Development and Screening of Transition Metal
Complexes as CXCR4 Antagonists
Dr. Tim Hubin
Department of Chemistry and Physics
1
CXCR4 chemokine receptor
•
Important role in embryonic
development:
– Organogenesis (liver, heart)
– Stem cell movement
– Cerebellar neuron migration
(formation of brain)
• Seven transmembrane Gprotein-coupled receptor
• 27% of amino acids are Asp, His
or Tyr.
• Expressed in :
»
»
»
»
Leukocytes
T-lymphocytes
Endothelial cells
Neuronal cells
Khan, A.; Greenman, J.; Archibald, S. J. Curr. Med. Chem. 2007, 14, 2257.
2
CXCL12
• 67 residue highly basic
protein
• Only known natural ligand
(chemokine) for CXCR4
• Secreted by stromal, lung
and liver cells, and lymph
nodes
• Attracts leukocytes to sites
of inflammation and
lymphoid organs
A
3
Disease states
• Role in disease
–
–
–
–
Human Immunodeficiency Virus (along with CCR5)
Tumour growth and metastasis
Stem cell mobilization
Autoimmune disorders (rheumatoid arthritis)
4
CXCR4-antagonists and HIV
• Inhibitors against 5
steps of HIV
replication cycle
• Still need for new
targets
• Prototype bicyclams
AMD3100
5
NH
I
Y
P
C
S I G E M -NH2
N
NH HN
N
HN
NH HN
CHO
T S D N Y T E E
M
G
E K M S D Y D G S
K Q G C
I
D D A
E
Y F G
R
I
E
F
W
A
Y
E
N
Y PN
E
N
N
F
I
L
F
D
V
F
A
N
D R
L
N
L
L C
N
W
ExtraT
V
K
I
A
V
H V K
A
cellular F I
K C
S F D
V
D
V
L
V
W
F
I
V A
P
171 F F Q
W A
I S
V H
262 I
F I
I T
I S
Q
I
P F
Y
H
I G
E T
P D
L
T Y V
I
Y
I S
I
T
A
M
Y
T
I
I
L
V
P
F
L
N
A
G
F
F V
Y L
T L
L L A
WL C
I L L
S
H F C
G
L L
A
S
I
P
D
V
V
I
P
C
F
A
G
F
L
L
V
I
I
N
N G
V W
L A
P
V
L S
L G
A L
V G
I
L I Y
L I
L H
V
I F
I L
S
R
V Y
A
Y
S
V
C
L I
V
T
F
Y
K
L
V
K
L
C
T
G
I
K
R D
A E
G M
D
IntraL
Y
I I S
KA
K A
L
T
F
L
cellular Y
L K
Q
L
K R
K
M
S
A
Q
K
S
H
K
T
I
K L R
H
S A QH
S
R
V
K G
P
H
V
R
A
S E S E T S
T N SQ
S
F R E E N
S
F H S S -COOH
Gerlach et al., 2001
6
Blocking receptor functions
Drug
CXCL12/HIV
Cell
7
Plerixafor/ AMD3100

The first bicyclams were
discovered as impurities
in a sample of cyclam.
Amongst the most active
anti-HIV agents in vitro.

Likely a prodrug;
complexation of Zn2+
will occur in plasma

Anti-HIV clinical testing
discontinued.

Stem cell mobilization
H
N
H
N
N
N
NH
HN
H
N
N
H
AMD3100
For example:
Mol. Pharm., 1999, 55, 67.
J. Med. Chem., 1995, 38, 366.
Biochemistry, 2003, 42, 715.
8
Molecular shape
H
N
H
N
N
NH
H
trans-I
trans-II
trans-III
trans-IV
trans-V
cis-V
Bosnich, B.; Poon, C. K.; Tobe, M. L. Inorg. Chem.,1965, 4,1102
9
Restrict to one configuration
N
H
N
N
H
N
R
N
N
N
trans-II
N
NH
Only trans-II
H
N
N
X
X
N
R
Only cis V
10
Side-Bridged Synthesis
Reagents: (a) acetonitrile, RT, 24 h (89%); (b) NaBH4, EtOH reflux, 1 h (65%).
11
Cross-Bridged Synthesis
(a)
(b)
Reagents: (a) acetonitrile, RT, 7 d (80%); (b) NaBH4, 95%EtOH, RT, 7 d (70%).
12
HN
N
MeH
N
N
NHH
N
N
N
H
H
N
N
N
HMe
Lewis, E. A.; Hubin, T. J.;AMD3100
Archibald, S. J. European Patent 1765826A2 .
13
Side bridged (SB)
Axial
Cu-O1
NH
N
HN
Equatorial
N
N
N
N
N
2.28(1) Å
Cross bridged (CB)
N
N
N
N
1.95(1) Å
N
N
N
Cu-O1
N
14
15
[Cu2(p-CB-cyclams)] = SJA5
16
CXCR4 Binding Assays
• Use anti-CXCR4
antibodies to screen
cell lines
• Two identified: Jurkat
and Molt-4
• Four anti-CXCR4
antibodies used
(variation in binding
epitopes)
A
17
Binding by flow cytometry
Fluorescent antibody
Receptor specific antibody
Drug molecule
Key
Name
- control.001
Parameter
FL1-H
+ Control 717.019 FL1-H
L2 717.010
FL1-H
L1 717.009
FL1-H
CXCR4
18
mAB Inhibition
[Zn2SB]4+
[Zn2AMD3100]4+
[Zn2CB]4+
Antagonist
[Cu2CB]4+
[Cu2AMD3100]4+
[Cu2SB]4+
AMD3100
SB
CB
0
20
40
60
80
100
120
mAb Inhibition / %
Summary of mAb 12G5 binding to CXCR4 in the presence of bound antagonists.
19
Competitive Binding Studies
Antagonist
Concentration (μM)
AMD3100
IC50
0.360 ± 0.030
EC50
0.134
CI (95 %)
0.067 - 0.267
Zn-AMD3100
0.278 ± 0.033
0.164
0.127 - 0.211
Cu-AMD3100
0.439 ± 0.021
0.303
0.240 - 0.382
Zn-Side Bridged
0.922 ± 0.161
0.740
0.457 - 1.200
Cu-Side Bridged
0.251 ± 0.074
0.251
0.191 - 0.330
Zn-Cross Bridged
0.230 ± 0.004
0.109
0.046 - 0.257
Cu-Cross Bridged
0.160 ± 0.004
0.100
0.086 - 0.116
IC50 and EC50 concentrations for CXCR4 antagonists in
competition with mAb 44717 in Jurkat cells.
20
% inhibition of antibody binding
Residence time
100
Copper 3
90
80
AMD3100
70
60
Copper AMD3100
50
40
30
20
10
0
1
24
48
72
96
hours
G. McRobbie, A. Khan, G. Nicholson, L. Madden, J. Greenman C. Pannecouque, E. De Clercq, T. J.
Hubin and S. J. Archibald, J. Am. Chem. Soc, 2009, 3416.
21
Compound
Concentration (16 nM)
(τ) (hr)
t1/2 diss (hr)
AMD3100
9.7
6.7
Cu2-AMD3100
23.5
16.3
Cu2-Cross
Bridged
49.0
33.9
Relative (τ) and t1/2 dissociation constants for
compounds binding to CXCR4 receptor in Jurkat cells.
22
HIV Infection Assays
Table 1. Anti-HIV activities, cytotoxicity and selectivity index in MT-4 cells.
Compound
HIV strain
Av EC50(μM)a
SDb
Av CC50(μM)c
SId
Zn2Side-Bridged
HIV-1 (IIIB)
HIV-2 (ROD)
0.0025
0.0040
0.0010
0.0013
60.56
60.56
29,215
16,077
Cu2Cross-Bridged
HIV-1 (IIIB)
HIV-2 (ROD)
0.00459
0.0171
0.0017
0.0011
>125
>125
>27,233
7,331
AMD3100
HIV-1 (IIIB)
0.011
>225
>20,455
Zn2-AMD3100
HIV-1 (IIIB)
0.008
>225
>28,125
a
average effective concentration to reduce the HIV-induced cytopathic effect by 50% in MT-4 cells. b standard deviation over
three assays. c Concentration required to have a cytotoxic effect reducing MT-4 cell viability by 50%. d Selectivity index.
AMD3100 data from Este, et al Mol. Pharmocol. 1999, 55(1), 67-73.
23
Ca2+ Ion Signaling Assays
8000
Fluorescence Change (counts)
Control
7000
1000 ng/ml
200 ng/ml
6000
40 ng/ml
5000
8 ng/ml
4000
3000
2000
1000
0
-1000
0
50
100
150
Time (sec)
Ca-signaling data for AMD3100 CXCR4
experiment by collaborator Schols.
24
Table 2. IC50 (ng/ml) values calculated from Ca-signaling experiments
U87.CD4.CXCR4
U87.CD4.CCR5
Cu2-Cross Bridged
4.33
>1000
Zn2-Cross Bridged
5.42
>1000
Cu2-Side Bridged
43.97
>1000
Zn2-Side Bridged
<1.6
>1000
AMD3100
16.7
>1000
Cu2-AMD3100
72.67
>1000
Zn2-AMD3100
<1.6
>1000
maraviroc
>1000
2.09
AMD3451
Micromolar
Micromolar
25
CXCR4 and Cancer Cell Metastasis
o CXCL12 is normally responsible for trafficking of lymphocytes
o CXCL12 is secreted by stromal, lung and liver cells, and lymph nodes
o The interaction at the cell membrane is through CXCR4, which is overexpressed in some cancers
o Potential mechanism of metathesis
Normal cell
Cancer cell
ANTI-CANCER ACTIVITY
Invasion assays
• Cell invasion assays in response to a
chemokine gradient.
• Initially used SJSA cells.
• Experiments run in presence and absence of
antagonist.
27
Control
Drug/
no CXCL12
CXCL12
Drug + CXCL12
28
Cancer Cell Invasion Assay
Invasion of SJSA cells in matrigel
with CXCL12 (12.5 nM) and CXCR4
antagonists (20-200 nM). Cells
were counted in five different
fields (x40 obj) in duplicates.
Mean of the values plotted.
Asterisk represents significance (p
< 0.01) from B. A = no CXCL12
and no antagonist; B = CXCL12
only; C = 20 nM Cu-Cross Bridged
antagonist; D = 200 nM Cu-Cross
Bridged antagonist; E = 20 nM
AMD3100; F = 200 nM AMD3100.
29
Stem Cell Mobilization—OMRF (Barlic)
• An acute administration of AMD3100 is known to rapidly mobilize
bone marrow stem cells and progenitors. This mobilization is due to
inactivation of the CXCR4-CXCL12 axis which holds progenitors in the
bone marrow.
• AMD3100 induces neutrophilia and leukocytosis, which reach their
maximum 2 hours post-injection.
• AMD3100 has not been noted to have an impact on monocytes.
• C57BL/6 strain = a common strain of lab mouse, probably the most
widely used "genetic background“ for use as models of human
disease. They are the most widely used lab mouse strain, due to the
availability of congenic strains, easy breeding, and robustness. 30
31
32
saline
16
14
12
10
8
6
4
2
0
Time (h):
AMD3100
Total neutrophil count (x103/ml)
18
-
2
4
Total monocyte count (x103/ml)
Total leukocyte count (x103/ml)
baseline
8
SAJ5
9
8
7
6
5
4
3
2
1
0
Time (h):
-
4
8
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
Time (h):
-
2
2
4
8
Current research group:
Courtney Garcia (Chemistry/Medicine)
Paul Won (Chemistry/Pharmacy)
Justin Le (Chemistry/Pharmacy)
Past members:
Robert Ullom—University of Kansas (Medicine)
Joe Blas—Creighton (Medicine)
Danny Maples—OSU (Chemistry)
Randall Maples—OSU (Chemistry)
Dallas Matz—Arizona State University (Chemistry)
Mike McClain—OU (Chemistry)
Amy Cain—U. British Columbia (Chemistry)
Orry Birdsong—UT Galveston (Medicine)
Kimberly Roewe—OSU (Chemistry)
Kiet Ngyuen—SWOSU (Pharmacy)
Josh Priddle—OSU (Medicine)
Desiray Cannon (Chemistry)
Katherine Coats (Chemistry)
Natalie Simpson (Chemistry)
34
Kevin Wilson (Chemistry)
Acknowledgements
Funding
– OK-INBRE (NIH)
– Research Corporation
– SWOSU
Dr. Steve Archibald (Hull)
Dr. Abid Khan (Hull)
Prof. Erik De Clercq (Leuven)
Dr. Christophe Pannecouque(Leuven)
Dr. Dominique Schols (Leuven)
Prof. Tony Ng (KCL)
Dr. Jana Barlic (OMRF)
United States
Weatherford—synthesis/characterization (Hubin)
Oklahoma City—stem cell mobilization, atheroregression, obesity (Barlic)
United Kingdom
Hull—synthesis/characterization, CXCR4 binding, imaging, cancer metastases (Archibald)
London—cancer cell imaging (Ng), PET Imaging, pharmacology (Blower)
Belgium
Leuven—anti-HIV properties (DeClercq, Pannecouque), Ca-Signaling (Schols)
35
Collaborations
• Steve Archibald, University of Hull
– Central connection between all other collaborators
• Expertise
– Synthetic Organic and Inorganic Chemistry
– Bioinorganic and Medicinal Chemistry
– X-Ray Crystallography
• Contributions
– Synthesis and Characterization of New Compounds
– Antibody binding studies on CXCR4 expressing cell lines
– Cancer Cell Invasion Assays
– X-Ray Crystal Structures
36
Collaborations
• Tim Hubin, Southwestern Oklahoma State University
• Expertise
– Synthetic Organic and Inorganic Chemistry
– Bioinorganic and Medicinal Chemistry
• Contributions
– Synthesis and Characterization of New Compounds
37
Collaborations
• Dominique Schols, Christophe Pannecouque, University of Leuven
– Eric De Clercq (retired, but still active)
• Expertise
– Virology
– Chemokine Receptors
• Contributions
– HIV Infection Assays—Pannecouque
– Ca2+ Ion Signaling Assays—Schols
– CXCR4 and CCR5 binding studies—Schols
– Viral mutation studies—Pannecouque
38
Collaborations
• Tony Ng, King’s College London
– Gilbert Fruhwirth
• Expertise
– Cancer
– Fluorescence Imaging of CXCR4 in vitro and in vivo
– Immunology
• Contributions
– Initial mouse toxicity studies
– Imaging of CXCR4 expression
– Confirmation of antagonism using fluorescence imaging
39