Abstract 4500: A high-throughput approach to discover and characterize glueable ligase-target pairs

Abstract Targeted protein degradation can be induced with monovalent small molecules, termed molecular glues, that modify the substrate-binding interface of E3 ligases and reroute them to novel targets. Despite there being over 600 E3 ligases encoded in the human genome, most glues have focused on only a few. Expanding to additional ligases and targets is challenging due to the low probability of successful 'glueing. ' Many have suggested that a weak basal interaction between a ligase and neo-substrate is an important prerequisite for the function of a molecular glue, but such weak interactions are challenging to discover. To address this need, we have harnessed AlphaSeq, a synthetic biology platform that measures protein-protein interactions at scale and with high sensitivity, to identify and characterize novel E3-target pairs. Our approach is twofold. First, we test for novel weak interactions (~100nM to 10uM) between a panel of E3 ligases and a library of disease relevant target proteins. Second, starting from a weakly interacting protein pair, we build mutational libraries of both proteins and measure their binding strengths. Mutations that strengthen binding serve to validate the weak interaction, provide structural insights into the nature of binding, and help to inform subsequent small molecule discovery. Here, we present our approach to discover and evaluate glueable ligase-target pairs using AlphaSeq, validation through proof-of-concept experiments involving a known interaction agonized by small molecules, and examples of novel glueable ligase-target pairs that we have recently discovered. Citation Format: Arpita Sen, Phil Burke, Ruchi Bansal, Dasha Krayushkina, David Noble, Kerry McGowan, Natasha Seelam Murakowska, Paxton Reed, Juliana Barrett, Randolph Lopez. A high-throughput approach to discover and characterize glueable ligase-target pairs abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts) ; 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84 (6Suppl): Abstract nr 4500.