Targeted protein degradation via intramolecular bivalent glues

Abstract

Targeted protein degradation is a drug modality represented by compounds that recruit a target to an E3 ubiquitin ligase to promote target ubiquitination and proteasomal degradation. Historically, the field distinguishes monovalent degraders from bifunctional degraders (PROTACs) that connect target and ligase via separate binding ligands joined via a linker1-4. Here, we elucidate the mechanism of action of a PROTAC-like degrader of the transcriptional coactivator BRD4, composed of a BRD4 ligand linked to a ligand for the E3 ligase CRL4DCAF15. Using orthogonal CRISPR/Cas9 screens we identify the degrader activity is independent of DCAF15, and relies on a different CRL4 substrate receptor, DCAF16. We demonstrate an intrinsic affinity between BRD4 and DCAF16, which is dependent on the tandem bromodomains of BRD4 and further increased by the degrader without physically engaging DCAF16 in isolation. Structural characterization of the resulting ternary complex reveals both BRD4 bromodomains are bivalently engaged in cis by the degrader and are bound to DCAF16 through several interfacial BRD4-DCAF16 and degrader-DCAF16 contacts. Our findings demonstrate that intramolecularly bridging domains can confer glue-type stabilization of intrinsic target-E3 interactions, and we propose this as a general strategy to modulate the surface topology of target proteins to nucleate co-opting of E3 ligases or other cellular effector proteins for effective proximity-based pharmacology.

Rational Chemical Design of Molecular Glue Degraders

“…Several recent independent studies with specific protein targets and compounds have also revealed the possibility for converting protein-targeting ligands into molecular glue degraders through subtle chemical changes. These examples include: (1) CR8, a close analog of the nondegradative CDK12 inhibitor (R)- roscovitine, that engages in a ternary complex between CDK12-cyclin K and the CUL4 adaptor protein DDB1, leading to cyclin K ubiquitination and degradation; (2) BI-3802, a BCL6 inhibitor that was discovered to be a degrader of BCL6 through recognition of BI-3802-mediated polymerization filaments by the SIAH1 E3 ligase; (3) GNE-0011, MMH1, and MMH2, reversibly and irreversibly acting analogs of the nondegradative BRD4 inhibitor JQ1, that led to BRD4 degradation through ternary complex formation with the cullin E3 ligase substrate receptor DCAF16. − …”

Proximity-Based Modalities for Biology and Medicine

“…In fact, most, if not all of the modalities are still explorative and are at their early stages of development, and caution should be taken before plunging into them, as they may not work solely or at all as intended. For example, multiple PROTAC-like molecules have been reported to work through mechanisms other than those originally anticipated. ,,, …”

“…Importantly, increasing evidence supports the emerging concept that MG degraders promote pre-existing E3 ligase-target interactions rather than creating new ones de novo. 47,51 interactions are challenging to identify or predict using conventional approaches. We anticipate that the development of novel computational and experimental methods to reveal and validate protein−protein interaction pairs will be fundamental to offer a step change in our ability to discover new MG degraders.…”

“…Several MGs are under clinical or preclinical evaluation, including (R)-CR8, NRX-252114, and BI-3820 . Beyond monomeric MGs, bifunctional PROTAC-like molecules were recently found to be able to glue the target protein Brd4 to the E3 ligase DCAF16 by intramolecularly bridging two domains of the target protein and anchoring an intrinsic Brd4-DCAF16 protein–protein interaction, without directly engaging DCAF16, leading to very efficient Brd4 degradation (see refs. − for related papers published around the same time).…”

“…Ushered by new assays and technologies, we anticipate that the discovery of MG degraders will shift greatly from fortuitous to intentional development. Importantly, increasing evidence supports the emerging concept that MG degraders promote pre-existing E3 ligase-target interactions rather than creating new ones de novo . , Such interactions are often of weak to intermediate binding affinity and hence inconsequential for effective protein ubiquitination/degradation; therefore, these interactions are challenging to identify or predict using conventional approaches. We anticipate that the development of novel computational and experimental methods to reveal and validate protein–protein interaction pairs will be fundamental to offer a step change in our ability to discover new MG degraders.…”