Discovery of Selective and Orally Bioavailable Heterobifunctional Degraders of Cyclin-Dependent Kinase 2

Abstract

Cyclin-dependent kinase 2 (CDK2) plays an important role in cell cycle regulation and has emerged as a compelling target for the treatment of cancer, largely because of its potential to overcome the resistance associated with CDK4/6 inhibition. Efforts to develop CDK2 inhibitors have historically proven challenging due to undesirable safety profiles associated with inhibiting off-target CDK isoforms. Herein, we describe the structure-guided discovery of a series of orally bioavailable and selective degraders of CDK2. Degrader 37 demonstrated improved phenotypic selectivity compared to a clinical CDK2 inhibitor, with greater specificity for disease-relevant cyclin E1 (CCNE1)-amplified cancer cells vs nonamplified cohort. The antitumor activity of 37 in mice bearing CCNE1-amplified HCC1569 tumors correlated with sustained >90% degradation of CDK2 and sustained 90% inhibition of Rb phosphorylation.

1

Reported nonselective inhibitors of CDK2.

2

Rationale for CDK4 and 6 sparing activity of TMX-3010 in a model of TMX-3010 and cpd 5 bound to CDK2 (PDB code: 1JSV). As seen in the binding pose, the tolyl methyl group makes a favorable van der Waals interaction with Phe82 of CDK2, which is a smaller, more polar histidine in CDK4 and 6.

3

Scaffold hopping from reported unselective CDK2 inhibitors to a selective CDK2 inhibitor 1, and identification of the CDK2 degrader 2.

4

Rational design of CDK2 ligands with improved physicochemical properties.

5

(a) Crystal structure of compound 3 (green)/CDK2 (PDB code: 9NYQ); (b) overlay of 3 with 1 (pink) in the crystal structure of 3/CDK2, with wire mesh representing the surface of the binding site (PDB code: 9NYQ).

6

(a) Cryo-EM structure of CDK2/Cyclin E1 – compound 24 – CRBN/DDB1 at 3.9 Å resolution (PDB code: 9NYR). Compound 24 induced non-native CDK2-CRBN interactions as highlighted in insets (b, c, and d). (b) Zoomed-in view of compound 24 (sticks) and density (transparent surface). His-353 of CRBN engages the backbone of CDK2 Ile-10 and the piperazine linker of compound 24. (c) CRBN Lon domain residue Phe150, residing in a β-hairpin loop, sandwiches between CDK2 Tyr77 and Tyr19, and (d) CDK2 Arg200 interacts favorably with the backbone of CRBN Lys392 and Ile393.

7

Volcano plot showing deep tandem mass tag proteomics analysis in human PBMC cells treated with 37 for 8 h. No off-target protein degradation was observed.

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(a) PK/PD and (b) efficacy of 37 in an HCC1569 xenograft model.

1. Synthesis of Amine 44 Needed for the Preparation of 37

2. Synthesis of 37