Protein degradation using heterobifunctional chimeras is a rapidly growing mechanism to alter protein function. Critical to this mechanism is the formation of a ternary complex in which the target protein is recruited to an E3 ligase. We report structural, biophysical, and cellular studies that expand our understanding of this process - shedding light on different degradation efficiencies for distinct recruitment mechanisms for Bruton’s Tyrosine Kinase.

The pace of progress in drug discovery is highly dependent on the availability of robust and quantitative assay platforms that allow for straightforward but accurate characterization of the underlying molecular interactions and target abundance. We have developed novel TR-FRET-based assay systems to facilitate the characterization of PROTACs and molecular glue degraders, including the facile measurements of endogenous protein levels, target affinity profiling, and quantitative determination of complex ternary cooperativity. It also allows kinetic and thermodynamic measurement of ligand binding with endogenous proteins, high-throughput quantification of endogenous target protein abundance in cell lysates, and measurement of absolute cooperativity of ternary complexes.

Cereblon is both a substrate adaptor protein for CRL4-mediated protein destruction, and the target of several novel clinical compounds which redirect this destructive activity to degrade "undruggable" substrates of interest. Though previous crystallographic structures existed, we use Cryo-EM to study the cereblon apoenzyme and directly compare it with several liganded and substrate-engaged states. This ensemble analysis reveals important allosteric changes to the mechanistic cycle of cereblon.

The BRG1 (SMARCA4) protein is mutated in a subset of NSCLC, and such alterations make the cancers highly dependent on the closely-related BRM (SMARCA2) paralog for growth. The detailed characterization of a potent, heterobifunctional, chimeric BRM degrader compound using NSCLC cells and tumor models will be discussed. The conjugation of a second chimeric BRM degrader molecule to monoclonal antibodies to enhance its in vivo activity will also be described.

Targeted protein degradation of disease-causing proteins is a promising new therapeutic modality. A novel approach has emerged for extracellular protein degradation that utilizes the asialoglycoprotein receptor (ASGPR) to recruit pathogenic proteins for endolysosomal degradation. We describe the discovery of novel bifunctional compounds called ATACs (ASGPR-Targeting Chimeras) containing Avilar’s proprietary, high-affinity, small-molecule, ASGPR-binding ligands.

Most strategies in protein degradation focus on co-opting E3 ligases directly. Another strategy would be to find a native protein which is potently degraded (naturally or induced) and then hitchhike on that protein. I will present our work on using such an approach to coax the DNA repair protein methylguanine methyltransferase (MGMT) into degrading other target proteins.

Methotrexate (MTX), a folate cofactor analog, is a potent inhibitor of dihydrofolate reductase (DHFR), where it is used as both an antineoplastic and an immunosuppressant therapeutic. MTX undergoes folylpolyglutamate synthetase (FPGS)-mediated g-glutamylation, which affects cellular retention and target specificity. Unfortunately, therapeutic resistance to MTX is a significant clinical barrier. Furthermore, the polypharmacological of MTX remains poorly understood. We developed a series of potent MTX-based PROteolysis TArgeting Chimeras (PROTACs) to investigate DHFR degradation pharmacology and one-carbon biochemistry.

The zinc finger transcription factor SALL4 is an ideal target in cancer, but small-molecule therapies against SALL4 are not yet clinically available. We have deduced the crystal structure of the complex of SALL4 bound to DNA via zinc finger cluster 4 (ZFC4), followed by computational predictions and cell-based drug screening identification of a new lead molecule that preferentially targets SALL4 positive cancer cells.

PROTACs as a nascent technology have shown early promises and the field witnessed an explosion of discovery and development activities recently. In this presentation, we will share some learnings from multiple programs over the last 6 years, including our understanding of potential resistant mechanisms and some unexpected findings on selectivity, toxicity, etc.

Three features separate the targeted protein degradation from previously drug discovery modalities; its catalytic mechanism to achieve higher efficacy, its ability to target previously undruggable proteins such as non-enzyme targets, and its potential to deliver drug activity to selective tissues. All three features depend on the E3 ligands. Human cells express estimated more than 600 E3 ubiquitin ligases, yet nearly all current advanced degraders use the same E3 ligand binding to cereblon. I will discuss our rationale and efforts in discovering novel E3 ligands for targeted protein degradation.     

The field of small molecule-mediated protein degradation has grown tremendously over the past few years. Nonetheless, only a handful of E3 ligases have been identified to support this process. We have developed a chemoproteomic method to discover additional E3 ligases capable of supporting small molecule-mediated protein degradation and identified two novel E3 ligases, DCAF16 and DCAF11, as the targets of electrophilic bifunctional degraders that engage diverse protein substrates.

A major challenge in the TPD field is the lack of accessible E3 ligase ligands for developing degraders. To expand the E3 ligase toolbox, we sought to convert the KEAP1 inhibitor into a recruitment handle for several targets. We characterize tool compounds to explore KEAP1-mediated ubiquitination and delineate the challenges of exploiting new E3 ligases for generating bivalent degraders.