The design of novel cyclin dependent kinase inhibitors
Simak Ali
Division of Cancer, Department of Surgery & Cancer, Imperial College London
Breast Cancer Statistics

Each year in the UK > 44,000 women are diagnosed with
breast cancer i.e. > 100 a day

In the last 10 years, breast cancer rates in the UK have
increased by 12%

8 in 10 breast cancers are diagnosed in women aged 50
and over

1 in 8 life time risk of developing breast cancer

Commonest cancer in UK & Europe (even though it
affects ~ only 1 gender) in 2006 outranked lung caner
(which affects both sexes) for the 1st time
Breast Cancer Treatment Based on Molecular
Sub-Type
BREAST CANCER
80%
ER -ve
Basal-like
ErbB2 +ve
Herceptin
Metaplastic / Medullary / Mucinous / Others
EGFR overexpression
c-kit
aß-crystallin BRCA1 deficiency
Gefitinib, erlotinib Imatinib/Gleevec MEK inhibitors DNA damage
Lapatinib
PI3K inhibitors PARP inhibitors
CI-1033
Possible novel treatment options for
Basal-like Breast cancer
ER +ve
Normal-like
Luminal B/C
Luminal A
Hormone
Therapy
Resistance to Hormone Therapy in Breast
Cancer

80% breast tumours are ER +ve  endocrine therapy
(SERMs such as tamoxifen, faslodex, raloxifene;
aromatase inhibitors)

Highly effective, but:

Intrinsic resistance (~65% cancers sensitive to Tamoxifen)

Acquired resistance (sequencing with another SERM or AI)

Determine mechanisms of endocrine resistance

Identify new markers of increased risk or therapy failure

New Therapeutic Strategies for the treatment of
endocrine resistant breast cancer
Estrogen Receptor Phosphorylation
P
Ser118
ER
Transcription DNA
(AF-1)
Binding
Hormone Binding
(AF-2)
Serine 118 Phosphorylation and Endocrine
Resistance
Immunodetection in breast tumours pre- and post-tamoxifen
(relapse) treatment:
P-Ser118 levels increased post-tamoxifen treatment
(z = -2.357; p = 0.02)
No significant increase in ER (z = -0.815; p = 0.42)
P
Ser118
ER
Transcription DNA
(AF-1)
Binding
Hormone Binding
(AF-2)
Ser118 is phosphorylated by TFIIH associated
CDK7
+CDK7
350
350
Reporter Gene Activity (%)
Reporter Gene Activity (%)
400
300
250
200
150
100
50
0
300
250
200
150
100
50
0
No
Ligand
+CDK7
Chen et al 2000 Mol Cell
-14
-12
-10
-8
-6
Estrogen Concentration (M)
Ser118 is phosphorylated by TFIIH associated
CDK7
Chen et al 2000 Mol Cell
CDK7 Function
Cyclin B
P
Cdk1
Cdk7
(cdc2)
Cyclin D
P
Cyclin A
P
Cell
Cycle
Cdk1
Cdk
4/6
Cyclin E
(cdc2)
P
Cyclin A
P
TFIIH
Cdk2
Cdk8
Cdk9
Cdk2
P
P P
P
P
P P
P
Cdk7
(Y1-S2-P3-T4-S5-P6-S7)52
PolII
Development of Selective CDK7 Inhibitors
A
roscovitine
B
DGsolv
-19.5
(kcal/mol)
-14.5 (1)
DGsolv
(kcal/mol) -22.4
-16.7
C
-21.4
-17.1
D
E
Development of Selective CDK7 Inhibitors

184 Compounds designed.

68 compounds synthesised and screened for inhibition of CDK7,
CDK2, CDK5, CDK9.

Primary screen: compounds at 100 nM

IC50 determined (to 1 µM) for all compounds
demonstrating >20% inhibition of CDK7 at 100 nM.

58 compounds inhibited CDK7 with IC50 <1000 nM

20 of these inhibited CDK7 with IC50 <100 nM. For these, IC50
determined for CDK2, CDK5, CDK9, if <1000 nM.

BS-194

BS-181
BS-181 is a highly selective CDK7
inhibitor
Kinase
µmol/L
CDK1
CDK2
CDK4
CDK5
CDK6
CDK7
CDK9
Roscovitine
IC50
(µmol/L) SD
1.8
0.3
0.1
0.02
15.3
6.6
0.24
0.1
28
4.9
0.51
0.1
1.2
0.8
BS-181
IC50
(µmol/L)
8.1
0.88
33.0
3.0
47.0
0.021
4.2
Kinase
µmol/L
CDK2/Cyclin A
CK1
DYRK1A
SD
0.6
0.08
1.5
0.5
4.0
0.002
0.5
BS-181
IC50 (µmol/L)
0.75
7.4
2.3
Ali et al 2009 Cancer Research
Protein Kinase
MKK1
ERK1
ERK2
JNK1
JNK2
JNK3
p38 MAPK
P38ß MAPK
p38g MAPK
p38s MAPK
ERK8
RSK1
RSK2
PDK1
PKBa
PKBb
SGK1
S6K1
PKA
ROCK 2
PRK2
PKCa
PKC zeta
PKD1
MSK1
MNK1
MNK2
MAPKAP-K2
MAPKAP-K3
PRAK
CAMKKa
CAMKKb
CAMK1
SmMLCK
PHK
% Activity
Remaining
96
108
86
100
90
128
95
115
108
96
32
67
55
80
82
81
42
95
110
76
90
98
114
53
65
105
105
88
119
115
57
67
48
35
49
Standard
Deviation
7
10
10
6
8
9
1
2
1
2
1
9
2
8
14
8
10
10
13
0
9
2
7
5
10
7
11
7
9
2
5
0
8
4
12
% Activity
Protein Kinase
Remaining
CHK1
80
CHK2
36
GSK3b
112
CDK2-Cyclin A
9
PLK1
100
PLK1 (Okadaic Acid) 108
AURORA B
95
AURORA C
98
AMPK
122
MARK3
104
BRSK2
98
MELK
63
CK1
29
CK2
108
DYRK1A
17
DYRK2
94
DYRK3
87
NEK2a
92
NEK6
85
NEK7
97
IKKb
96
PIM1
88
PIM2
102
PIM3
78
SRPK1
44
MST2
112
EFK2
119
HIPK2
88
HIPK3
90
PAK4
75
PAK5
80
PAK6
95
Src
88
Lck
95
CSK
89
Standard
Deviation
4
2
8
1
13
13
7
5
8
0
6
0
6
8
1
7
1
9
2
9
1
6
3
10
2
8
13
1
8
8
8
9
8
9
9
BS-181 is a highly selective CDK7
inhibitor
Table 2. In vitro growth inhibitory activity of BS-181
Breast
Cell line
MCF-7
MDA-MB-231
T47D
ZR-75-1
BT474
BT20
Colorectal
COLO-205
Lung
A549
NCI-460
Osteosarcoma U2OS
SaSO2
Prostate
PC3
LNCaP
Liver
HepG2
Roscovitine BS-181
IC50 (µM)
IC50 (µM)
13
18
25
33.5
30.5
32.5
8
17
9.5
10.5
13.9
10.8
8.4
12.3
20
15
16.5
25.5
30.5
19
11.5
37
21
14.5
20
23
32
24
50% inhibition:
Roscovitine
BS-181
Relative Tumour Volume
Tumour Type
MCF-7
4.5
4
3.5
3
2.5
2
1.5
1
0.5
0
* p<0.05
** p<0.01
*** p<0001
Control (N=11)
*
*
*** ***
*
***
***
***
***
Tumour Volume
0
2
4
6
8
Days
Ali et al 2009 Cancer Research
P-Ser2
P-Ser5
7.5 µmol/L >50 µmol/L
14.5 µmol/L 9.0 µmol/L
10
12
14
BS-181 10mg/kg/day (N=10)
BS-181 20mg/kg/day (N=13)
80
*
60
40
*
20
0
DMSO
1µM
10µM 25µM
BS-181 Concentration
50µM
Ali et al 2009 Cancer Research
MCF-7
100
80
60
40
*
20
*
0
DMSO
1µM
10µM 25µM
BS-181 Concentration
50µM
p53
ß-actin
HCT116 p53+/+
HCT116 p53-/-
100
Percentage of apoptosis
(n=3, +/- SEM)
Percentage of apoptosis
(n=3, +/-SEM)
BS-181 promotes cell cycle block
and p53-dependent apoptosis
Summary




Initial work identified Cdk7 as a protein kinase that
regulates ER activity in breast cancer
CADD using reported crystal structures and
inhibitors allowed the identification of potential
scaffolds for drug design towards selective Cdk7
inhibitors
BS-181 identified as a highly selective Cdk7 inhibitor
with anti-tumour activity in vivo
However, BS-181 has poor ADMET/PK properties,
which make its development as a cancer drug
difficult
BS-194, a potent CDK inhibitor
Kinase
µmol/L
CDK1
CDK2
CDK4
CDK5
CDK6
CDK7
CDK9
BS-181
Roscovitine
IC50
SD
1.8
0.3
0.1
0.02
15.3
6.6
0.24
0.1
28
4.9
0.51
0.1
1.2
0.8
BS-194
IC50
0.033
0.003
20.0
0.03
35.5
0.25
0.09
SD
0.01
0.001
1.3
0.006
1.3
0.004
0.04
100
NCI 60 Cancer Cell Lines:
Mean GI50 = 2.81E-07 mol/L
80
BS-194
Percentage Growth
60
40
20
0
-20
-40
-60
-80
-100
-9
-8
-7
-6
-5
Log10 of Sample Concentration (Molar)
Heathcote et al 2010 J Med Chem
-4
BS-194, an orally bioavailable CDK inhibitor
Relative tumor volume
6
HCT116 Xenograft
5
Vehicle
BS194- 50mg/kg
BS194- 25mg/kg
4
3
** p<0.001
* p<0.01
2
*
**
**
1
0
1
4
*
**
**
7
11
**
14
Days
BS-194
Heathcote et al 2010 J Med Chem
BS-194 crystallisation with Cdk2
Heathcote et al 2010 J Med Chem
BS-181 and BS-194 as lead compounds for
further development
BS-181
BS-194
• CDK7 selective compound (IC50 18 nM
• CDK2/pan inhibitor (IC50 2.4 nM (CDK2), IC50 25
(CDK7), IC50 880 nM (CDK2))
nM (CDK1), IC50 30 nM (CDK5))
• modest activity in cells (MCF-7):
• good activity in cells (MCF-7):
GI50 (21 M ), TGI (32 M), LC50 (48 M)
GI50 (0.06 M ), TGI (0.1 M), LC50 (>100 M)
• low bioavailability (PO: 2%, IP: 37%)
• high bioavailability (PO: 88%, IP: 73%)
and poor cell permeability (0.8 x 106 cm/sec)
and high cell permeability (9.7 x 106 cm/sec) and
metabolically stable (97% parent after 24h)
Merge properties
BS-181 and BS-194 as lead compounds for
further development
IC50 (nmol/L)
CDK7
CDK2
CDK1
CDK5
CDK9
CDK4
CDK6
GI50 (nmol/L)
MCF-7 GI50
HCT116 GI50
Oral Bioavailability
(R)-Roscovitine
BS-181
BS-194
ICEC0574
ICEC0768
ICEC0829
ICEC0510R
ICEC0942
510
100
2100
160
950
13,500
23,500
21
880
8,100
3,000
4,200
33,000
47,000
250
3
33
30
90
20,000
35,500
27
1,290
4,000
7,900
55
19,410
51,000
76
1,620
2,200
4,300
200
15,100
26,000
20
1,100
1,400
8,340
260
1,000
ND
31
1,800
3,200
8,200
523
21,000
20,000
40
580
1,521
9,000
1,100
42,000
32,100
13,000
20,000
300
4,100
12,720
2,800
6,190
1,500
5,070
5,600
9,180
960
1,130
2%
88%
40%
30%
60%
Summary




Initial work identified Cdk7 as a protein kinase that
regulates ER activity in breast cancer
CADD using reported crystal structures and inhibitors
allowed the identification of potential scaffolds for drug
design towards selective Cdk7 inhibitors
Lead compounds identified through in vitro assays and
confirmed using biomarker and in vivo evaluation
Reiterative design using CADD and chemistry at Imperial
has now been used to identify further compounds with
improved drug-like properties whilst maintaining target
selectivity
Acknowledgements
Surgery & Cancer
ESTROGEN SIGNALLING
Dongsheng CHEN
Manikandan PERIYASAMY
Ross THOMAS
Laki BULUWELA
CDK INHIBITORS
Sean DELANEY
Dean HEATHCOTE
Hetal PATEL
Zahida ZAHOOR
ADMET/PK
Richard STARKEY
Maciej KALISZCZAK
Eric ABOAGYE
Charles COOMBES
Chemistry
Basti KROLL
Bodo SCHEIPER
Alekasandra
SIWICKA
Robert PACE
Alexander BONDKE
Brian SLAFER
Matt FUCHTER
Tony BARRETT
CADD, Emory
Ashutosh JOGALEKAR
Dennis LIOTTA
Jim SNYDER
IGBMC, Strasbourg
Pierre CHAMBON
Jean-Marc EGLY
Molecular Biosciences
Pascale HAZEL
Paul FREEMONT
Drug Discovery Centre
Cathy TRALAU-STEWART
Albert JAXA-CHAMIEC
MD Anderson
Tim MADDEN
Garth POWIS
Mount Sinai
Stephane LAROCHELLE
Robert FISHER