Modulating protein-protein interactions with multicovalent COUPLrs

Abstract

Proteins function as integral components of complex molecular networks, orchestrating a wide array of biological processes, including signal transduction, gene regulation, and metabolic control. Pathogenic mutations frequently perturb these protein-protein interaction (PPI) networks, compromising biochemical function. Consequently, modulation of PPIs with small molecules provides a highly promising framework for the development of targeted strategies in disease treatment. In this thesis, I describe our efforts to develop a novel class of multicovalent PPI modulators called COUPLrs. Using mobility shift proteomics, we identified multiple COUPLrs capable of selectively oligomerizing lineage-specific cancer mutations including NPM1c and EML4-ALK. We further highlight that molecular COUPLrs can systematically and unbiasedly identify proteins amenable to chemical complexation, binding to a variety of protein classes classically considered “undruggable.” In Chapter 1, I provide a brief outlook of the critical role PPIs play in biological systems and disease, outlining traditional approaches to discovering small-molecule PPI modulators. I then focus on PROTACs and molecular glues as emerging therapeutic strategies capable of modulating PPIs by recruiting E3 ligases to disease-relevant proteins, discussing the advantages and restrictions of rationally designing and screening for new scaffolds. In Chapter 2, I discuss our usage of COUPLrs in tandem with CONNECT proteomics to streamline the identification of hundreds of proteins amenable to covalent complexation with other proteins. We profiled our library of COUPLrs in 13 cancer lines representative of 10 tissue lineages, discovering differentially essential proteins which are highly promising for coupling. Finally, we optimized an advanced COUPLr for the oncogene EML4-ALK which led to the formation of a dimer of trimers and subsequent degradation of EML4-ALK. In Chapter 3, I describe our recent efforts to take highly specific and potent small molecule ligands of TEAD and convert them into COUPLrs capable of modifying TEAD’s native PPIs. I provide an overview of the design considerations and subsequent evaluations of TEAD COUPLr treatments in a YAP oncogenic cancer cell line. Finally, I discuss proteomic evaluation of TEAD COUPLrs to identify potential binding partners. Collectively, this thesis defines a novel class of PPI modulators which seek to streamline screening readouts and enhance the probability of identifying small molecule modulators capable of coupling proteins together. These COUPLrs provide a unique advantage over PROTACs due to their favorable drug-like properties. Compared to molecular glues, COUPLrs can be more readily rationally designed and screened for through a combination of mobility shift assays and proteomics.