CRISPR-based oligo recombineering prioritizes apicomplexan cysteines for drug discovery

H. J. Benns, M. Storch, J. A. Falco, F. R. Fisher, Fabio Tamaki, E. Alves, C. J. Wincott, Rachel Milne, Natalie Wiedemar, G. Craven, Beatriz Baragaña, Susan Wyllie, Jake Baum, G. S. Baldwin, Eranthie Weerapana, Edward W. Tate, M. A. Child

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Abstract

Nucleophilic amino acids are important in covalent drug development yet underutilized as anti-microbial targets. Chemoproteomic technologies have been developed to mine chemically accessible residues via their intrinsic reactivity towards electrophilic probes but 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 rapid 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 anti-malarial lead molecule and validating the apicomplexan translation machinery as a target for ongoing covalent ligand development.
Original languageEnglish
Pages (from-to)1891-1905
Number of pages30
JournalNature Microbiology
Volume7
Issue number11
DOIs
Publication statusPublished - 20 Oct 2022

Keywords

  • Mutagenesis
  • Parasitology
  • Proteomics
  • Target identification

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