The design of novel cyclin dependent kinase inhibitors Simak Ali Division of Cancer, Department of...
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Transcript of The design of novel cyclin dependent kinase inhibitors Simak Ali Division of Cancer, Department of...
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)
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-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