Discovery of novel, targetable covalent ligand-protein interactions using chemoproteomics

Division Of Basic Sciences - Nci

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Abstract

We are expanding the scope of electrophilic chemotypes with defined selectivity and tunability to advance the development of covalent ligands. Our initial efforts focused on exploring unique and underexplored electrophilic moieties inspired by natural product scaffolds. To this end, we developed a modular synthetic strategy to rapidly incorporate a natural product-derived electrophilic warhead, 3-bromo-4,5-dihydroisoxazole (BDHI), into a diverse fragment library. This approach enabled the synthesis of an initial collection of ~350 compounds (J. Am. Chem. Soc., 2023). Additionally, we characterized a reactive heterocyclic scaffold, 4-chloro-pyrazolopyridine (CPzP), demonstrating its potential for selective protein modification (ACS Chem. Biol., 2024). Applying chemoproteomic methods (competitive SDS-PAGE analysis and quantitative LC-MS/MS profiling), we showed that fragments bearing these unique electrophiles can selectively engage distinct subsets of cysteines within the human proteome, supporting their utility in site-specific covalent targeting. These findings highlight their promise for enabling inverse drug design strategies and late-stage functionalization in covalent ligand discovery. Our ongoing work involves further refining the reactivity of these electrophilic warheads and optimizing ligand scaffolds to enhance potency, selectivity, and overall drug-like properties. For example, based on the structural data from crystallography and computational modeling, we designed and synthesized several CPzP analogs that showed improved in vitro and cellular activity against prolyl endopeptidase (PREP), a serine protease that is implicated in neurodegenerative diseases and cancer. Our study demonstrates that CPzP chemotype serves as a novel medicinal chemistry element useful for developing covalent PREP inhibitors targeting cysteine in the active site, providing a strategy to gain compound selectivity and a means to investigate how structurally diverse PREP inhibitors affect different functions of PREP. A manuscript for this work is in an advanced stage of preparation to be submitted.