Discovery of a molecular glue promoting CDK12-DDB1 interaction to trigger Cyclin K degradation [WES]

Molecular glues are small molecules that exert their biologic or therapeutic activities by inducing gain-of-function interactions between pairs of proteins. In particular, molecular-glue degraders, which mediate interactions between target proteins and components of the ubiquitin proteasome system to cause targeted protein degradation, hold great promise as a unique modality for therapeutic targeting of proteins that are currently intractable. Here, we report a new molecular glue HQ461 discovered by high-throughput screening of small molecules that inhibited NRF2 activity. Using unbiased loss-of-function and gain-of-function genetic screening followed by biochemical reconstitution, we show that HQ461 acts by promoting interaction between CDK12 and DDB1-CUL4-RBX1 E3 ubiquitin ligase, leading to polyubiquitination and proteasomal degradation of CDK12’s interacting protein Cyclin K (CCNK). Degradation of CCNK mediated by HQ461 compromised CDK12 function, leading to reduced phosphorylation of CDK12 substrate, downregulation of DNA damage response genes, and cell death. Structure-activity relationship analysis of HQ461 revealed the importance of a 5-methylthiazol-2-amine pharmacophore and resulted in an HQ461 derivate with improved potency. Our studies reveal a new molecular glue that engages its target protein directly with DDB1 to bypass the requirement of a substrate-specific receptor, presenting a new strategy for targeted protein degradation. Parental HCT-116 were plated sparsely on 10-cm plates to allow the isolation of ten individual clones. Each of these ten clones were expanded to one confluent 15-cm plate to establish ten independent cell populations. One million cells from each population were then plated on one 10-cm plate and treated with 20 μM HQ461 continuously for two weeks, with media change every 3 to 4 days. HQ461 resistant clones emerged from 5 out of the 10 populations. These clones were isolated, expanded, and tested for their sensitivity to HQ461 using cell viability assay. Five HQ461-resistant clones selected from independent populations were subjected to genomic DNA isolation as described in the previous session. Their genomic DNAs were then pooled in equal amounts for whole-exome sequencing at 200x average coverage (Novogene). As a control, genomic DNAs isolated from the corresponding cell populations before HQ461 selection were also pooled for whole-exome sequencing.