1. The design of novel cyclin dependent kinase inhibitors
Simak Ali
Division of Cancer, Department of Surgery & Cancer, Imperial College London

2. 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

3. Breast Cancer Treatment Based on Molecular Sub-Type
80%
ER -ve
ER +ve
Basal-like
ErbB2 +ve
Normal-like
Luminal B/C
Luminal A
Herceptin
Hormone
Therapy
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

4. 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

5. Estrogen Receptor Phosphorylation
Ser118
ERa
Transcription DNA Hormone Binding
(AF-1) Binding (AF-2)

6. Serine 118 Phosphorylation and Endocrine Resistance
Immunodetection in breast tumours pre- and post-tamoxifen (relapse) treatment:
P-Ser118 levels increased post-tamoxifen treatment
(z = ; p = 0.02)
No significant increase in ER (z = ; p = 0.42) P
Ser118
ERa
Transcription DNA Hormone Binding
(AF-1) Binding (AF-2)

7. Ser118 is phosphorylated by TFIIH associated CDK7
Estrogen Concentration (M)
+CDK7
Chen et al 2000 Mol Cell

8. Ser118 is phosphorylated by TFIIH associated CDK7
Chen et al 2000 Mol Cell

9. CDK7 Function Cell Cycle TFIIH (Y1-S2-P3-T4-S5-P6-S7)52 PolII M G1 G2
Cyclin H
MAT1
Cdk7
Cell
Cycle M G1 S G2
Cyclin B
Cdk1
(cdc2) P
Cyclin E
Cdk2
Cyclin D
Cdk
4/6
Cyclin A
TFIIH
Cyclin T
Cyclin C
Cyclin H
MAT1
Cdk7
Cdk9
Cdk8 P
PolII
(Y1-S2-P3-T4-S5-P6-S7)52

10. Development of Selective CDK7 Inhibitors
DGsolv
(kcal/mol)
(1)
roscovitine A B C D E

11. 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

12. BS-181 is a highly selective CDK7 inhibitor
Kinase
Roscovitine
BS-181
µmol/L
IC50 (µmol/L) SD
IC50 (µmol/L)
CDK1
1.8
0.3
8.1
0.6
CDK2
0.1
0.02
0.88
0.08
CDK4
15.3
6.6
33.0
1.5
CDK5
0.24
3.0
0.5
CDK6 28
4.9
47.0
4.0
CDK7
0.51
0.021
0.002
CDK9
1.2
0.8
4.2
Protein Kinase
% Activity Remaining
Standard Deviation
MKK1 96 7
CHK1 80 4
ERK1
108 10
CHK2 36 2
ERK2 86
GSK3b
112 8
JNK1
100 6
CDK2-Cyclin A 9 1
JNK2 90
PLK1 13
JNK3
128
PLK1 (Okadaic Acid)
p38a MAPK 95
AURORA B
P38ß MAPK
115
AURORA C 98 5
p38g MAPK
AMPK
122
p38s MAPK
MARK3
104
ERK8 32
BRSK2
RSK1 67
MELK 63
RSK2 55
CK1 29
PDK1
CK2
PKBa 82 14
DYRK1A 17
PKBb 81
DYRK2 94
SGK1 42
DYRK3 87
S6K1
NEK2a 92
PKA
110
NEK6 85
ROCK 2 76
NEK7 97
PRK2
IKKb
PKCa
PIM1 88
PKC zeta
114
PIM2
102 3
PKD1 53
PIM3 78
MSK1 65
SRPK1 44
MNK1
105
MST2
MNK2 11
EFK2
119
MAPKAP-K2
HIPK2
MAPKAP-K3
HIPK3
PRAK
PAK4 75
CAMKKa 57
PAK5
CAMKKb
PAK6
CAMK1 48
Src
SmMLCK 35
Lck
PHK 49 12
CSK 89
Kinase
BS-181
µmol/L
IC50 (µmol/L)
CDK2/Cyclin A
0.75
CK1
7.4
DYRK1A
2.3
Ali et al 2009 Cancer Research

13. BS-181 is a highly selective CDK7 inhibitor
Table 2. In vitro growth inhibitory activity of BS-181
Tumour Type
Cell line
Roscovitine
BS-181
IC50 (µM)
Breast
MCF-7 13 20
MDA-MB-231 18 15
T47D 25
16.5
ZR-75-1
33.5
25.5
BT474
30.5
BT20
32.5 19
Colorectal
COLO-205 8
11.5
Lung
A549 17 37
NCI-460
9.5 21
Osteosarcoma
U2OS
10.5
14.5
SaSO2
13.9
Prostate
PC3
10.8 23
LNCaP
8.4 32
Liver
HepG2
12.3 24
50% inhibition: P-Ser2 P-Ser5
Roscovitine µmol/L >50 µmol/L
BS µmol/L 9.0 µmol/L
MCF-7
Control (N=11)
BS mg/kg/day (N=10)
BS mg/kg/day (N=13)
Tumour Volume *
***
* p<0.05
** p<0.01
*** p<0001
Ali et al 2009 Cancer Research

14. BS-181 promotes cell cycle block and p53-dependent apoptosis
MCF-7
HCT116 p53+/+
HCT116 p53-/-
p53
ß-actin
HCT116 p53+/+ *
HCT116 p53-/- * *
Ali et al 2009 Cancer Research

15. 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

16. BS-194, a potent CDK inhibitor
Kinase
Roscovitine
BS-194
µmol/L
IC50 SD
CDK1
1.8
0.3
0.033
0.01
CDK2
0.1
0.02
0.003
0.001
CDK4
15.3
6.6
20.0
1.3
CDK5
0.24
0.03
0.006
CDK6 28
4.9
35.5
CDK7
0.51
0.25
0.004
CDK9
1.2
0.8
0.09
0.04
BS-181
NCI 60 Cancer Cell Lines:
Mean GI50 = 2.81E-07 mol/L
BS-194
Heathcote et al 2010 J Med Chem

17. BS-194, an orally bioavailable CDK inhibitor
Heathcote et al 2010 J Med Chem

18. BS-194 crystallisation with Cdk2
Heathcote et al 2010 J Med Chem
Figure 2 Heathcote et al.

19. BS-181 and BS-194 as lead compounds for further development
CDK7 selective compound (IC50 18 nM (CDK7), IC nM (CDK2))
modest activity in cells (MCF-7):
GI50 (21 M ), TGI (32 M), LC50 (48 M)
low bioavailability (PO: 2%, IP: 37%)
and poor cell permeability (0.8 x 106 cm/sec)
CDK2/pan inhibitor (IC nM (CDK2), IC50 25 nM (CDK1), IC50 30 nM (CDK5))
good activity in cells (MCF-7):
GI50 (0.06 M ), TGI (0.1 M), LC50 (>100 M)
high bioavailability (PO: 88%, IP: 73%)
and high cell permeability (9.7 x 106 cm/sec) and metabolically stable (97% parent after 24h)
Merge properties

20. BS-181 and BS-194 as lead compounds for further development
(R)-Roscovitine
BS-181
BS-194
ICEC0574
ICEC0768
ICEC0829
ICEC0510R
ICEC0942
IC50 (nmol/L)
CDK7
510 21
250 27 76 20 31 40
CDK2
100
880 3
1,290
1,620
1,100
1,800
580
CDK1
2100
8,100 33
4,000
2,200
1,400
3,200
1,521
CDK5
160
3,000 30
7,900
4,300
8,340
8,200
9,000
CDK9
950
4,200 90 55
200
260
523
CDK4
13,500
33,000
20,000
19,410
15,100
1,000
21,000
42,000
CDK6
23,500
47,000
35,500
51,000
26,000 ND
32,100
GI50 (nmol/L)
MCF-7 GI50
13,000
300
4,100
2,800
1,500
5,600
960
HCT116 GI50
12,720
6,190
5,070
9,180
1,130
Oral Bioavailability 2%
88%
60%
40%
30%

21. 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

22. Acknowledgements Surgery & Cancer Chemistry Molecular Biosciences
Basti KROLL
Bodo SCHEIPER
Alekasandra SIWICKA
Robert PACE
Alexander BONDKE
Brian SLAFER
Matt FUCHTER
Tony BARRETT
Pascale HAZEL
Paul FREEMONT
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
Drug Discovery Centre
Cathy TRALAU-STEWART
Albert JAXA-CHAMIEC
CADD, Emory MD Anderson
Ashutosh JOGALEKAR
Dennis LIOTTA
Jim SNYDER
Tim MADDEN
Garth POWIS
IGBMC, Strasbourg
Mount Sinai
Pierre CHAMBON
Jean-Marc EGLY
Stephane LAROCHELLE
Robert FISHER