Breaking Bad Proteins-Discovery Approaches and the Road to Clinic for Degraders

Abstract

Proteolysis-targeting chimeras (PROTACs) describe compounds that bind to and induce degradation of a target by simultaneously binding to a ubiquitin ligase. More generally referred to as bifunctional degraders, PROTACs have led the way in the field of targeted protein degradation (TPD), with several compounds currently undergoing clinical testing. Alongside bifunctional degraders, single-moiety compounds, or molecular glue degraders (MGDs), are increasingly being considered as a viable approach for development of therapeutics, driven by advances in rational discovery approaches. This review focuses on drug discovery with respect to bifunctional and molecular glue degraders within the ubiquitin proteasome system, including analysis of mechanistic concepts and discovery approaches, with an overview of current clinical and pre-clinical degrader status in oncology, neurodegenerative and inflammatory disease.

Keywords: PROTAC; TPD; heterobifunctional degrader; molecular glue degrader; proteasome; targeted protein degradation; ubiquitin.

Figure 1

Schematic representation of the mechanisms of proteolysis-targeting chimeras (PROTACs) and molecular glue degraders (MGDs). (A) The UPS consists of a cascade involving three enzymes that sequentially attach ubiquitin molecules to target proteins, marking them for proteasomal degradation. Degraders exploit this system by inducing proximity between a target protein (POI) and an E3 ligase, facilitating ubiquitination and subsequent degradation of the target protein. PROTACs are heterobifunctional molecules comprising a POI warhead and an E3 connected by a linker while molecular glue degraders are monomeric molecules. (B) Representation of the hook effect illustrating that at high concentrations, bifunctional degraders form binary complexes that reduce target degradation. This figure was created with BioRender.com.

Figure 2

Ubiquitin ligase families and examples of degraders directly recruiting E3 ligases or alternative UPS components. (A) The homologous to E6-AP C terminus (HECT) E3 family forms a thioester ubiquitin intermediate with a HECT domain cysteine, before transferring it to the substrate protein. Really interesting new gene (RING) finger E3 ligases utilize a RING domain to facilitate direct transfer of ubiquitin from E2 enzymes to substrate proteins. RING-in-between-RING (RBR) E3 ligases combine features of both RING and HECT ligases; the RING1 domain first recruits the E2–Ub complex before transferring ubiquitin onto a RING2 catalytic cysteine residue, ultimately facilitating its transfer onto the substrate protein. (B) The cullin–RING ligases (CRL) are modular RING-family E3 ligases that utilize exchangeable adaptors (adpt) and receptors (recept) organized around a CUL domain to recruit specific substrates. Exemplified are the CUL2-RBX1-ElonginB-ElonginC-VHL (CRL2^VHL) and the CUL4–RBX1–DDB1–CRBN (CRL4^CRBN) complexes, with PROTAC and MGD recruiters as indicated. (C) MGDs can induce ubiquitination by triggering oligomerization of a protein. For example, MGD BI-3802 induces the dimerization of BCL6, facilitating its recognition by the E3 ligase SIAH1, which then mediates polyubiquitination of BCL6, ultimately resulting in its degradation. (D) Degraders can also recruit alternative UPS components to induce POI degradation such as E3 adaptors, E2 enzymes or directly the 26S proteasome. For example, the adaptor DDB1 can be recruited by MGD dCeMM3, forming a ternary complex with CDK12-Cyclin K conjugate, leading to Cyclin K ubiquitination and degradation. The E2 enzyme UBE2D can form a ternary complex with the MGD EN450 and NFKB1, leading to NFKB1 ubiquitination and subsequent degradation. MG EN67 can also be converted into a PROTAC. The proteasome can be directly recruited with compounds such as chemical inducers of degradation (CIDE). These degraders can be used to target a protein of interest (POI), such as BRD4, to the 26S proteasome for ubiquitin-independent proteasomal degradation. This figure was created with BioRender.com.

Figure 3

Overview of concepts and considerations for degrader discovery, characterization, and validation. This figure was created with BioRender.com.