Functional screening prioritizes apicomplexan cysteines for covalent drug discovery

Nucleophilic amino acids are important in covalent drug development yet underutilized as antimicrobial targets. Chemoproteomic technologies have been developed to mine chemically-accessible residues via their intrinsic reactivity toward electrophilic probes. However, these approaches cannot discern which chemically reactive sites contribute to protein function and should therefore be prioritized for drug discovery. To address this, we have developed a CRISPR-based Oligo Recombineering (CORe) platform to support the expedient identification, functional prioritization, and rational targeting of chemically reactive sites in haploid systems. Our approach couples protein sequence and function with biological fitness of live cells. Here, we profile the electrophile sensitivity of proteinogenic cysteines in the eukaryotic pathogen Toxoplasma gondii and prioritize functional sites using CORe. Electrophile-sensitive cysteines decorating the ribosome were found to be critical for parasite growth, with target-based screening identifying a parasite-selective antimalarial lead molecule and validating the apicomplexan translation machinery as a target for ongoing covalent ligand development.