Targeted protein degradation (TPD) using heterobifunctional chimeras holds the potential to expand target space and grow the ‘druggable proteome.'  Most acutely, this provides an opportunity to target proteins that lack enzymatic activity or have otherwise proven intractable to small molecule inhibition. Bridging covalent ligand discovery with chimeric degrader design has emerged as a potential mechanism to advance both fields.  Here, the authors, employ a set of biochemical and cellular tools to deconvolute the role of covalent modification in TPD using Bruton’s tyrosine kinase.  Results reveals that covalent target modification is fundamentally compatible with the protein degrader mechanism of action.

Mass spectrometry-based targeted proteomics approaches have emerged as a promising technology to monitor absolute abundance of therapeutic target proteins that cannot be achieved via traditional protein detection technologies. We developed and applied these high-throughput strategies to address key MoA questions on PROTACs projects, including target protein and E3 abundance, protein turnover rates, and also for compound profiling. Case studies will be shown to demonstrate the technology and the impact on several PROTAC projects.

Using our proprietary chemoproteomics platform, we identified site-selective covalent ligands for the CRL4^DCAF2 E3 ligase. DCAF2 is a substrate receptor upregulated in specific cancers. A ligand was developed into hetero-bifunctional degraders that exhibited robust cellular DCAF2-dependent BRD2/4 degradation. Our work validates DCAF2 as a novel E3 ligase substrate receptor amenable to targeted protein degradation and demonstrates the utility of the Frontier Platform to discover novel ligands enabling proximity-inducing therapeutics.

Thalidomide and its derivatives, known as immunomodulatory drugs (IMiDs), bind to the E3 ligase substrate adapter cereblon (CRBN), resulting in lifesaving anti-cancer treatments or horrific teratogenicity. E3 ligase complexes recognize degrons, specific amino acid sequences sufficient to promote ubiquitination and degradation when embedded in a protein. I will discuss chemical approaches to finding a degron for the thalidomide-binding domain of CRBN and its implications for CRBN's physiological function and therapeutic engagement.

Pin Therapeutics has created PinGLUE, a multifaceted discovery platform that integrates two primary components. The first is a novel degron display assay for screening of potential substrates against reconstituted cellular degradation machinery. The second is a streamlined E3 ligand discovery effort that extends current knowledge of heterobifunctional molecules with the intention of creating new therapeutic heterobifunctional molecules or uncovering new possible starting points for the degron display assay. We will discuss the challenges faced while building this platform and share some of our successful outcomes from these efforts.

Target protein degradation (TPD) technology provides promising therapeutic strategies for the treatment of human diseases. However, almost all of the advanced degraders recruit the same cereblon E3 ligase. Discovery of new E3 ligase ligands will help to realize the full potential of the TPD technology. In my talk, I will discuss our rationale and efforts in discovering novel E3 ligands for TPD.

PROTACs are becoming an important therapeutic modality and a great tool for target validation. However, making a target-specific PROTAC can be challenging. To overcome this problem we use genetic engineering to modify target proteins in order to enable potent, selective, and reversible protein degradation with generic, degron-specific PROTACs. This presentation will focus on genetic and chemical tools for targeted protein degradation with a highlight on novel opportunities from our perspective.

Small molecule degrader development is hampered by a low complexity matrix of E3 recruitment modules featuring a high degree of POI-specificity. High-throughput phenotypic screening using programmable PROTEINi exploits novel and unprecedented E3 ligases. Modular PROTEINi library design allows the discovery of bifunctional degraders scanning a vast diversity of new tissue- and POI-selective mechanisms. PROTEINi provide a phenotypically validated molecular inspiration as the foundation for degrader drug design and development.

WD40 repeat proteins constitute one of the largest protein families, characterized by 44-60 amino acid repeats terminating in tryptophan and aspartate (WD).  WD repeat proteins act as scaffolding proteins and play an important role in many cellular functions and as such have become an interesting family to drug. We will describe the discovery and optimization of small molecule binders to two members of this family (WDR5 and DCAF1).

SyntheX created empirical drug discovery platforms to identify protein interaction modulators. The ToRNeDO platform discovers molecular glues that can bridge a productive interaction between an E3 ubiquitin ligase and a neosubstrate of interest to achieve targeted protein degradation. Using genetically engineered circuits, the platforms rely on intracellular drug selection, bypassing many bottlenecks that exist with canonical in-vitro screening assays. This presents a novel approach to discover functional molecular glues from a first pass screen.

Circulating PCSK9 was degraded by various formats of heterobifunctional molecules that simultaneously bind to PCSK9 and the asialoglycoprotein receptor (ASGPR). Various formats, including bispecific antibodies, antibody-small molecule conjugates, and heterobifunctional small molecules demonstrate binding in vitro and accelerated PCSK9 clearance in vivo. These molecules showcase a new approach to PCSK9 inhibition, targeted plasma protein degradation (TPPD), and demonstrate the feasibility of heterobifunctional ligands to accelerate the clearance of circulating pathogenic proteins.