Bettayeb et al. CDK inhibitor CR8
Oncogene
SUPPLEMENTARY MATERIAL
CR8, a potent and selective, roscovitine-derived inhibitor of cyclin-dependent kinases
Karima BETTAYEB, Nassima OUMATA, Aude ECHALIER, Yoan FERANDIN, Jane A. ENDICOTT, Hervé GALONS and Laurent MEIJER
5 Tables + 1 Figure
Supplementary Table 1. PROQINASE Kinase Selectivity Panel (32 kinases). ProQinase kinase preparations and assays are described in Bach et al., 2005. (www.proqinase.com). IC50 values (expressed in µM) were determined for each kinase. IC50 values <10 µM are underlined in yellow.
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Bettayeb et al. CDK inhibitor CR8
Kinase / IC50 value (µM)ABL1 / > 100
AKT1 / > 100
Aurora-A / 93.0
Aurora-B / > 100
Aurora-C / > 100
BRK / > 100
CDK1 /cyclin B / 3.8
CDK2 /cyclin E / 0.88
CDK3 /cyclin E / 1.9
CDK4 /cyclin D1 / 12.0
CDK5 /p25 / 2.1
CDK6 /cyclin D1 / 13.0
CDK7 /cyclin H/ Mat1 / 3.0
CDK9 /cyclin T / 1.4
CHK1 / > 100
CSK / 79.0
FAK / 19.0
FLT3 / > 100
IKK-β / > 100
IKK-ε / > 100
Ins-R / > 100
IRAK4 / > 100
JNK 3 / > 100
LCK / > 100
MST4 / > 100
NEK6 / > 100
PLK1 / > 100
S6K / > 100
SGK1 / > 100
SNK / > 100
TGF-R1 / > 100
VEGF-R2 / > 100
Supplementary Table 2. CEREP Kinase Selectivity Panel (44 kinases). Preparation and assay of kinases are described in Bach et al. (2005). (www.cerep.com). Enzymes were assayed in the presence of 10 µM CR8, and kinase activities expressed as % of control kinase activity, i.e. in the absence of inhibitor. >80 % inhibition at 10 µM is underlined in yellow.
at 10 µM
Abl kinase (h) / 95.2
Akt1/PKBa (h) / 104.6
AMPKa / 97.0
BMX kinase (h) (Etk) / 104.6
Brk (h) / 108.3
CaMK2a (h) / 93.7
CaMK4 (h) / 88.5
CDK1/cyclin B (h) / 19.5
CDK2/cyclin E (h) / 5.4
CHK1 (h) / 108.9
CHK2 (h) / 69.0
c-Met kinase (h) / 99.9
CSK (h) / 93.2
EphB4 kinase (h) / 111.6
ERK1 (h) / 73.9
ERK2 (h) (P42mapk) / 63.2
FGFR2 kinase (h) / 72.0
FGFR4 kinase (h) / 99.4
FLT-1 kinase (h) (VEGFR1) / 104.0
FLT-3 kinase (h) / 86.1
Fyn kinase (h) / 104.2
IGF1R kinase (h) / 101.2
IRK (h) (InsR) / 94.4
JNK 2 (h) / 69.2
KDR kinase (h) (VEGFR2) / 111.0
Lck kinase (h) / 95.6
Lyn kinase (h) / 87.5
MAPKAPK2 (h) / 101.3
MEK1/MAP2K1 (h) / 93.7
p38a kinase (h) / 91.5
p38 δ kinase (h) / 90.8
p38 γ kinase (h) / 83.5
PDGFR β kinase (h) / 106.0
PDK1 (h) / 95.7
PKA (h) / 85.8
PKCa (h) / 101.3
PKC β1 (h) / 102.9
PKC γ(h) / 96.6
Ret kinase (h) / 98.3
ROCK2 (h) / 100.9
RSK2 (h) / 89.4
Src kinase (h) / 114.9
Syk (h) / 102.8
TRKA (h) / 86.5
Supplementary Table 3. Prof. Philip COHEN’s laboratory Kinase Selectivity Panel (76 kinases). Enzymes were prepared and assayed as described in Bain et al. (2007). Enzymes were assayed in the presence of 10 µM CR8, and kinase activities expressed as % of control kinase activity, i.e. in the absence of inhibitor. >80 % inhibition at 10 µM is underlined in yellow.
at 10 µM / Standard error
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Bettayeb et al. CDK inhibitor CR8
AMPK / 75 / 1BRSK2 / 80 / 8
BTK / 88 / 5
CAMKKb / 47 / 15
CAMK1 / 50 / 8
CDK2-Cyclin A / 3 / 1
CHK1 / 112 / 6
CHK2 / 27 / 1
CK1 / 33 / 3
CK2 / 87 / 1
CSK / 110 / 15
DYRK1A / 4 / 0
DYRK2 / 72 / 7
DYRK3 / 68 / 2
EFK2 / 107 / 1
EPH A2 / 82 / 5
EPH-B3 / 69 / 1
ERK1 / 40 / 3
ERK2 / 29 / 3
ERK8 / 20 / 4
FGF-R1 / 75 / 5
GSK3β / 53 / 10
HIPK2 / 62 / 3
IGF1-R / 49 / 15
IKKβ / 80 / 0
IKKε / 107 / 15
IR-HIS / 73 / 1
IRR / 28 / 12
JNK1 / 87 / 5
JNK2 / 97 / 1
Lck / 68 / 1
MAPKAP-K2 / 96 / 3
MARK3 / 91 / 0
MELK / 49 / 7
MKK1 / 66 / 8
MNK1 / 77 / 5
MNK2 / 74 / 3
MSK1 / 90 / 12
MST2 / 65 / 2
MST4 / 87 / 6
NEK2a / 85 / 6
NEK6 / 90 / 15
p38a MAPK / 102 / 5
P38 β MAPK / 102 / 5
p38 γ MAPK / 122 / 15
p38s MAPK / 90 / 8
PAK4 / 58 / 15
PAK5 / 58 / 15
PAK6 / 11 / 0
PDK1 / 81 / 8
PHK / 39 / 1
PIM1 / 103 / 5
PIM2 / 79 / 15
PIM3 / 96 / 10
PKA / 96 / 15
PKB a / 94 / 6
PKB β / 97 / 1
PKC a / 90 / 15
PKC zeta / 76 / 4
PKD1 / 47 / 1
PLK1 / 47 / 15
PLK1 (okadaic Acid) / 67 / 8
PRAK / 89 / 6
PRK2 / 102 / 8
ROCK 2 / 83 / 4
RSK1 / 86 / 9
RSK2 / 40 / 1
S6K1 / 66 / 5
SGK1 / 80 / 1
SmMLCK / 57 / 0
Src / 93 / 6
SRPK1 / 92 / 13
VEG-FR / 90 / 12
SYK / 102 / 7
TBK1 / 85 / 1
YES1 / 103 / 1
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Bettayeb et al. CDK inhibitor CR8
Supplementary Table 4. CDK2/cyclin A - (R)-Roscovitine and CDK2/cyclin A - (R)-CR8 co-crystal structures. Statistics of the dataset used and of the refined structures.
Cell dimensions (Å) / 74.3, 135.2, 168.9 / 74.4, 133.4, 147.4
Maximal Resolution (Å) / 2.70 / 1.80
Observations / 154,060 / 781,599
Unique reflections, completeness (%) / 44,226
93.6 / 122,692
90.2
Rmerge1 / 0.09 / 0.07
mean I / s(I) / 11.4 / 16.4
Highest resolution bin (Å): / 2.85-2.70 / 1.90-1.80
Completeness (%) / 94.2 / 84.1
mean I / mean s(I)
Rmerge / 2.8
0.422 / 3.3
0.285
Resolution range (Å) / 20.00-2.70 / 20.00-1.80
Rconv2 / 21.0 / 19.5
Rfree3 / 27.0 / 22.9
Mean protein temperature factors (Å)2 / 56.8 / 21.4
Mean ligand temperature factors (Å)2 / 52.8 / 18.4
Coordinates, pdb file # / 3DDP / 3DDQ
1 where Ih,j is the intensity of the jth observation of unique reflection h.
2 where Foh and Fch are the observed and calculated structure factor amplitudes for reflection h.
3 Rfree is equivalent to Rconv, but is calculated using a 5% disjoint set of reflections excluded from the maximum likelihood refinement stages.
Supplementary Table 5. Effects of (R)-roscovitine and (S)-CR8 on the survival of various cell lines. Roscovitine and CR8 were tested at various concentrations for their effects on 19 different cell lines: Cell survival was estimated 72 h after the addition of each purine using the MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) reduction assay as described in Echalier et al. (2008). IC50 values (expressed in mM) were calculated from the dose-response curves.
Cell line / Mean / SD / Mean / SD
BxPC-3 (pancreas adenocarcinoma) / 20.19 / 1.96 / 1.45 / 1.31
Calu-6 (anaplastic lung carcinoma) / 13.09 / 2.47 / 0.97 / 0.77
CCD-112CoN (normal colon) / 27.06 / - / 7.93 / -
CCRF-CEM (acute lymphoblastic leukaemia) / 31.53 / 23.28 / 10.38 / 13.23
FaDu (squamous cell carcinoma) / 11.52 / 3.38 / 0.32 / 0.27
HT1080 (fibrosarcoma) / 43.68 / 5.49 / 7.28 / 4.28
HT-29 (colorectal adenocarcinoma) / 13.24 / 1.83 / 0.49 / 0.65
HUV-EC-C (umbilical vein endothelium) / 21.44 / 0.62 / 2.62 / 3.36
IGROV-1 (ovarian carcinoma) / 24.96 / 0.31 / 3.16 / 1.06
KG-1 (acute lymphoblastic leukaemia / 26.25 / 17.55 / 2.32 / 1.01
LoVo (metastasis of colorectal adenocarcinoma) / 6.82 / 0.09 / 0.25 / 0.13
MCF-7 (metastasis of invasive ductal mammary carcinoma) / 22.50 / 3.34 / 4.79 / 2.11
MDA-MB-231 7 (metastasis of invasive ductal mammary carcinoma) / 12.21 / 2.44 / 1.67 / 0.94
NCI-H460 Metastasis of lung carcinoma) / 9.08 / 2.48 / 2.94 / 1.43
PANC-1 (pancreas carcinoma) / 33.92 / 3.63 / 2.30 / 1.36
PC-3 (bone marrow metastasis of prostate adenocarcinoma) / 18.38 / 5.32 / 0.58 / 0.22
Ramos (Burkitt’s lymphoma) / 14.71 / 8.48 / 0.57 / 0.52
RPMI 8226 (myeloma B cells) / 13.98 / 5.24 / 0.42 / 0.27
SW872 (liposarcoma) / 19.49 / 3.80 / 0.40 / 0.25
Average 19 cell lines / 20.21 / 5.09 / 2.68 / 1.84
Figure S1. Comparative diagram of the binding modes of roscovitine (left) and CR8 (right) to the active form of CDK2 in complex with cyclin A.
Supplementary References
Bach, S., Knockaert, M., Lozach, O., Reinhardt, J., Baratte, B., Schmitt, S., Coburn, S.P., Tang, L., Jiang. T., Liang, D.C., Galons, H., Dierick, J.F., Totzke, F., Schächtele, C., Lerman, A.S., Carnero, A., Wan, Y., Gray, N. and Meijer, L. (2005). Roscovitine targets: protein kinases and pyridoxal kinase. J Biol Chem 280: 31208-31219.
Bain J, Plater L, Elliott M, Shpiro N, Hastie CJ, McLauchlan H, Klevernic I, Arthur JS, Alessi DR, Cohen P. (2007). The selectivity of protein kinase inhibitors: a further update. Biochem J. 408: 297-315.
Echalier, A., Bettayeb, K., Ferandin, Y., Lozach, O., Clement, M., Valette, A., Liger, F., Marquet, B., Morris, J.C., Endicott, J.A., Joseph, B. and Meijer, L. (2008). Meriolins (3-(pyrimidin-4-yl)-7-azaindoles): synthesis, kinase inhibitory activity, cellular effects and structure of a CDK2/cyclin A/meriolin complex. J Med Chem 51: 737-751.
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