JNJ-3738
Probe criteria
| Inhibitor/agonist potency: goal is < 100 nM (IC50, Kd) | Surpasses criterion:Biochemical assay using CDK7/cyclinH/MAT1 complex (500 μM ATP; Kapp ~30 μM): KIapp 31 nM; kinact = 0.0052/s; kinact/ KIapp 2603 M-1s-1 |
| Selectivity within target family: > 30-fold | Good CDK familiy selectivity: Closest family member is CDK9 (45 % inh.) vs CDK7 (95 % inh.) (activeXScreeen) > 100 times selectivityActivX screen (combined output THP-1 & H1975 cell lysates (~280 kinases)): Closest off-targets at 1 µM [% inhibition]: CAMKK2 (70), STK16 (65).DiscoverX KinaseScreen at 1 µM: CDK7 (> 99 % inh., pIC50 = 8.42), CDK9 (36 % inh., pIC50 = 6.03) |
| Selectivity outside target family | CEREP panel at 10 µM: Clean except for HRH1 (antagonist radioligand) 77% inh.Proteome-wide Selectivity-activity-based protein profiling (ABPP) (Jurkat): Closest off-target: PTGES2 (67 % inh.) |
| On target cell activity for cell-based targets: goal is < 1 µM IC50/EC50 | Surpasses criterion:Pharmacodynamics (PD): Detection of p-RNA pol II Ser 5 phosphorylation in A549 (WT CDK7) cells after 3 hrs incubation with probe: IC50 =58 nM ; CDK7 C312S mutant: IC50 > 10 µM (QuantiGene Plex Gene Expression Assay: downregulation at the messenger level of superenhancer driven genes. The level of phosphorylation of Serine 5 on RNA pol II Vs totalRNA pol II is determined.)Antiproliferation assay using OCI AML3 cells (4 day): IC50 = 4 nM, C312S mutant: IC50 = 3.98 µM |
| Control compound (100 times less potent than the probe) | JNJ-6240: IC50 ~ 200 nM (KMATP app ~30 µM; fluorescent end-point assay); PD: Detection of p-RNA pol II Ser 5 in A549 cells after 3 hrs incubation with probe: IC50 > 10 µM; C312S mutant: IC50 > 10 µM |