Discovery of bivalent small molecule degraders of cyclin-dependent kinase 7 (CDK7)

Abstract

Cyclin-dependent kinase 7, along with cyclin H and MAT1, forms the CDK-activating complex (CAK), which directs cell cycle progression via T-loop phosphorylation of cell cycle CDKs. Pharmacological inhibition of CDK7 leads to selective anti-cancer effects in cellular and in vivo models, motivating several ongoing clinical investigations of this target. Current CDK7 inhibitors are either reversible or covalent inhibitors of its catalytic activity. We hypothesized that small molecule targeted protein degradation (TPD) might result in differentiated pharmacology due to the loss of scaffolding functions. Here, we report the design and characterization of a potent CDK7 degrader that is comprised of an ATP-competitive CDK7 binder linked to a CRL2^VHL recruiter. JWZ-5-13 effectively degrades CDK7 in multiple cancer cells and leads to a potent inhibition of cell proliferation. Additionally, compound JWZ-5-13 displayed bioavailability in a pharmacokinetic study conducted in mice. Therefore, JWZ-5-13 is a useful chemical probe to investigate the pharmacological consequences of CDK7 degradation.

Keywords: CDK7; PROTAC; Protein degrader; Selective.

Figure 1.

Chemical structures of representative CDK7 inhibitors

Figure 2.

(A) Chemical structure of covalent CDK7 inhibitor YKL-5–124 and noncovalent inhibitor YKL-5–167 (B) Docking model of YKL-5–124 in the ATP-binding site of CDK7 (PDB : 6XD3).

Figure 3.

Immunoblot analysis of CDK7 in Jurkat cells treated with CRBN-based compounds for 16 h. Quantified data represents mean ± SEM from three independent biological replicates.

Figure 4.

Immunoblot analysis of CDK7 in Jurkat cells treated with VHL-based compounds for 16 h. Quantified data represents mean ± SEM from three independent biological replicates.

Figure 5

(A) Chemical structure of compound 17 (B) Immunoblot analysis of CDK7 in Jurkat cells treated with 0.1 μM of 17 at the indicated time points. (C) Immunoblot analysis of CDK7 in Jurkat cells treated with 17 at the indicated concentration for 6h. Quantified data represents mean ± SEM from three independent biological replicates. (D) Immunoblot analysis of CDK7 in different cells treated with 17 for 6 h.

Figure 6.

(A) Chemical structure of 17-Neg (B) Immunoblot analysis of CDK7 in Jurkat cells treated with 17 or 17-Neg for 6 h. (C) Immunoblot analysis of CDK7 in Jurkat cells pretreated for 2 h with MG132, MLN-4924, YKL-5–167 and VHL ligand, then treated with 17 for 6 h. (D) Western blotting analysis of CDK7 in Jurkat and VHL-knockout Jurkat cells treated with 17 for 6 h.

Figure 7.

(A) Western blotting analysis of CDKs in Jurkat cells treated with 17 for 6 hours. (B) Quantitative proteomics of OVCAR3 cells following treatment with 0.1 μM 17 for 6 hours (C) Western blotting analysis of CDK7, MAT1, Cyclin H treated with 17 for 6 h in OVCAR3 and Jurkat cells.

Figure 8.

Proliferation assay of 17, 17 −Neg, or YKL-5–167 in different cell lines.

Figure 9.

Plasma concentrations of 17 over 24 h, following a single 10 mg/kg IP or 3 mg/kg IV injection. The compound concentration shown at each time point is the mean ± SD from three test mice.

Scheme 1.

Synthesis of Compounds 1–9 ^a Reagents and conditions: (a) Et₃N, THF, 0 °C (b) Methyl 4-(chlorocarbonyl)benzoate, DIPEA, DCM, 0 °C (c) TFA, DCM, rt (d) (S)-2-isocyanato-N,N-dimethyl-2-phenylethan-1-amine, DIPEA, DCM, 0 °C (e) 2M NaOH, THF, 60 °C (f) HATU, DIPEA, DMF, rt

Scheme 2.

Synthesis of VHL-based Compounds 10 – 22.