6
n)
IC5
IC0(M
TGFP1
I,0(M
IC0(M
2.7
63
1,910
Sect
M_
.. ......
..
..
9.2
Fold
eet
HM!M M.NIK
.:M
-H~
22
10
ATm *C Thermal Shift
LOMe
."OMe
16
~~~~~0
19
0
NOMe
OMe
2=
6
0
360±10
53,800±6,00
16
Figue
seectiityof
64. Pteny ancmpond 5 deivaivesbasd onthemal hif, bicheica
kinase~~~~~~~~~~~~~~~~~~~~~..
acivty and. liadidcdtasrpinlatvt.say.()Te2aioyiiesaflf1
(b)Moifcaton t the1 AT-bidin pocket hydophbi domai (TI2
of copud1.()qhahf
NNA
m), bichmia enzymati inhbiio ..
for ALK2 an....knae.rtens.ndiniitono
cel-asd MP ad GFf31-ndce transcipioa acivt
I.
50 by.ompund15.eriaties ...
notdetrmied) () Crreatin o terml.siftan
celbae ......
f inibti nasa..
met-poitonsof.hepheyl.in
. Futur stde will seek tootiiebidn.ih
hydrophobic:
poke byNreplcin th ,45timtoxpeyetreywihadiesestofay
and
moieties.
heeoay
84
6.3.3
Structure-activityrelationship(SAR) of hinge bindingposition
To further explore the SAR of 15, we modified the primary amine residue of the 2aminopyridine (R3 , Figure 6.5a,b), a region that was previously shown to interact with the hinge
region of the ALK2 kinase domain. Here, the amine was within hydrogen bonding distance of to
the backbone carbonyl of H284, as well as the gatekeeper residue T283 of ALK2 (Figure 6.1).
Both residues are changed in ALK5 (D281 and S280, respectively).
Secondary and tertiary
amines such as 24 and 25, respectively, exhibited reduced potency in both the thermal shift and
cell-based assays.
Similarly, 28, in which the primary amine is replaced with a methoxy
substituent, exhibited decreased potency, suggesting that bulky substituents are not well tolerated
at this position. Notably, these compounds exhibited negligible thermal shift despite detectable
albeit low activity in cell-based assays. Despite the high degree of correlation between the
thermal shift and cell-based assays (Figure 6.3b), compounds that exhibit very low ATm may
exhibit measurable inhibition in cells at moderately high concentrations, potentially due to
cytotoxicity at high concentrations (>50 pM, Figure 6.9).
Replacing the primary amine with
hydrogen (26) resulted in only a modest decline in potency and significantly increased selectivity
for BMP6 versus TGF-P signaling, suggesting that the primary amine hydrogen bond to the
hinge backbone is more critical for binding to ALK5 (D281) than ALK2 (H284). Finally, we
used two other small substituents, e.g. chlorine (27) and methyl (10) groups to explore the
possibility that ALK2 is less sensitive than ALK5 to substituents at this position. Although both
compounds lost potency relative to 26, there was a significant increase in selectivity for BMP
over TGF-P signaling with both showing greater than 150-fold selectivity in cell-based assays.
In particular, 10 remained relatively potent with a biochemical IC 50 of 24 nM for ALK2, a cellbased ICso for BMP6 of approximately 100 nM and 164-fold selectivity for BMP6 versus TGF-
p1.
The activities of compounds 15, 26, and 10 in the thermal shift kinase assay as well as two
different cell-based assays (ligand induced transcription and constitutively active type I receptor
transcriptional activity) are summarized in Supplementary Table 2. Across these various assays,
the results are consistent with increased selectivity for compound 10 (LDN-214117) albeit with a
reduction in potency. These results highlight that the 2-position of the pyridine in the K02288
series can be exploited to achieve reasonably potent and highly selective BMP inhibitors,
presumably via optimization of hinge domain interactions.
85
(a)
HN
scaffold
i
OMe
N
0
I0
OMe
OMe
(b)
ALK2
ATm( 0 C)
NH 2
15
ALK5
ATm( 0C)
C-,
1,000
C)
(U
.0
100
24
0.3
0.9
oenyan
0
*ALK5 vs TGFb1
0
5
10
NMe 2
H
CI
Me
OMe
24
25
26
27
10
28
ALK2
(nM)
ALK5
(nM)
.
.....
-0.5
elcivt o
15
ATM *C Thermal Shift
NHMe
K
Fiue ..
OALK2 vs BMP6
10
BMVP6
IC50 (nM)
...........
...... .....
10,000
R3
AT
Duff.
50
100,000
C)
N
R3 =
Thermal Shift vs Cell-based IC
(d)
2-aminopyridine
TGFI3I
IC50 (nM)
Fold
Slc
.
.
....
~~~~w...
...
.
nd
nd
**..
.........
280 ±60
I
28,000 ±5,300
99
........
0
...............
028 drvaiesbse
n hrmlshf,
ichmcaMins
activity, and ligand induced transcriptional activity assays. a) The 2-aminopyridine scaffold of 15 b)
Modifications to the primary amine kinase hinge binding domain (R 3) of compound 15. c) Thermal shift
(ATm), biochemical enzymatic inhibition (IC 5o) for ALK2 and ALK5 kinase proteins, and inhibition of
cell-based BMP6 and TGF-P1-induced transcriptional activity (IC 5o) by compound 15 derivatives (nd
not determined). d) Correlation of thermal shift and cell-based BMP/TGF-P inhibition assays.
6.3.4
=
Structure-activityrelationship(SAR) of K02288 and LDN-193189 hybridmolecules
We previously described a highly selective pyrazolo[1,5-a]pyrimidine based BMP type I
receptor kinase inhibitor LDN-212854 that exhibited biased activity for ALK2 [141]. This
selectivity was achieved by a 5-quinoline moiety that interacts with the same hydrophobic pocket
as the 3,4,5-trimethoxy group of K02288. With this in mind, we synthesized several derivatives
of 15 that replaced the 3,4,5-trimethoxyphenyl with 4- or 5-quinolines (Figure 6.6). As expected,
the 5-quinoline (31) demonstrated substantially increased selectivity for BMP versus TGF-P
inhibition (Figure 6.6b). However, all of these compounds were substantially less potent than 15.
Modeling of these 5-quinoline substituted compounds in the ATP-binding pocket suggested that
86
binding to the kinase hinge by the 2-aminopyridine scaffold may constrain the quinoline moiety
to a suboptimal position as compared to the pyrazolo[l,5a]pyrimidine scaffolds (Figure 6.12).
Conversely, replacing the 5-quinoline of LDN-212854 with the 3,4,5-trimethoxyphenyl of
K02288 yielded 32 that demonstrated potent BMP6 inhibitory activity, but with less selectivity.
Finally, hypothesizing that two individual changes yielding improvements in selectivity might
synergize, we combined the substitutions of the 2-amino group with hydrogen and the 3,5dimethoxy group found in 26 and 17 to yield 33.
Although this molecule demonstrated
improved selectivity it had considerably less potency.
ALK2
ATm(*C)
ALK
15
15.1
13.9
26
10
14.1
13.7
10.4
9.7
iff.
3.7
IC50 (nM)
Cell Based Assay
Ligand Induced
TGF-P
58
Ratio
15
BMP6
1
caALKI
24
caALK2
5
caALK3
6
caALK4
25
caALK5
23
26
15
952
65
202
43
105
427
215
10
67
14,650
778
186
382
5,535
4178
Ratio5/2
j
Table 6.1. Comparison of compounds 15, 26, and 10 across multiple assays including thermal shift kinase
assay, ligand induced transcriptional assay, and constitutively active ALKI-5 transcriptional activity
demonstrates increased selectivity for ALK2 for compound 10, albeit with a reduction in potency.
87
'-
I
29
HN
ON
N
N
N
Thermal Shift vs Cell-based IC50
100,000
29
S 10,000
N
N
N
U(-
1,000
100
~
31
HN
N
NH 2
N
M
.L
N
10
U
HN
1
N
N
N
as~~
15
30
OMe
OMe
ALK5
ATm( 0 C)
~
10
OMeN
MeO
N
ALK2
ATm( 0 C)
5
ATm *C Thermal Shift
NH
N
(b)
0
N
NH2
33
N
*ALK2 vs BMP6
*ALK5 vs TGFb1
ATm
Duff.
~~~(M
OMe
H
N
HN_
ALK2
(nM)
ALK5
(nM)
BMVP6
1050 (nM)
~
10Q
(W)
41~
N
TGFP1
1050 (nM)
NH2
H
Fold
Select.
~±P
30
9.9
7.4
25
110
5,000
520±6.2.80±300.
.....
1...
...............
3
..4................
.......
.........
4 ?O....
O .~*
...
..
.....
7
32.....
142
116..26..10.30
.20±2..760±80..41
.....
....
......
......
...
..
....
1....
....
...
1.2
Figur 6 6 oteny andseletivit of
08drvtvsbsdo
hra
hfbohmclknsb
aciiyasy. () tutr.fhbiddrvtvs
a :Tiit!
andTligand inue trascrptina
The.....al
shift...
(.m)
biohem
ca
enym
ti
ini
itio
....
for...............
AL...and.AL...kinase.proteins.and
0
inhibition~~~~~~~~~~~~
BMP an TG- 1-nue
of celbae
trncrpioa.ctvty....bhbidmleue
f
(nd = not deermined). (c Corlai noftemlThf
n celbae BM/G -11 inhibition assays..
6.3.5Kinome
.
seetvt of....
.DN2183
an LD-]1 7
We ~
reotdteknm-ie
~
preiosl
SE
eetvt o ohK08adLN1319
showin thtK...ha..or.eecieprfl.wt.fwrof-age.iassihiie.a o
(0 ............
...ad.ih(10.M)ihbio.onetatos[9,111.esogttodtn.n
h
kinome-wide seetvt
.........
of....
dervaive co pouds10.LD.....7)
.nd
15,vi
enzymtic of
inaspproimatly
proilin
20 kinsessummrize in.he.knomedendog.a
(Fiur 6.) The kinas most highly inibte bycmon60 (L20 14117 wa5L2
followed..
by...
TNI..........
PK2, an ABL..
1.dtie.eut
...
o io epoiig
rvddi
al
(L N- 1417 de ontrte significant......
Although....
les
potent tha 15,. compound.. 10..
6.2)......
88
improvement in selectivity across the kinome. At 100 nM and 1 pM compound 10 inhibited
only 0.5% and 3.6% of kinases profiled by more than 50%, whereas compound 15 inhibited
2.1% and 14.4% of kinases profiled, respectively.
(a)
LDN-212838
PDGFR
TK
LCK
-13
IPK2
4R
(b)
LDN-214117
TK
RIPK2
ALK5 K3
ALKi
CK1
ALK3
et
ABL *-
ALK1
ALK2
ABL
0.
~C
OK1
AGC
AGC
IC 50
0 < 1000 nM
CAMK
o
<100 nM
O
<10nM
CAMK
Figure 6.7. Kinome dendrogram plot for (a) LDN-212838 (15) and (b) LDN-214117 (10) showing an
improved selectivity profile for LDN-214117, albeit with reduced potency for BMP type I receptor
kinases.
% > 50% inhib.
5.4%
17.6%
1.6%
4.8%
# > 50% inhib.
12
39
4
12
% Inhibition
LDN-212838
6
7
8
Kinase
ABL-M351T
ABL-Q252H
ABL-H396P
ABL-Y253F
ABL-E255K
BRK
PDGFRa-D842V
MAP4K4
9
1
2
3
4
5
100
nM
Kinase
ALK2
TNIK
RIPK2
ABL1
MAP4K4
MAP4K5
LCK
PDGFR-BETA
MINK
ARG
10
11
PDGFRa-V561 D
12
DDR2
MAP4K2
ALK6
PRKD2
LCK
89
LDN-214117
100 |
i 1M
nM
ABL1__
LYNA
_
LYNB
LOK
9.2
41.3
YES
10.9
34.9
SLK
11.8
PDGFR-a
SNF1LK2
YES
21.7
41.5
FGR
8.9
8.6
HCK
21.1
ALK4
8.8
BRK
7.5
40.2,
FYN
7.5
39.7
MER
8.3
PDGFRP___
47.1
TNK2
5.9
MAP4K2
35.6
FMS
4.6
25.8
ALK5
3.3
MER
30.0
LYNB
7.6
PDGFRa
38.7
LYNA
3.0
FGR
36.2
SRC
7.5
TYR3
23.3
PRKD1
3.9
LOK
31.6
TYR03
3.8
EPHB2
21.7
PRKD3
1.6
TXK
21.1
ERB-B4
3.9
_
16.8
DDR2
-0.9
_
23.4
FLT-3
3.3
16.4
PKC-IOTA
11.9
LTK
13.5
FAK
25.7
LRRK2-G2019S
ABL-T3151
15.7
12.7
FLT-1
7.8
EGFR
3.2
PRKD2
12.1
EPH-A1
3.0
PRKD1
11.0
HCK
3.4
MRCK-a
6.3
EPH-B2
4.8
15.1
MARK3
4.1
TXK
12.0
13.7
EPH-A4
-0.9
P70S6K2
14.9
13.2
EPHB4
6.7
CLK3
9.1
12.4
P38P
TNK2
9.5
6.4
BLK
1.1
12.2
KIT
5.1
11.5
MARK4
5.3
MST4
8.7
11.5
PRKD3
7.2
EGFR
7.7
PTK5
LRRK2-G2019S
0.8
4.3
11.4
11.0
ALK
6.2
BMX
6.5
10.9
CSK
10.1
LRRK2
6.5
EGFR-L861Q
SRMS
6.3
11.4
P38-BETA
SGK3
PI3K-y
LTK
0.7
3.3
14.5
3.6
10.7
10.6
10.5
10.4
ARG
SRC
FYN
EGFR-L858R__
PTK5__
FMS__
BLK
90
54
MARK1
3.2
55
EPHA3
56
7.9
EPH-A8
CK1-GAMMA3
3.0
1.2
6.6
FES
7.1
57
CK1-y3
BMX
5.3
58
59
60
ERBB4
BRAF
TEC
9.1
7.8
4.6
FLT-4
MEK2
2.8
4.1
EPH-B4
4.6
CHEK2
0.8
61
TBK1
5.7
STK16
3.5
62
63
KDR
6.3
HIPK4
5.3
KIT
4.8
MELK
-2.9
64
MRCK-P
2.5
ALK
1.9
65
66
67
MET
6.6
IKK-E
0.6
CLK4
CK1
BTK
3.1
AXL
1.0
1.9
1.4
7.1
68
PAR-1 Ba
1.2
LATS1
4.4
7.1
69
70
71
CK1 a
4.7
TTK
12.8
BRAF-V599E
5.1
CK1-y1
RON
3.7
2.0
7.8
4.8
9.7
1.1
2.5
-2.5
7.0
7.0
6.4
6.4
6.0
5.9
75
TNK1
EGFR-T790ML858R
17.2
16.1
PI3K-6
P13K-a
PKN1
CK1-GAMMA1
PDK1
P13KB
0.4
14.3
MNK2
5.8
5.8
76
PYK2
10.2
13.5
EPH-B1
4.9
5.7
77
RET-Y791 F
3.1
12.8
RON
2.2
5.5
78
88
89
90
CK1-y2
MST1
P38a
FGFR1
RET
INSR
AURORA-B
ARK5
KIT-D816V
CHEK2
ROS
MNK2
EPHB3
3.0
2.7
4.0
2.5
8.4
3.9
2.6
2.5
0.9
2.4
0.9
1.0
4.6
11.9
10.0
9.4
8.1
7.9
7.9
7.8
7.8
7.8
7.5
7.4
7.3
7.0
SPHK2
CSK
ZAP70
DYRK1B
MKNK1
PKC-GAMMA
PAK6
PYK2
EPH-A4
DYRK1A
FGFR1
CK1-GAMMA2
TTK
12.6
3.8
4.2
2.5
2.9
2.8
1.0
1.4
7.4
2.8
4.5
0.5
3.0
5.3
5.2
5.0
4.9
4.9
4.8
4.8
4.7
4.7
4.6
4.6
4.5
4.4
91
AURORA-C
3.2
6.7
JAK2
5.2
4.2
92
93
P13-K-6
MEK1
NEK9
2.6
2.9
2.8
6.6
6.5
6.4
PAK4
TRKC
PAR-1 B-a
1.4
4.1
2.1
4.1
4.1
4.1
72
73
74
79
80
81
82
83
84
85
86
87
94
3.4
91
95
CDK6/cyclinD3
3.2
6.4
96
PAK1
0.3
6.4
97
106
PKC-a
MAPK1
TIE2
FGFR3
FER
CHEK1
PAK5
DYRK1A
FGFR2
FLT-1
1.2
3.3
1.1
1.5
0.4
2.1
9.0
2.2
1.6
2.6
6.2
6.1
6.0
6.0
5.8
5.7
5.7
5.5
5.4
5.4
107
MKNK1
2.6
5.4
108
109
TSSK1
MUSK
0.7
1.5
110
TRKA
111
GRK7
2.7
4.0
TBK1
1.4
4.0
MST3
IKK-ALPHA
TSSK2
GSK-3-BETA
PRKG2
PIM3
EPH-A5
PASK
MAPKAPK-2
PLK1
-1.9
2.5
4.3
1.6
-0.4
3.4
4.9
3.4
4.4
4.2
4.0
3.9
3.9
3.9
3.9
3.8
3.8
3.8
3.7
3.5
ERB-B2
-0.4
3.5
5.4
5.1
JNK3
AURORA-B
1.4
1.9
3.5
3.3
4.9
5.0
PHK-GAMMA1
2.9
3.3
112
RET-V804L
FLT-3
1.8
1.4
4.9
4.8
INSR
PKA
2.6
1.3
3.3
3.3
113
AMP-A1B1G1
0.3
4.8
TRKB
3.1
3.3
114
KIT-V560G
3.6
4.8
PRAK
0.5
3.2
98
99
100
101
102
103
104
105
CDK9-
118
ERB-B2
RSK1
PHKy1
MST2
-0.2
1.4
3.3
2.9
4.8
4.6
4.5
4.5
CYCLINT1
PKACB
FER
GRK6
2.9
2.4
2.9
8.2
3.2
3.2
3.2
3.2
119
RSK2
0.5
4.4
EPH-A3
1.8
3.2
120
PDGFRa-T674
2.2
4.4
CDK5-P25
1.8
3.1
121
125
126
PKC-y
EPH-A2
EGFR-T790M
PRKG1
FLT-4
P13-K-a
0.8
-2.5
2.2
-0.2
1.0
3.9
4.3
4.2
4.1
3.8
3.8
3.7
PKC-ZETA
RSK3
PAK1
MARK3
TAOK3
AURORA-A
4.8
3.2
1.9
0.4
2.8
1.9
3.1
3.1
3.0
3.0
3.0
3.0
127
IGF1R
2.4
3.7
CDK2-CYCLINE
-1.0
2.9
128
KIT-T6701
2.5
3.7
NEK7
3.7
2.9
129
DYRK1B
-0.7
3.6
JAK3
3.1
2.8
130
FES
NEK6
2.7
0.4
3.5
3.5
ROCK2
MST1
3.7
3.3
2.8
2.8
PAK6
4.6
3.5
CDK1
2.2
2.7
2.0
2.6
115
116
117
122
123
124
131
132
133
AURORA-A
2.3
3.3
PKC-ALPHA
134
DCAMKL2
-0.6
3.2
KDR
-0.5
2.6
135
JAK3
0.0
3.2
HASPIN
2.9
2.5
92
SGK3
CK2
CDK4/cyclinD
TRKB
PDK1
PHKy2
IKK-P
.7.8
3.2
STK25
0.8
2.5
7.9
1.1
2.2
3.0
2.0
0.6
3.1
2.9
2.9
2.9
2.8
2.7
HIPK2
MET
ROS
JNK2
TNK1
MEK1
1.5
-0.7
1.8
-0.2
4.9
-0.5
2.5
2.4
2.4
2.3
2.2
2.2
SGK2
1.9
2.7
CDK3-CYCLINE
1.7
2.2
147
JNK2
CAMK26
TRKC
IRR
2.0
1.4
2.9
2.6
2.6
2.6
2.6
2.5
TSSK1
JNK1
MSSK1
CDK7
2.3
-0.5
1.8
0.1
2.2
2.2
2.2
2.1
148
RSK3
2.0
2.5
CLK2
1.7
2.1
149
NEK2
3.7
2.5
FGFR4
1.4
2.1
150
AKT2
1.8
2.4
PAK3
2.4
2.0
151
HIPK1
BRSK2
FLT-3-D835Y
AKT1
AKT3
CDK2/cyclinE
PKA
ROCK2
1.2
0.6
-4.4
0.4
0.4
1.0
1.2
-0.6
2.4
2.3
2.2
2.2
2.1
2.1
1.9
1.9
MRCK-ALPHA
TAOK2
MUSK
CAMK1D
PRKG1
SRPK2
RET
IKK-EPSILON
-0.8
1.9
2.0
1.2
3.4
2.2
2.0
1.5
2.0
1.9
1.9
1.9
1.9
1.9
1.8
1.8
CDK2/cyclinA
1.5
1.8
MAPK3
1.1
1.8
ITK
CRAF
NEK7
IRAK4
RSK4
HIPK4
PKCP2
SGK1
PAK3
0.3
1.8
8.8
1.5
0.6
0.8
0.3
2.6
0.8
1.8
1.8
1.7
1.7
1.6
1.6
1.5
1.4
1.4
PKC-THETA
MARK4
CDK5
SRPK1
NEK2
TAK1-TAB1
IKK-BETA
LATS2
HIPK3
1.3
0.8
2.3
2.2
2.9
1.1
1.9
1.3
1.2
1.8
1.8
1.7
1.7
1.7
1.6
1.6
1.6
1.6
PLK1
3.1
1.3
CDK4-CYCLIND
-3.3
1.6
NEK1
1.0
1.3
DAPK3
0.7
1.6
173
PRAK
P38y
PKC-E
0.8
1.6
0.5
1.2
1.2
1.1
MARK1
JAK1
P38-ALPHA
-0.1
2.4
-2.2
1.5
1.5
1.5
174
P14-K-P
1.4
1.1
DYRK4
1.5
1.5
175
PASK
1.7
1.0
IRR
1.6
1.4
176
ZAP70
0.9
0.9
CDK6-CYCLIND3
2.8
1.4
136
137
138
139
140
141
142
143
144
145
146
152
153
154
155
156
157
158
159
160
161
162
163
164
165
166
167
168
169
170
171
172
93
177
MAPKAPK3
0.6
0.9
MAPK1
1.5
1.3
178
PKC-i
TSSK2
1.2
0.7
AMP-A1B1G1
5.5
1.3
1.0
0.6
ARK5
1.4
1.3
MST4
PRKG2
PAK2
P386
JAK
GRK6
MSSK1
0.2
0.3
0.2
1.8
1.3
0.4
-2.0
0.6
0.6
0.6
0.5
0.5
0.4
0.4
DYRK2A-SGC
P38-DELTA
HIPK1
PAK5
TRKA
DYRK3
BRSK1
1.5
0.6
0.8
1.1
1.7
1.1
0.9
1.3
1.3
1.2
1.2
1.2
1.2
1.1
PKC-p1
0.5
0.4
EPH-B3
1.0
1.1
PIM-2
P70S6KB1
BRSK1
0.0
0.2
0.1
0.3
0.2
0.1
RSK1
RSK2
BRSK2
1.1
0.8
1.7
1.1
1.1
1.1
DAPK1
0.6
0.0
PRKX
0.3
1.1
CLK3
MAPK3
0.4
1.5
0.0
0.0
AKT3
NEK9
1.3
1.2
1.1
1.1
JAK2
-0.2
0.0
AMP-A2B1G1
0.8
1.0
195
PRKX
0.7
-0.1
CAMK1A
1.1
1.0
196
CDK5/p35
0.2
-0.1
NEK1
1.9
1.0
197
198
199
200
201
MSK1
0.3
-0.1
SGK1
2.0
1.0
DYRK2
1.6
-0.2
CDK2
3.9
1.0
ROCK1
CDK3/cyclinE
0.3
0.1
-0.2
-0.2
P70S6K1
CAMK2D
1.5
1.2
0.9
0.9
179
180
181
182
183
184
185
186
187
188
189
190
191
192
193
194
TYK2
-0.1
-0.2
PKC-BETA1
0.4
0.9
202
GRK7
1.8
-0.3
PLK3
3.9
0.9
203
FGFR4
1.3
-0.3
IRAK4
0.5
0.8
204
CDK1/cyclinB
-0.6
-0.4
MST2
0.4
0.8
205
GSK3a
1.4
-0.5
MSK2
0.0
0.8
206
211
SRPK1
GSK3P
AXL
CAMK2a
CAMK4
MSK2
1.4
0.3
-3.7
0.6
21.1
0.9
-0.5
-0.8
-0.9
-1.0
-1.0
-1.0
SRMS
PKN2
CAMK2B
BRAF
EPH-A2
RSK4
-0.2
3.6
-3.8
0.0
-1.5
1.2
0.8
0.8
0.8
0.8
0.8
0.7
212
CAMK16
0.2
-1.0
TYK2
1.6
0.7
213
PKC-r
2.9
-1.0
AURORA-C
1.0
0.7
214
PIM-1
0.5
-1.2
IRAK1
2.3
0.6
215
CLK2
0.6
-1.3
CLK1
0.5
0.6
216
PIM3
1.0
-1.4
PAK2
0.0
0.6
217
IKK-a
-1.0
-1.9
GSK-3-ALPHA
1.0
0.5
207
208
209
210
94
218
MAPKAPK2
0.1
219
SYK
220
221
222
223
224
225
226
227
228
229
230
231
1
1
-3.6
TEC
0.0
0.9
-3.6
MSK1
0.7
0.4
SPHK2
5.6
-6.1
PIM-1-KINASE
1.1
0.4
SPHK1
1.8
-12.2
MRCK-BETA
-0.2
0.4
TIE2
1.1
0.4
NDR2
0.1
0.3
NEK6
ROCK1
DCAMKL2
PHK-GAMMA2
SPHK1
0.9
1.2
1.4
1.4
0.2
0.3
0.3
0.2
0.2
0.2
AKT1
0.5
0.1
IGF1R
MAPKAPK-3
0.3
-0.5
0.0
0.0
PIM2
0.5
-0.1
FGFR3
FGFR2
AKT2
NDRG1
CAMK4
DAPK1
SGK2
CAMK2G
PLK2
P38-GAMMA
SYK
CAMK2A
FRAP1
ITK
BTK
CHEK1
PKC-ETA
PERK
P14-K-BETA
0.1
-1.3
-0.6
1.0
0.0
-1.1
1.5
0.6
7.1
-2.3
0.2
-0.8
-2.3
0.0
1.5
3.8
1.9
-10.9
-10.2
-0.5
-3.1
-4.3
-14.5
-14.5
PLK4
-26.9
-26.2
232
233
234
235
236
237
238
239
240
241
242
243
244
245
246
247
248
249
250
251
252
0.5
-0.5
-0.6
-0.6
-0.6
-0.7
-0.8
-0.8
-0.8
-0.9
-1.0
-1.0
-1.1
-1.2
-2.4
Table 6.2. Kinome profiling for LDN-212838 and LDN-214117 at 100 nM and 1 M for >200 kinases
representing a wide sampling of the human kinome ranked by kinases with the greatest inhibition of
enzymatic activity.
95
6.3.6
FOP mutations, inhibitorbinding affinity, and implicationsfor therapeutics
The majority of individuals with FOP harbor the R206H germline mutation affecting the
glycine-serine (GS-) rich regulatory domain of ALK2 [66, 148-150]. While several of the other
known FOP-causing mutations also involve residues of the GS-domain (i.e., L196P, R2021 and
Q207E), several affect important regulatory sites within the kinase domain (i.e., G328E/R/W,
R258S, G356D and R375P) [68-70, 73, 98, 140].
We and others have shown that much of the enhanced cellular activity of various FOPcausing ALK2 mutants is attributable to differential regulation of the signaling pathway, i.e.,
impaired interactions with kinase regulatory protein FKBP12, and differential basal versus
ligand-induced signaling activity [73, 151]. However, there is the possibility that ALK2 mutants
have intrinsic differences in their enzymatic function, which could manifest as differences in
affinity for ATP and altered Km, with implications for their cellular activity and susceptibility to
inhibitors. We tested this directly by measuring the Km for ATP of wild type ALK2 and four
FOP-causing ALK2 mutants (L196P, Q207E, G328E, and R258S). The Km values for wild type
and mutant ALK2 were between 16 ptM - 48 pM (Table 6.3). Importantly, none of the FOPcausing mutants exhibited enhanced affinity for ATP as compared with wild-type ALK2. Since
ATP concentrations within cells vary from 1 - 10 mM [152], far in excess of the calculated Km
values, these slight differences in Km would likely be inconsequential in cells.
Kinase
Km ATP
ALK2 WT
16± 1
ALK2 Q207E
40 ± 5
ALK2 R258S
45 ± 6
ALK2 L196P
48± 3
ALK2 G328E
35 ± 5
Table 6.3. Km values for wild type ALK2 and various constitutively active mutant versions of ALK2 seen
in FOP
96
(a)
20 -
ATm(*C) ALK2 vs
GS Mutants
Mutant
316 -
M
.::u
TX
R2
.;.'=:fX"
=11TITM
IN; K11,11-11IN
IH
114 E.4111HATIPH,
00207D 1.1
QE 12 -
E
M.
x
0.99
Ebji-M
....
..
.....
....
...........
..........
.. ..
....
...
.
...
..
...
.....
RM
I:it
XL196P 1.3
0.96
8
8
ATM
(b)
20
,
16
12
0C
ALK2 WT
ATm( 0C) ALK2 vs Kinase
S
Mutants
Mutant
. ..
16 -C~4
-.
1 ...
R-144115ml
KE-KK
i-t-MM p--M M
H.......
: I.:
M:...
:.:..
- ...
...
.....
KH:
0.97
R375P 1 .0
.VV: ...........
VM i i:M
"'
:
Hn
1:1
IR
NFIXTUT
1:17T.F.:
j1j:q
if-1
±1 4'.i j. .............
+
-J
R2
M
............
...........
......................
'U.M.
MT11.
T1:5.
Wh-H-imi-ni
12 E
<-
8 '
8
12
16
ATm *C ALK2 WT
Figure 6.8. FOP causing ALK2 mutations do not affect inhibitor binding. a) Strong correlation of
thermal shift data for ATP competitive kinase inhibitors binding to wild-type ALK2 versus known FOP
causing GS domain mutations of ALK2 and b) known FOP causing kinase domain mutations suggests the
potency of ATP competitive inhibitors are not affected by these disease causing mutations. m = slope, R 2
= correlation coefficient.
A related, long-standing and clinically relevant question in the FOP field has been whether
mutant ALK2 proteins might exhibit differential inhibition by, or distinct affinity for particular
kinase inhibitors, and if so, whether highly-specific inhibitors could be engineered to target
selectively the activity of these activated mutant proteins. We sought to answer this question by
probing a panel of seven representative mutant ALK2 proteins with the library of K02288
97
derivatives displaying varying potency against wild-type ALK2 using a thermal shift kinase
assay. We found a highly linear correlation (r2 = 0.94-0.99) between the thermal shift induced
by these derivatives with wild-type vs. mutant ALK2 proteins (Figure 6.8).
These results
strongly suggests that ATP-competitive kinase inhibitors developed against wild-type ALK2 will
be effective in blocking signaling by all FOP variants, and likely precludes the possibility that
ATP-competitive ALK2 kinase inhibitors can be devised to selectively target only mutant ALK2
proteins.
6.3.7
Cytotoxicity of kinase inhibitors
We next sought to determine the cytotoxicity of these derivatives and to compare them to
many of the current FDA approved kinase inhibitors (Figure 6.9). Since hepatotoxicity is one of
the most common reasons for withdrawal of approved drugs, we used HEPG2 cells for
evaluation of cytotoxicity [153]. Compounds were tested in a large concentration range (1 to
100 pM) for 4 and 24 hours. Based upon residual cell viability after treatment, compounds were
categorized as having low (>75%), medium (25-75%), or high (<25%) cytotoxicity. Although
cell viability provides a simple quantitative measure of the tolerability of these compounds, other
approaches
measuring
cell
membrane
stability, apoptotic
activity,
cell
proliferation,
mitochondrial function, oxidative stress, and DNA fragmentation can be used to provide a
broader picture of overall drug tolerability.
Of the twelve approved kinase inhibitors tested only one exhibited high cytotoxicity at 100
pM after 4 hours of incubation, whereas 6 of the 28 derivatives in our K02288 library exhibited
high cytotoxicity after 4 hours. Over a 24 hour period 4 of 12 approved kinase inhibitors showed
high cytotoxicity at 100 pM, whereas 23 of the 28 K02288 derivatives showed high toxicity.
However, 10 which demonstrated good potency and high BMP selectivity exhibited very low
cytotoxicity.
In fact, cytotoxicity was not correlated with BMP signaling inhibition, TGF-p
inhibition, nor selectivity for BMP versus TGF-p signaling (Figure 6.10).
For example, the
highly potent BMP inhibitor 11 was also non-cytotoxic, suggesting that the mechanisms of
cytotoxicity within this series of compounds do not result from effects on BMP or TGF-p
signaling.
98
[c] pM
24 hr
4 hr
Compound
1
10
100
1
Imatinib
4 hr24h
Compound
10
100
[C] JAM
1
10
100
1
10
10
X16
Gefitinib
Sorafenib
17
18
Dasatinib
Sunitinib
Nilotinib
Lapatinib
Pazopanib
Ruxolitinib
Cdizotinib
Vemurafenib
193189
20
21
22
23
C
24
(Q25
26
bi
i27
10
212854
28
K02288a
11
29
30
12
13
31
32
33
14
15
Figure 6.9. Cell viability. HepG2 cells were exposed to 1, 10, and 100 PM of compounds for 4 or 24
hours. The average cell viability of three experiments is shown with green indicating >75%, orange
indicating 25-75%, and red <25%.
63.8
Structuralbasis of inhibitor binding
A number of the most promising derivatives were tested for co-crystallization with ALK2 to
further understand the binding mode and SAR. Diffraction quality crystals were obtained in the
presence of 26 and the structure of the complex was solved at 2.6 A resolution (Figure 6.11).
In the co-crystal structure, 26 was bound to the kinase hinge region as shown previously for
the parent molecule K02288 [98]. Both molecules established an ATP-mimetic hydrogen bond
between the pyridine nitrogen and the amide of H286. Replacement of the 3-phenol and primary
amine with 4-phenylpiperazine and hydrogen, respectively, did not alter the overall position of
26, but resulted in the loss of the hinge hydrogen bond interaction between the primary amine
and the carbonyl of H284.
The conserved 3,4,5-trimethoxyphenyl provided hydrophobic
interaction as well as a water-mediated hydrogen bond to the catalytic lysine K235. Docking of
10 produced a similar binding mode, with no significant change resulting from the introduction
of the methyl group. Overall, the ATP pocket occupied by these 3,5-diarylpyridines was similar
99
(a)
Cell Viability at 100pM vs Cell-based IC
50
*4 hr M24 hr
0 100%
0. 0
I.
0
.0
M0
50%
0
0
()
-U
0%
1
10
100
1,000
10,000
Cell-based IC50 (nM)
(b)
Cell Viability at 100pM vs Cell-based IC
0
@4 hr 024 hr
50
100%
0
0
0
50%
.0
MAMA
-~mi
0%
10
100
1,000
10,000
100,000
Cell-based IC50 (nM)
Figure 6.10. Plots of cell-based BMP (a) and TGF-p (b) IC 5 o versus cell viability show no correlation
between potency and toxicity
to the pyrazolo[1,5-a]pyrimidine scaffold of LDN-193189.
However, the two series differed
slightly in their hinge binding orientation resulting in shifts in the position of their respective
hydrophobic pocket groups as well as the shared 4-phenylpiperazine (Figure 6.12).
The selectivity of these molecules for ALK2 over ALK5 likely results from dynamic
conformational differences between these kinases as well as the modest number of sequence
changes in the ATP pocket. Perhaps as a result of its smaller serine gatekeeper residue, the ATP
pocket in many ALK5 co-crystal structures shows a more open conformation than those of
100
ALK2 with a noticeable movement of the N-lobe away from the C-lobe (Figure 6.1).
Such
conformational differences are expected to change the shape, volume and dynamics of the ATP
pocket to impact inhibitor binding.
(a)
(b)
Y25 H2
L281
H286
2.
A
D354
V222
H284
E248
L263
3-A
%248
K235
V222
E287
K25
T283
A243L..3
Wat
2. A
T283)
A35
M282.
M
4340
88L343
QH286
G2
N4
Y2
G28
V
S290
fN341
V214
(D293
Figure 6.11. Binding mode of 26. (a) The inhibitor (yellow) forms a single hydrogen bond to the hinge
amide of H286 as well as a water-mediated bond to the catalytic lysine K235. (b) Plot of the interactions
of the inhibitor (purple) in the binding pocket of ALK2. The plot was generated by LigPlot+.[154]
LDN-214117 model
LDN-1 93189
Y285,8
E4
KE24
T283
K235
E287
M288
H286
A353
G28
D354
N41
S290
Figure 6.12. Docking model for 10. Docking was performed using the ICM-Pro software package
(Molsoft) and the ALK2-26 structure as a template. Compound 10 (cyan) is predicted to bind similarly to
the parent molecule K02288 (PDB 3MTF) as well as the close derivative 26 (PDB 4BGG). The hinge
binding orientation of this 2-aminopyridine series differs compared to the pyrazolo[l,5-a]pyrimidine
scaffold of LDN-193 189 (dark blue thin sticks; PDB 3Q4U).
101
6.4
Conclusion
We have developed a library of BMP receptor kinase inhibitors based on the 2-
aminopyridine scaffold of K02288.
This library allowed us to explore the SAR of various
functional groups and resulted in the creation of several potent derivatives.
Several of these
compounds demonstrated improved activity, selectivity or both, measured using a thermal shift
assay, an enzymatic assay, and cellular assays of BMP/TGF-P-induced transcription, thus
overcoming the limited potency of the parent compound in cells.
We determined that the solvent-exposed 3-phenol substituent of K02288 was responsible for
its unexpectedly low activity in cells as compared to kinase assay IC 5 o. By replacing this group
with either a 4-phenol or 4-phenylpiperazine we were able to improve potency in cellular assays
compared to K02288 by 20- and 100-fold, respectively. We previously reported that the 3,4,5trimethoxyphenyl
occupies
the rear hydrophobic pocket
to provide
excellent
shape
complementarity, and forms water-mediated hydrogen bonds to the catalytic lysine residue
(K235) [98]. Here we found that the 4-methoxy group was largely dispensable, while the 3- or
5-methoxy groups were more critical for maintaining potency. The balance of selectivity and
potency found in the 3,5-dimethoxy derivative 17 suggests further medicinal chemistry
optimization is possible and could yield further insights into the determinants of activity in the
hydrophobic pocket. Within the 2-aminopyridine core, we found that the primary amine was
more critical for TGF- than BMP binding affinity, and could be replaced with a nonpolar
methyl group to generate a highly BMP selective compound 10 (LDN-214117).
Finally, we
concluded that replacing the 3,4,5-trimethoxyphenyl with quinolines as previously described for
pyrazolopyrimidine compounds (LDN-193189 and LDN-212854) was not an effective strategy
and resulted in a substantial loss of potency.
We used this structurally diverse compound series with varying degrees of potency to
explore the effect of FOP-causing mutations on inhibitor binding affinity. These compounds
exhibited nearly identical binding affinity for wild-type ALK2 and each of the FOP-causing
mutants tested, demonstrating that ATP-competitive inhibitors active against wild-type protein
will effectively target diverse FOP mutants. While this result would also suggest that ATPcompetitive inhibitors cannot specifically target mutant versus wild-type ALK2, one could
envision molecules targeting allosteric sites unique to mutant proteins to potentially achieve
specificity.
102
The novel series of compounds reported here constitutes an alternative pharmacophore with
discrete properties, including distinct kinome selectivity, as compared to the pyrazolopyrimidine
class of BMP inhibitors [98]. Several of these compounds, including 10 (LDN-214117), may be
useful as highly selective probes of BMP-mediated cellular physiology that may provide a useful
complement to the dorsomorphin class of compounds. Furthermore, this class of BMP inhibitors
offers a structurally distinct template for the development of therapeutics for the treatment of
BMP signaling-mediated diseases such as FOP.
103
Chapter 7 Development of a potent dual BMP/TGF-P inhibitor
Our laboratorywas selected,for its expertise in developing BMP kinase inhibitorsfor the
treatment of FOP, as one of the drug development projects for the Therapeuticsfor Rare and
Neglected Diseases (TRND) program at the National Center for Advancing Translational
Sciences (NCATS) at the National Institutes of Health (NIH). The purpose of TRND is to
advance drug development programs through the pre-clinicalprocess for rare and neglected
diseasesfrom both academia and private industry. In this chapter we describe the discovery,
resulting from our TRND collaboration, of a novel derivative based on the pyrazolo[1,5a]pyrimidine core, TRND-343765, that is a potent inhibitor of both BMP and TGF-3 type I
receptor kinases. In this chapter we characterize the properties of TRND-343765 and
demonstrate improved pharmacokinetics.Additionally we show a proof-of-concept using TRND343765 and inhibitor resistant mutant kinases as a platform for studying BMP and TGF-3
signaling in vitro.
7.1
Background and Motivation
Dorsomorphin, the original BMP type I receptor inhibitor "hit", was discovered in a high-
throughput high-content zebrafish embryo screen of 7,500 compounds looking for a dorsalization
phenotype that results from BMP signaling inhibition [63]. A modest medicinal chemistry effort
led to the synthesis of approximately 30 derivatives and a structure activity relationship looking
at inhibitory potency of BMP4-induced phosphorylation of SMAD 1/5/8 resulted in the
discovery of LDN-193189 a highly potent inhibitor of BMP signaling [107]. LDN-193189 was
shown to significantly, but not completely, inhibit heterotopic ossification (HO) in a mouse
model of fibrodysplasia ossificans progressiva (FOP) when given IP at 3 mg/kg BID [108]. In
Chapter 4 we described the development of potent and highly selective BMP type I receptor
kinase inhibitor, LDN-212854, that demonstrated a bias towards ALK2 with improved in vivo
tolerability [141]. In this study we demonstrated that IP administration twice daily of 6 mg/kg of
either LDN-212854 or LDN-193189 completely prevented HO in a mouse model of FOP, but
both compounds, LDN-212854 to a lesser degree, resulted in a significant negative impact on the
growth of mouse pups (Figure 5.5).
Using this relative weight loss, as compared to vehicle
treated pups, as a measure of compound toxicity suggested that the therapeutic index (toxic dose
divided by efficacious dose) for both compounds was less than desirable for a clinical drug
candidate [155].
Thus our laboratory's collaboration with TRND sought to take our lead compounds, LDN193189 and LDN-212854, and optimize them for various properties including potency,
104
selectivity, in vitro and in vivo toxicity, pharmacokinetics, pharmacodynamics, and metabolism.
TRND synthesized over 100 derivatives of LDN-193189 and LDN-212854 by varying both the
solvent exposed group (phenylpiperazine) and the deep hydrophobic binding group (quinoline)
while leaving the core scaffold (pyrazolo[1,5-a]pyrimidine) intact. Compounds were screened in
kinase assays, cell-based ligand induced transcriptional assays, and cellular viability assays.
During this process we identified a novel compound, TRND-343765, that is a potent inhibitor of
both BMP and TGF-P signaling and possesses desirable properties for both in vitro and in vivo
studies.
7.2
Experimental Methods
7.2.1
Kinase assay
Compounds were assessed in ALKl-6 enzymatic assays. Specifically, compounds were
assayed using LANCE® Ultra ULightTM technology (Perkin Elmer) against human ALK1-6
enzymes (ALKI: Life Technologies, ALK2-6: Carna Biosciences). Briefly, ALK enzyme (10
nM) was prepared in kinase buffer (50 mM HEPES, pH 7.5, 10 mM MgCl2, 0.005% Tween-20
and 2 mM DTT) and dispensed at 2.5pL/well into a 1536 well, white, solid bottom, microtiter
plate (Greiner, 789175-F). Negative controls for the assay were generated by adding one column
containing kinase buffer only. Compounds in DMSO solution were transferred to the assay plates
at 23 nL/well via a NX-TR pin tool workstation (WAKO, San Diego, CA) and incubated with
enzyme for 10 minutes at ambient temperature. ULight Topo Ila Substrate (50 nM) was prepared
in kinase buffer containing either 10 ptM or 1000 pM ATP and dispensed at 2.5 p.L/well into the
assay plate. Following a 1 hour incubation at ambient temperature, Europium anti-phospho DNA
Topoisomerase 2-alpha antibody (4 nM) was prepared in IX LANCE detection buffer containing
12 mM EDTA and dispensed at 5 p.L/well into the assay plate. Plates were measured using the
EnVision plate reader (Perkin Elmer), with excitation 320 nM and emissions of 615 nm and 665
nm.
7.2.2
Luciferase reporterassay
C2C12 myofibroblasts cells stably transfected with BMP responsive element from the Idl
promoter fused to luciferase reporter gene (BRE-Luc) were generously provided by Dr. Peter ten
Dijke (Leiden University Medical Center, NL)[1 11]. Human embryonic kidney 293T cells stably
105
transfected with the TGF-P responsive element from the PAI-1 promoter fused to luciferase
reporter gene (CAGA-Luc) were generously provided by Dr. Howard Weiner (Brigham and
Women's Hospital, Boston, MA)[1 12]. C2C12 Bre-Luc and 293T CAGA-Luc cells were seeded
at 20,000 cells in DMEM supplemented with 2% FBS per well in tissue culture treated 96-well
plates (Costar@ 3610; Corning). The cells were incubated for 1 h (37 0 C and 10% C0 2) and
allowed to settle and attach. Compounds of interest or DMSO were diluted in DMEM and added
at final compound concentrations of 1 nM to 10 pM. Cells were then incubated for 30 min.
Adenovirus expressing constitutively active BMP and TGF- type I receptors (Ad.caALK1-5),
generously provided by Dr. Akiko Hata (University of California at San Francisco), were added
to achieve a multiplicity of infection (MOI) of 100. Plates were incubated overnight at 37 0 C.
Cell viability was assayed with an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-dipheny
tetrazolium
bromide) colorimetric assay (Promega) per the manufacturer's instructions.
Media was
discarded, and firefly luciferase activity was measured (Promega) according to manufacturer's
protocol. Light output was measured using a Spectramax L luminometer (Molecular Devices)
with an integration time of one second per well. Data was normalized to 100% of incremental
BRE-Luc activity due to adenoviruses specifying caALK1, 2, or 3, or the incremental CAGALuc activity due to adenoviruses specifying caALK4 or 5. Graphing and regression analysis by
sigmoidal dose-response with variable Hill coefficient was performed using GraphPad Prism
software.
7.2.3
Cell viability assay
C2C12 and HEK293T cells used in the luciferase reporter assay were seeded in DMEM
supplemented with 10% FBS at 25,000 cells per well in tissue-culture treated 96-well plates
(Costar® 3610; Corning). The cells were incubated for 2 h (37 0 C and 5% CO 2 ) and allowed to
settle and attach. Compounds of interest or DMSO were diluted in DMEM and added at final
compound concentrations between InM and 100 pM. Cells were incubated for 24 hours after
which the media was discarded. Cells were lysed by adding 30 pL of passive lysis buffer
(Promega) and shaken at RT for 15 min. Cell viability was determined by quantifying the ATP
present in each well by adding 10 pL of CellTiter-Glo (Promega) per well and measuring the
light output Spectramax L luminometer (Molecular Devices) with an integration time of one
106
second per well. Data was normalized to 100% viability for cells receiving only DMSO without
any concurrent compound.
7.3
Results and Discussion
7.3.1
Lead optimization of LDN-193189 to improve metabolic stability
Although LDN-193189 is a potent inhibitor of BMP signaling, it has a short half-life in vivo of
less than 4 hours thus limiting its efficacy and requiring twice daily dosing for complete efficacy
[108, 141]. TRND identified the two main metabolic pathways for LDN-193189 to be aniline
formation at the phenylpiperazine and oxidation of the quinoline (Figure 7.1a). Aniline is a
known pro-mutagenic reactive metabolite that when activated can covalently modify DNA [156,
157]. Reactive metabolites are well known to cause toxicity the best example being paracetamol
(acetaminophen, e.g. Tylenol®) which is metabolized into the hepatotoxic compound N-acetylp-benzoquinone imine and is responsible for 80% of drug associated liver failure cases [158,
159].
Several strategies to prevent aniline formation were employed including changing the
phenylpiperazine group to an ethoxy-linked piperidine or adding non planar functional groups
such as in 2,6-dimethylpiperazine to provide steric clash with metabolic enzymes [156]. The
oxidized metabolite of LDN-193189, NIH-Q55, was identified to by in vitro analysis to be the
product of aldehyde oxidase (AO), an enzyme whose importance has been increasingly
recognized in drug development [160, 161].
Pharmacokinetic analysis revealed that LDN-
193189 is quickly metabolized by the liver into NIH-Q55, which demonstrated plasma Cmax
levels and total exposure levels (AUC) many times greater than that of LDN-193189 (Figure
7.1b).
We tested the cell-based potency of NIH-Q55 against BMP6-induced transcriptional
activity and found that it was significantly weaker (ICso = 935 nM versus 14 nM) than LDN193189. The fact that LDN-193189 is quickly converted into a low-potency metabolite suggests
that its efficacy could be significantly improved by overcoming AO oxidation of the quinoline.
One strategy to prevent AO oxidation was to methylate the 2-position on the quinoline.
107
(a)
aniline
H2N
~
HNN
HNIH-Q55
HN
HN
ON
N
O
N-N
N
N
N
N
LDN-193189
aldehyde
oxidation (AO)
N
0
ACa (ngh/mL)
11530
224,00
AUC (ghr/)
550
9 00
Muscle AUC* (ng.hr/mL)
8,130
37,500
N
H
* Accumulation in muscle tissue after 7 days dosing at 10mg/kg/day
Figure 7.1. (a) In vitro studies of metabolism for LDN-193189 revealed major metabolism via oxidation
of the quinoline (NI.H-Q55) mediated by aldehyde oxidase. Analine formation at the phenylpiperazine
position was also observed. (b) In vivo studies confirmed the in vitro findings and found that LDN193189 is quickly metabolized by the liver into a low potency metabolite, NIH-Q55. Furthermore, the
metabolite accumulates in tissues (e.g. muscles) at concentrations far exceeding those of LDN- 193189.
7.3.2
SA R of LDN-193 189 derivatives reveals 2-methyiquinoline reduces selectivity
In order to prevent oxidation of the quinoline by AO, several derivatives of LDN- 193189
were synthesized with 2-methyl quinolines. In each case, methylation at the 2-position of the
quinoline resulted in a significant (>90%) loss of selectivity between BMP and TGF-3 signaling
inhibition (Figure 7.2). The quinoline group interacts with the deep hydrophobic pocket of
108
(a)
(b)
HN
TRND-319114
ON.
Compound
BMP6
TGFbl
Selec.
(nM)
(nM)
.............
MM
U.M.
.............
..........
. ......................
...........
..
...
...
...
...
...
......
.
*P A...................
titNT1i::M-1IT1.
...
...............
.....
I-........
MIT
FPV:
..............
............
......
....
.
............
!ijj V:M.4
..........
...
..........
N
LDN-193189
M
:
TRND-343765
N
i; : : : :1:
M. .; : ..
::::::i:::
.:
TE1
rr
...
.....
..
...........
.
.
....
::I::.
H-V ......................
.... HTM
.....................................
...
........................................
.............
!.!:.:: ..........................
3
36
96
TRND- 319114
.....................
........................................
............
............
4................................
....................
M ............
....
...
...
...
...
...................
..........................
......
.....................
.......
............
IN :
...
...
...
M
...
...
...
...
...
...
..
...
...
....
MM:
......
-..
'...
:............
...
..
....
...
.
.....
J .;.............
..
....
.........
.....
...
...
...
...
P:
............
. ....
...
...
...
.
........ ...
.............
..............
0 11
...................
..............
.......
........
4. ..
:: .....
..
...........
.........................
..........................
....................
..
...
...
;:.:::.
;Wj :1M.HTM!
................
..............
...............
............
V.
.................
.................................
...........................................
N
TRND-263025/\/
NN
TRND-347968
N
1.5
21
31
TRND-343765
........... ....
...
...
...
...
...
...
...
...
...
...
..
...
...
...
...
...
...
...
...
...
...
...
.
..........................
...................
...
...................
................
.....
....
::.......................
.............
..................
..............
4....
.......
...............
::.........................
...
...
...
...
...
.....
...
...
...
...
...
...
...
...
.
.............
M.M.M.M.M.Uh..
r::::::
......
.....
............
...
....
...
...............
.7
..
P
...
................
...
XM-V
4
-3
MI ..............
..
.
..
...
....
.......
.................
.............
.
...
...
.........
.
...
...
..
...
...
..
..
...
...
...
...
...
...
.
..
...
...
...
...
...
...
...
..
..........
............
....................
...
:...
.....
l.....
.......
......
...
.................
t:::
9.
.....
.....................
............
..
.............
.......
..........
...............
...................
.................................
N
TRND-344860
TRND-347968
/\/
614
226
0
N
Figure 7.2. (a) Methylation at the 2-position of the quinoline in LDN-193189 and derivatives was used
as a strategy to block aldehyde oxidase metabolism at this position. (b) In each case there was a dramatic
decrease in BMP versus TGF- selectivity
ALK2 and the methyl group at the 2-postion could potentially interact with the gatekeeper
residue Thr283. It is possible that the methylation at the 2-position completely alters the binding
mode of LDN-193189 derivatives to either ALK2 or ALK5 or potentially both resulting in
reduced selectivity.
Further crystallographic studies of 2-methyl quinoline derivatives co-
crystallized with ALK2 and ALK5 may reveal unique binding interactions that result in reduced
selectivity.
Of the 2-methyl quinoline compounds screened, TRND-343765 was of particular interest.
TRND-343765 maintained low nanomolar potency against both BMP and TGF-P signaling
(Figure 7.2b) and was optimized to prevent aniline formation with a 2,6-dimethylpiperazine
solvent exposed group. Given these changes, we hypothesized that TRND-343765 would have
an optimal pharmacokinetic profile with improved half-life and exposure levels as compared
with LDN-193189.
7.3.3
In vivo metabolism of TRND-343 765
TRND analyzed the pharmacokinetic properties of TRND-343765 and as hypothesized the
compound had significantly improved pharmacokinetic properties as compared with LDN109
193189 suggesting it could serve as a useful in vivo probe. In fact, TRND-343765 had almost
twice the half-life, more than four times the exposure, and improved bioavailability as compared
with LDN- 193189 given the same 3 mg/kg P0 dose (Figure 7.3).
(a)
H NNK
N
HN
N
N
N
N
N.
-
N
N
-N
N
LDN-1 93189
TRND-343765
........
..............
..................
.................
....... 4.....
9....V
nL
...
...
...
...
9...
I6
130
>300
I --. ......... W
CStbltyxicty (pm)
(b)
)
Pharmacokinetics, PO at 3mg/kg
--
TRND-343765
-e- LDN-1 93189
500
400
E 300
C
200
100
0
0
6
12
Time (hours)
18
24
Figure 7.3. Significant improvements to in vivo metabolic stability seen with TRND-343765. (a) TRND343765 had improved half-life, AUC, and oral bioavailability (F) as compared with LDN-193189. (b)
TRND-343765 demonstrated superior pharmacokinetics to LDN-193189 with high plasma concentrations
at 7 hours after administration, while LDN- 193189 was almost completely cleared.
110
7.3.4
Characterizationof TRND-343 765 as a potent dual BMP and TGF-3 inhibitor
(
I
I
LDN-193189
TRND-343765
190nM,
'U]
*ALK1
190nM
ALK3
X
ALK4
ALK5
ALK6
*
1_11
I
St
0
2
UI
4
-V-
2
0
logJJ) nM
4
logJcJ) nM
LDN-1 93189
IC, (nM)
TRND-343765
IC. (nM)
LDN-1 93189
IC. (nM)
TRND-343765
ALK2
10.4
7.9
27
32
ALK3
29.3
ALK4
388
28.9
1921
159
1,138
66.9
33.5
26.2
(b)
ALK6
IC., (nM)
280
183
LDN-193189
153
TRND-343765
.4
IC50 = 9 nM
\ TGFb1 IC50 = 290 nM
-BMP6
* BMP6 IC50 = 12 nM
* TGFb1I I5 =46 nM
0
-
02
4
2
I"&OID nN
logac3 nN
Figure 7.4. Characterization of the inhibitory profile of TRND-343765 compared with LDN-193189. (a)
Kinase assay inhibition curves for ALK1-6 showing that at 200 nM TRND-343765 completely inhibits all
BMP and TGF-P type I receptor kinases, while LDN-193189 only inhibits BMP type I receptor kinases.
(b) IC 5o and 1C 90 values for LDN-193189 and TRND-343765 showing potent dual BMP and TGF-P
inhibition. (c) Cell-based ligand induced transcriptional activity inhibition curves.
111
TRND-343765 demonstrated both improved pharmacokinetics and lower cytotoxicity than
LDN-193189 with no detectable cytotoxicity up to 100 pM as compared to significant cell death
at 10 pM for LDN-193189. Given these improved properties and preliminary results from in
vitro screening, we sought to characterize the inhibition profile of TRND-343765 compared to
LDN-193189 against both BMP and TGF-P signaling. We profiled TRND-343765 in cell free
kinase assays against all BMP (ALKI, ALK2, ALK3, and ALK6) and TGF-P (ALK4 and
ALK5) type I receptor kinases and found that TRND-343765 potently inhibited all six kinases
with complete inhibition at approximately 200 nM. Although in Chapter 4 we showed that
LDN-193189 was not as selective as LDN-212854, in this case it is significantly more selective
than TRND-343765 further demonstrating that TRND-343765 is a potent dual inhibitor.
(a)
Inhibition of BRE-Luc Transcriptional Activity at 100nM
1.00
(d)
Inhibition of CAGA-Luc Transcriptional Activity at 100nM
1.00
BMP2
oTGFb1
TGFb2
BMP4
BMP6
E0
0
*TGFb3
N
BMP7
0.50
- caALK4
scaALK5
0.50
BMP9
caALK1
caALK2
caALK3
0.00
0.00
DMSO
LDN-1 931 89
TRND-343765
DMSO
mE
LDN-1 93189
TRND-343765
mE
(b)
Inhibition of BRE-Luc Transcriptional Activity at 25OnMmE
(e)
Inhibition of CAGA-Luc Transcriptional Activity at 25OnM
mc
1.00
I
BMP2
T
oTGFb1
TGFb2
1.00
BMP4
N
BMP6
N
E
BMP7
E
0.5
BMP9
mTGFb3
UcaALK4
050
. caALK5
caALK1
caALK2
caALK3
0.00
DMSO
LDN-193189
DMSO
Inhibition of BRE-Luc Transcriptional Activity at 250nM
(C)
1.00
L
I
0.00
TRND-343765
T T
TRND-343765
Inhibition of CAGA-Luc Transcriptional Activity at 500nM
M
1.00
BMP2
LDN-193189
-TGFbI
BMP4
.N
TGFb2
a BMP6
TGFb3
N
o caALK4
* BMP7
0
Z
050
.BMP9
z
0.50
caALK5
scaALK1
-*caALK2
T
000o
DMSO
m.-...I
LDN-193189
T
T
I.L
caALK3
TRND-343765
000
DMSO
LDN-193189
TRND-343765
Figure 7.5. Cell-based inhibition of BMP and TGF-P transcriptional activity. (a-c) LDN-193189 and
TRND-343765 both potently inhibited all BMP ligand and constitutively active type I receptor
transcriptional activity (d-f) Only TRND-343765 potently inhibited all TGF-p ligand and constitutively
active type I receptor transcriptional activity.
112
We further characterized TRND-343765 against both ligand-induced and constitutively
active type I receptor induced transcriptional activity (Figure 7.5).
Both LDN-193189 and
TRND-343765 potently inhibited a broad array of BMP ligands (BMP2, BMP4, BMP6, BMP7,
and BMP9) and constitutively active type I receptors (caALK1, caALK2, and caALK3) with
complete inhibition seen at 250 nM, as measured by activity of the BMP responsive element
transcriptional reporter BRE-Luc.
Interestingly caALK1 and BMP9 were not as potently
inhibited as the other BMP ligands and receptors, showing only partial inhibition at 100 nM by
either compound. TRND-343765 completely inhibited all three TGF-P ligands (TGFbl, TGFb2,
and TGFb3) and constitutively active type I receptors (caALK4 and caALK5) at 250 nM while
LDN-193189 showed only partial inhibition, as measured by the TGF responsive reporter
CAGA-Luc. Taken together, these data demonstrate that TRND-343765 when used at 250 nM
in cell-based assays can effectively inhibit both BMP and TGF-P type I receptor kinase activity
and ligand induced signaling. Thus, TRND-343765 can serve as a useful in vitro tool whenever
potent inhibition of both BMP and TGF-P signaling is required.
7.3.5
Mutant type I receptors and TRND-343 765 as in vitro probes of signaling
We sought to exploit the unique properties of TRND-343765 in a novel engineered mutant
receptor system. Since this compound effectively silences BMP and TGF- pathways at low-tomoderate, and non-cytotoxic concentrations, endogenous signaling via either arm of these
pathways can be experimentally abrogated in cells by a single manipulation. In such a context,
the function of specific receptors could be studied by expressing engineered mutant type I
receptors that are resistant to the pan-BMP/TGF-$ inhibitor. A panel of such inhibitor-resistant
receptors could be developed to reconstitute discrete elements of the BMP/TGF-P transduction
machinery in the presence of TRND-343765 or similar "pan-SMAD" inhibitor molecules. This
system could be used to complement commonly used siRNA reagents that result in knock-down
of specific receptors expression (Figure 7.6a). Here we use TRND-343765 to abrogate the
kinase activity of all wild type BMP and TGF-P type I receptor kinases and then transfect our
cell type of interest with inhibitor resistant mutants that would function normally, including
transducing ligand-induced signals. Given the complex relationship and functional redundancy
between receptors of the BMP/TGF- signaling pathways, the ability to reconstruct this pathway
113
by its constituent parts may be critical for understanding how this transduction machinery
functions by a reductionist approach.
Kinase inhibitor resistance is commonly seen to develop in cancers because kinase
inhibition places selection pressure on cancer cells which eventually develop resistance-causing
mutations [162].
One such mutation occurs at the gatekeeper residue which forms the rear
hydrophobic binding pocket just past the highly conserved hinge region which binds ATP [79].
The gatekeeper residue is a conserved threonine (Figure 7.6b) in 20% of kinases, including
the BMP type I receptors (ALKI, ALK2, ALK3, and ALK6) and while in the TGF-p type I
receptors (ALK4 and ALK5) it is the less common but still similar serine [163]. The activated
kinase Bcr-Abl is responsible for the vast majority of chronic myelogenous leukemia cases and
when treated with imatinib can develop gatekeeper mutations that prevent inhibitor binding but
do not affect ATP binding such as a threonine to isoleucine (T3151) [164]. Replacement of the
small gatekeeper residue such as threonine or serine with larger amino acids such as isoleucine
or methionine can lead to steric clash with inhibitors that access this hydrophobic pocket and
mediate resistance.
We utilized this understanding of gatekeeper mutations to generate a proof-of-concept
inhibitor resistant ALK2 and its constitutively-active counterpart caALK2.
We mutated the
gatekeeper threonine to isoleucine (T2831) and transfected HEK293T cells with plasmids
expressing this receptor. As expected, ALK2 wild type receptor signaled only in the presence of
ligand (BMP6) while caALK2 wild type signaled without ligand and both were effectively
inhibited by 100 nM of TRND-343765 (Figure 7.6c,d). However, when stimulated with BMP6
ALK2 T2831 mutant continued to signal even in the presence 100 nM of TRND-343765
indicating resistance to this inhibitor. Similarly, caALK2 T2831 signaled in the presence and
absence of ligand despite 100 nM of TRND-343765, also demonstrating inhibitor resistance.
Thus, by using commonly observed gatekeeper mutations in cancer we have been able to
generate ALK2 and caALK2 that are completely resistance to TRND-343765.
We are thus able
to expose cells to 100 nM of TRND-343765 and transfect them with inhibitor resistant ALK2
T2831, for example, and know that all of the downstream BMP signaling is occurring through
our resistant type I receptor. This is a powerful tool that will enable us to very selectively study
BMP signaling phenomenon
114
BMP6
(a)
TGFb1
(b)
TRND-343766
TRND-343765
283
s
Type
receptors
Bre-Luc Transcriptional Activity
(C)
200,000
1
TRND
-TRND
J 100,000
TRND
TRND
0
ALK2 T2831
ALK2 WT
m-BMP6 -TRND
a -BMP6 +TRND
m +BMP6 -TRND
+BMP6 +TRND
Bre-Luc Transcriptional Activity
(d)
30O0.0
TRND
TRND
TRND
TRND
100.000
0E
caALK2 WT
m -BMP6 -TRND
caALK2 T2831
a -BMP6 +TRND
* +BMP6 -TRND
*+BMP6 +TRND
Figure 7.6. (a) Abstract representation TRND-343765 used in conjunction with inhibitor resistant type I
receptors. (b) Deep hydrophobic pocket of ALK2 showing threonine gatekeeper residue that was mutated
to isoleucine to generate inhibitor resistant mutants. (c) Baseline and ligand-induced signaling of wild
type ALK2 and caALK2 is inhibited by TRND-343765 while T2831 mutants show inhibitor resistance in
both cases.
115
7.4
Conclusion
In this chapter we described the discovery of a novel inhibitor, TRND-343765, with potent
activity against both BMP and TGF-
type I receptors and with significantly improved
pharmacokinetic properties making it ideal for use in vivo. TRND-343765 potently inhibited
BMP and TGF-P induced transcriptional activity in cells at 250 nM and showed no cytotoxicity
up to concentrations of 100 pM. We used TRND-343765 as the basis for a novel approach to
studying BMP and TGF-P signaling. We demonstrated how we could generate inhibitor resistant
mutant kinases capable of rescuing signaling in cells. These mutant kinases can be used together
with TRND-343765 to specifically understand the contribution of one or multiple type I
receptors towards signaling and functional phenomenon in cells. Future work will expand this
tool kit to include a full complement of inhibitor resistant BMP and TGF-P type I receptor
kinases that can be used in concert to carefully dissect signaling and function. Although careful
monitoring of potential cardiotoxicity would be necessary, TRND-343765 might serve as a
useful therapeutic where both BMP and TGF-p inhibition are desired such as in cancer tumor
progression.
116
Chapter 8 Conclusions
In this chapter we conclude the thesis with a summary of the work presentedemphasizing
the novelfindings and their implications. We also suggestfuture work that can be built on the
findings in this thesis and questions that are raisedas a result of the work
8.1
Summary of the thesis
The goal of this thesis was to develop a deeper understanding of the structural features
that modulate potency and selectivity of BMP type I receptor kinase inhibitors with the goal of
generating highly selective inhibitors of ALK2 for the treatment of FOP and the study of the
BMP signaling pathways. Although there is still much work to do before a compound is ready
for clinical trials, the work described within this thesis has made a significant contribution to the
development of highly selective and potent ALK2 inhibitors for FOP.
We have shown that a hydrophobic domain-interacting 5-quinoline moiety confers far
more selectivity than the previously described 4-quinoline moiety in the pyrazolo[1,5a]pyrimidine class of kinase inhibitors.
This new compound, LDN-212854, showed several
orders of increased selectivity for BMP versus TGF-P type I receptors, placing it on par with
selective TGF-s inhibitors developed by the pharmaceutical industry.
This compound
demonstrated efficacy in a mouse model of FOP and caused significantly less weight loss as
compared with the previously synthesized parent compound LDN-193189. We also showed how
the selectivity for ALK2 by this compound could be exploited to elucidate the role of ALK2 and
ALK3 in particular signaling phenomenon.
LDN-212854 provides an important proof-of-
concept for the development of highly-selective ALK2 inhibitors for FOP.
We worked closely with our collaborators Dr. Alex Bullock and Dr. Greg Cuny to
develop a set of novel BMP type I receptor kinase inhibitors based on a 2-aminopyridine core
structure. An alternative core scaffold to pyrazolo[1,5-a]pyrimidine offers the opportunity to test
a unique chemical space, provide further insight into the determinants of selectivity, and provide
another set of compounds that could have clinical utility. We synthesized and characterized a
wide variety of derivatives based on K02288 and were able to increase the cellular potency of
this compound by 100-fold through changes to the solvent exposed group. We were also able to
significantly increase the selectivity of these compounds by modifying the 2-aminipyridine core
to favor binding to BMP over TGF-P type I receptor kinases.
117
We used this unique set of
derivatives to answer and important question in FOP biology. We showed that ATP competitive
kinase inhibitors had equal binding affinity to wild type ALK2 and various FOP-causing ALK2
mutant kinases.
These results strongly suggest that any ATP competitive kinase inhibitor
developed against ALK2 will work equally well as a treatment for FOP regardless of the specific
mutation affecting that patient.
Finally, as a result of our collaboration with the NIH TRND program we made the
fortuitous discovery of a pyrazolopyrimidine compound that was a potent inhibitor of both BMP
and TGF-
type I receptor kinases.
While kinase inhibitor selectivity is desirable for the
treatment of most diseases, with the possible exception of certain cancers, this compound
provides a valuable scientific tool for the study of the BMP and TGF-
signaling pathways.
Additionally, this compound has been metabolically optimized and has ideal characteristics for
in vivo use. We demonstrated that inhibitor-resistant mutant type I receptor kinases could be
created by mutating the gatekeeper residue, analogous to the naturally occurring gatekeeper
mutations that arise in cancers under kinase inhibitor therapy. By treating cells with this dual
inhibitor we could effectively knock-down all BMP and TGF- signaling and rescue the very
specific signaling of one or a combination of inhibitor-resistant mutant type I receptor kinases.
This system will allow us to examine BMP and TGF-P receptor-mediated signaling by more
reductionistic approaches, and could reveal insights that have previously been inaccessible due to
the functional redundancy and complexity of this pathway.
In summary this thesis has generated significant understanding of BMP type I receptor
kinase inhibitors and has generated unique compounds that are very useful as scientific tools and
have potential to become therapeutic molecules for the treatment of FOP and other diseases.
8.2
Suggested future work
8.2.1
Optimization of 5-quinoline position
Our investigations revealed that a 5-quinoline moiety on the pyrazolo[1,5-
a]pyrimidine core was significantly more selective for BMP versus TGF-P type I receptor
kinases than a 4-quinoline. To better understand if this observation is generalizable and holds
true for all hydrogen bond acceptors at this position, bioisosteres of 5-quinoline should be
synthesized and tested to determine if they maintain or potentially improve upon the potency and
selectivity of LDN-212854. Additionally, other hydrogen acceptor groups could replace the 5118
quinoline nitrogen to determine what is optimal for potency, selectivity, and metabolic stability.
These further investigations will complete the understanding of how selectivity between BMP
and TGF-P type I receptors is achieved despite the almost complete sequence homology within
the ATP binding pocket. Finally, 5-quinoline derivatives with optimized pharmacokinetic and
drug-like properties could potentially serve as clinical candidates for the treatment of FOP and
other diseases of aberrant ALK2 signaling.
8.2.2
Role of BMP signaling in heterotopic ossification in Bmall-We used LDN-212854 to reduce heterotopic ossification (HO) in a novel model of
progressive arthropathy and joint ankylosis due to loss of a diurnal clock regulating gene in limb
tissues. An inhibitor of BMP ligands, ALK3-Fc, failed to reduce HO in this model, suggesting
that ligands which have relatively lower affinity for ALK3 may be responsible for this process.
Additional evidence for the involvement of BMP signaling in this process by a complementary
strategy, such as genetic ablation of responsible receptors, or an alternate ligand inhibition
strategy, would help to confirm this interpretation. To corroborate the idea that enhanced BMP
signaling is contributing to HO formation, immunohistochemistry and western blotting of tendon
tissues prior to the formation of HO might reveal evidence of enhanced levels of phosphoSMADl/5/8.
Also, expression levels for the BMP type I receptors, various BMP ligands, and
endogenous inhibitors such as noggin should be determined in these same tissues from Bmall-and wild-type mice. These studies may reveal specific components of the BMP signaling pathway
dysregulated by the absence of circadian cycling in limb tissues that may contribute to enhanced
heterotopic ossification.
These studies could reveal novel targets for preventing progressive
entheseal ossification commonly seen in diseases such as ankylosing spondylitis.
8.2.3
Improved selectivity for TRND-343 765
Compound TRND-343765 exhibits several excellent drug-like properties including high
oral bioavailability, metabolic stability, optimized pharmacokinetics, and low cytotoxicity.
However, this compound is not selective for BMP versus TGF-P type I receptors. This poses a
potential problem for use as a therapeutic considering the cardiovascular toxicities previously
identified with ALK5 inhibition. It is possible, however, that balanced inhibition of BMP and
119
TGF-P might be more tolerable than inhibition of TGF-P signaling alone, a possibility that could
be explored in follow-up toxicology studies.
Additional medicinal chemistry could also be
pursued to reintroduce selectivity while preserving the optimized metabolism of TRND-343765.
In particular, the 2-methyl substituent could be replaced by many other groups such as chlorine,
amine, fluorine, or ethyl, potentially retaining metabolic stability but altering selectivity.
8.2.4
ResistantBMP and TGF-3 type I receptors to study signaling
We demonstrated that signaling via engineered inhibitor-resistant BMP type I receptors is
preserved in cells treated with TRND-343765, while signaling of endogenous BMP and TGF-P
receptors is completely blocked.
The success of this approach demonstrates that inhibitor-
resistant BMP or TGF-P type I receptors can be used with pan-BMP and TGF-P inhibitor
compounds such as TRND-343765 to examine the function of specific receptors alone or in
combination, without interference from the array of other endogenous receptors which frequently
serve redundant functions.
A full complement of inhibitor-resistant ALKl-7 and caALKl-7
receptors would provide the field with a powerful method for studying this complex signaling
pathway by providing the ability to study some of its individual components in isolation.
120
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