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

ErbB2 +veBasal-like Luminal B/C Luminal A

ER -ve ER +ve

Normal-like

BREAST CANCER

Herceptin

80%

HormoneTherapy

Possible novel treatment options forBasal-like Breast cancer

Metaplastic / Medullary / Mucinous / Others

EGFR overexpression c-kit aß-crystallin BRCA1 deficiency

Gefitinib, erlotinib Imatinib/Gleevec MEK inhibitors DNA damageLapatinib PI3K inhibitors PARP inhibitorsCI-1033

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

Transcription DNA Hormone Binding (AF-1) Binding (AF-2)

ERa

Ser118

Serine 118 Phosphorylation and Endocrine Resistance

P

Transcription DNA Hormone Binding (AF-1) Binding (AF-2)

ERa

Ser118

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)

Ser118 is phosphorylated by TFIIH associated CDK7

Chen et al 2000 Mol Cell

No Liga

nd

Estr

ogen

Tam

oxife

n

Faslod

ex

No Liga

nd

Estr

ogen

Tam

oxife

n

Faslod

ex0

50

100

150

200

250

300

350

400

Report

er

Gene A

ctiv

ity (

%)

+CDK7

No Ligand

-14 -12 -10 -8 -60

50

100

150

200

250

300

350

Report

er

Gene A

ctiv

ity

(%)

+CDK7

Estrogen Concentration (M)

Ser118 is phosphorylated by TFIIH associated CDK7

Chen et al 2000 Mol Cell

CDK7 Function

TFIIHCyclin

HMAT1

Cdk7

P

PPPP P P

P

PolII

(Y1-S2-P3-T4-S5-P6-S7)52

Cyclin C

Cdk8

Cyclin T

Cdk9

CellCycle

M

G1

SG2

Cyclin B

Cdk1(cdc2)

P

Cyclin E

Cdk2P

Cyclin D

Cdk4/6

P

Cyclin A

Cdk2P

Cyclin A

Cdk1(cdc2)

P

Cyclin

HMAT1

Cdk7

Development of Selective CDK7 Inhibitors

DGsolv

(kcal/mol)-19.5 -14.5 (1) -21.4

roscovitine

-22.4 -16.7 -17.1DGsolv

(kcal/mol)

A B

C 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

Ali et al 2009 Cancer Research

Kinase Roscovitine BS-181

µmol/L

IC50 (µmol/L) SD

IC50 (µmol/L) SD

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 0.1 3.0 0.5

CDK6 28 4.9   47.0 4.0

CDK7 0.51 0.1 0.021 0.002CDK9 1.2 0.8   4.2 0.5

Protein Kinase% Activity Remaining

Standard Deviation Protein Kinase

% Activity Remaining

Standard Deviation

MKK1 96 7   CHK1 80 4ERK1 108 10 CHK2 36 2ERK2 86 10   GSK3b 112 8JNK1 100 6 CDK2-Cyclin A 9 1JNK2 90 8   PLK1 100 13JNK3 128 9 PLK1 (Okadaic Acid) 108 13p38a MAPK 95 1   AURORA B 95 7P38ß MAPK 115 2 AURORA C 98 5p38g MAPK 108 1   AMPK 122 8p38s MAPK 96 2 MARK3 104 0ERK8 32 1   BRSK2 98 6RSK1 67 9 MELK 63 0RSK2 55 2   CK1 29 6PDK1 80 8 CK2 108 8PKBa 82 14   DYRK1A 17 1PKBb 81 8 DYRK2 94 7SGK1 42 10   DYRK3 87 1S6K1 95 10 NEK2a 92 9PKA 110 13   NEK6 85 2ROCK 2 76 0 NEK7 97 9PRK2 90 9   IKKb 96 1PKCa 98 2 PIM1 88 6PKC zeta 114 7   PIM2 102 3PKD1 53 5 PIM3 78 10MSK1 65 10   SRPK1 44 2MNK1 105 7 MST2 112 8MNK2 105 11   EFK2 119 13MAPKAP-K2 88 7 HIPK2 88 1MAPKAP-K3 119 9   HIPK3 90 8PRAK 115 2 PAK4 75 8CAMKKa 57 5   PAK5 80 8CAMKKb 67 0 PAK6 95 9CAMK1 48 8   Src 88 8SmMLCK 35 4 Lck 95 9PHK 49 12   CSK 89 9

Kinase BS-181

µmol/L IC50 (µmol/L)

CDK2/Cyclin A 0.75CK1 7.4DYRK1A 2.3

BS-181 is a highly selective CDK7 inhibitor

Ali et al 2009 Cancer Research

Table 2. In vitro growth inhibitory activity of BS-181

Tumour Type Cell line Roscovitine BS-181

    IC50 (µM) IC50 (µM)

Breast MCF-7 13 20MDA-MB-231 18 15T47D 25 16.5ZR-75-1 33.5 25.5BT474 30.5 30.5BT20 32.5 19

Colorectal COLO-205 8 11.5Lung A549 17 37

NCI-460 9.5 21Osteosarcoma U2OS 10.5 14.5

SaSO2 13.9 20Prostate PC3 10.8 23

LNCaP 8.4 32Liver HepG2 12.3 24

Control (N=11)

BS-181 10mg/kg/day (N=10)

BS-181 20mg/kg/day (N=13)

Tumour Volume

**

*

****** *** ******

***

* p<0.05** p<0.01*** p<0001

MCF-7

50% inhibition: P-Ser2 P-Ser5Roscovitine 7.5 µmol/L >50 µmol/LBS-181 14.5 µmol/L 9.0 µmol/L

BS-181 promotes cell cycle block and p53-dependent apoptosis

Ali et al 2009 Cancer Research

DMSO 1µM 10µM 25µM 50µM0

20

40

60

80

100

BS-181 Concentration

Per

cen

tag

e o

f ap

op

tosi

s (n

=3,

+/-

SE

M)

*

*

HCT116 p53+/+

DMSO 1µM 10µM 25µM 50µM0

20

40

60

80

100

BS-181 Concentration

Per

cen

tag

e o

f ap

op

tosi

s (n

=3,

+/-

SE

M)

**

HCT116 p53-/-

MC

F-7

HC

T11

6 p5

3+/+

H

CT

116

p53-

/-

p53

ß-actin

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

Heathcote et al 2010 J Med Chem

Kinase Roscovitine BS-194

µmol/LIC50 SD 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 0.1 0.03 0.006

CDK6 28 4.9   35.5 1.3

CDK7 0.51 0.1 0.25 0.004CDK9 1.2 0.8   0.09 0.04

BS-194

BS-181

-9 -8 -7 -6 -5 -4-100

-80

-60

-40

-20

0

20

40

60

80

100

Log10 of Sample Concentration (Mo-lar)

Perc

en

tag

e G

row

th

NCI 60 Cancer Cell Lines:Mean GI50 = 2.81E-07 mol/L

BS-194, an orally bioavailable CDK inhibitor

Heathcote et al 2010 J Med Chem

1 4 7 11 140

1

2

3

4

5

6 HCT116 Xenograft

VehicleBS194- 50mg/kg

Days

Rela

tive t

um

or

volu

me

****** **

***

*

** p<0.001* p<0.01

BS-194

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

• CDK7 selective compound (IC50 18 nM

(CDK7), IC50 880 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)

BS-194

• CDK2/pan inhibitor (IC50 2.4 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

(R)-Roscovitine BS-181 BS-194 ICEC0574 ICEC0768 ICEC0829 ICEC0510R ICEC0942IC50 (nmol/L)CDK7 510 21 250 27 76 20 31 40CDK2 100 880 3 1,290 1,620 1,100 1,800 580CDK1 2100 8,100 33 4,000 2,200 1,400 3,200 1,521CDK5 160 3,000 30 7,900 4,300 8,340 8,200 9,000CDK9 950 4,200 90 55 200 260 523 1,100CDK4 13,500 33,000 20,000 19,410 15,100 1,000 21,000 42,000CDK6 23,500 47,000 35,500 51,000 26,000 ND 20,000 32,100

GI50 (nmol/L)MCF-7 GI50 13,000 20,000 300 4,100 2,800 1,500 5,600 960HCT116 GI50 12,720 6,190 5,070 9,180 1,130

Oral Bioavailability 2% 88% 60% 40% 30%

BS-181 and BS-194 as lead compounds for further development

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

ESTROGEN SIGNALLING

Dongsheng CHENManikandan PERIYASAMYRoss THOMASLaki BULUWELA

CDK INHIBITORSSean DELANEYDean HEATHCOTEHetal PATELZahida ZAHOOR

ADMET/PKRichard STARKEYMaciej KALISZCZAK Eric ABOAGYE

Charles COOMBES

Surgery & Cancer ChemistryMolecular Biosciences Basti KROLL

Bodo SCHEIPERAlekasandra

SIWICKARobert PACEAlexander

BONDKEBrian SLAFERMatt FUCHTER Tony BARRETT

Pascale HAZEL

Paul FREEMONT

CADD, Emory MD AndersonAshutosh JOGALEKAR

Dennis LIOTTAJim SNYDER

IGBMC, Strasbourg

Tim MADDENGarth POWIS

Pierre CHAMBONJean-Marc EGLY

Mount SinaiStephane LAROCHELLERobert FISHER

Drug Discovery CentreCathy TRALAU-

STEWARTAlbert JAXA-CHAMIEC