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

Abstract

Molecular-glue degraders mediate interactions between target proteins and components of the ubiquitin-proteasome system to cause selective protein degradation. Here, we report a new molecular glue HQ461 discovered by high-throughput screening. Using loss-of-function and gain-of-function genetic screening in human cancer cells followed by biochemical reconstitution, we show that HQ461 acts by promoting an interaction between CDK12 and DDB1-CUL4-RBX1 E3 ubiquitin ligase, leading to polyubiquitination and degradation of CDK12-interacting protein Cyclin K (CCNK). Degradation of CCNK mediated by HQ461 compromised CDK12 function, leading to reduced phosphorylation of a 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 recruits its target protein directly to DDB1 to bypass the requirement of a substrate-specific receptor, presenting a new strategy for targeted protein degradation.

Major advances in targeted protein degradation: PROTACs, LYTACs, and MADTACs

“…Rather they showed that CDK12, which normally does not act as a E3 substrate receptor, binds DDB1 in the presence of CR-8, positioning cyclin K into the ubiquitination zone (radius from E3 ligase where accessible lysine residues on substrate can be marked with ubiquitin). Interestingly, similar results have now been published by the Winter ( 149 ) and Wang ( 150 ) groups. These studies show that other parts of the E3 ligase complex than the substrate recognition subunit may be amenable to hijacking for TPD.…”

Ubiquitin‐dependent regulation of transcription in development and disease

“…Molecular glues can also elicit transcription factor degradation by means other than directly attracting an E3 ligase: Compounds that bind BCL6, for example, trigger formation of BCL6 fibers that recruit the E3 ligase SIAH1 and counteract BCL6‐dependent gene repression in lymphoma cells (Kerres et al, 2017; Slabicki et al, 2020b). Moreover, molecular glues can affect the activity of RNA Pol II itself, by controlling the stability of a cyclin‐dependent kinase that regulates transcript elongation (Mayor‐Ruiz et al, 2020; Lv et al, 2020; Slabicki et al, 2020a).…”

The Potential of Proteolytic Chimeras as Pharmacological Tools and Therapeutic Agents

“…Oncology: Her2+ and triple-negative breast tumors [111] Covalent protac BTK and BLK CRBN Oncology: chronic lymphocytic leukemia [112] Covalent protac Platform for Halo-tagged proteins. VHL Research use [115] Covalent protac Platform for GFP-tagged proteins VHL Research use [117] Click protac BRD4 and ERK1/2 CRBN Oncology [119] Molecular glue RBM39 E3 ligase receptor DCAF15 Oncology [122] Molecular glue RBM39, cyclinK CRBN Oncology [123] Molecular glue DDB1-CDK12 molecular glue CRBN Oncology [124] Molecular glue CDK12-cyclin K CRBN Oncology [125] Allosteric modulator Aspartate decarboxylase PanD E3-independent Research use, anti-microbial agents [131] Ubiquitin-independent degrader Androgen receptor (AR) (F876L) ND (indirectly, CHIP) Oncology: prostate cancer [137] Ubiquitin-independent degrader Her3 (ErbB3)…”

“…The fact that the discovery of molecular glues has been so far the result of serendipity has prompted researchers to define rational methods to discover novel molecules acting as glues [ 123 ]. Two recent papers have used distinct approaches, bioinformatics and molecular screening, to establish new molecular glue candidates, somehow converging in identifying and characterizing outstanding cyclin K degraders [ 124 , 125 ] ( Figure 2 ). The Ebert lab bioinformatically analyzed the data of more than 4500 drugs tested against close to 600 cancer cell lines with mRNA levels of 500 E3 ligases, determining more than 67,000 correlations, and established a link between the Cul4 adaptor protein DDB1’s expression levels and the CDK inhibitor CR8’s [ 126 ] sensitivity.…”

“…The binding affinity and the tightness of the formed ternary complex directly correlate with the efficiency in ubiquitination, and thus with degrader glue capacity, as shown by testing distinct CDK family members, CDK12 mutants and CDK inhibitors similar to CR8 [ 125 ]. Remarkably, in another work, Qi’s group, by using a different approach, defined a distinct DDB1-CDK12 molecular glue, named HQ461, exhibiting some similarities to CR8 [ 124 ]. Lv et al performed a high-throughput screening of small molecules showing NRF2 inhibitory activity.…”