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

Abstract

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.

Inducible mismatch repair streamlines forward genetic approaches to target identification of cytotoxic small molecules

“…42 Subsequent studies demonstrated that CR8 recruits CDK12 to DDB1, which leads to the ubiquitylation and proteasomal degradation of the CDK12 heterodimeric partner, cyclin K. In two independent studies, at least four more structurally diverse small molecules were shown to have the same activity, raising the possibility that CDK12/cyclin K recruitment to DDB1 is promiscuous. 43,44 Based on our observations, and the results of prior studies, we hypothesized that SW394703 may act through a mechanism similar to CR8. Treatment with SW394703, but not its stereoisomer, led to a reduction in cyclin K protein levels at concentrations consistent with the anti-proliferative activity of SW394703 (Figure 6E).…”

“…Surprisingly, this mechanism is shared with three recently reported compounds which all have unique chemical structures. [42][43][44] Thus, our discovery of the SW394703 MoA not only provides a unique scaffold with which to optimize a cyclin K degrader, but also provides a starting point for the design of neo-functional molecules that recruit alternative proteins to DDB1-containing cullin complexes for degradation. This idea draws inspiration from the IMiDs, a class of molecular glue degraders for which different chemical derivatives are able to selectively recruit diverse neo-substrates to the CRL4-CRBN E3 ubiquitin ligase.…”

“…These genetic and biochemical data suggest that SW394703 is a new chemical scaffold that recruits CDK12 and cyclin K to DDB1, leading to cyclin K degradation in a similar mechanism to the previously reported molecular glue degraders CR8, HQ461, and dCeMM2/3/4. [42][43][44] Development of an inducible forward genetic system in a cell line with intact mismatch repair Initially, forward genetic studies with cancer cells were restricted to HCT116 or other cells that have intrinsic mismatch repair deficiencies. We previously demonstrated that silencing of MSH2, an essential component of DNA mismatch repair, promotes increased mutagenesis and enables forward genetic screening in cancer cell lines that are otherwise mismatch repair proficient.…”