CRISPR-based oligo recombineering prioritizes apicomplexan cysteines for drug discovery

• Published in: Nature microbiology Vol. 7; no. 11; pp. 1891 - 1905
• Main Authors: Benns, H. J., Storch, M., Falco, J. A., Fisher, F. R., Tamaki, F., Alves, E., Wincott, C. J., Milne, R., Wiedemar, N., Craven, G., Baragaña, B., Wyllie, S., Baum, J., Baldwin, G. S., Weerapana, E., Tate, E. W., Child, M. A.
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 01.11.2022; Nature Publishing Group
• Subjects: 14/63; 38/70; 45; 45/41; 631/154/555; 631/326/417; 631/61/191/1908; 631/92/475; 82/58; 96/106; Amino Acid Sequence; Antiparasitic agents; Biomedical and Life Sciences; CRISPR; Cysteine - chemistry; Drug development; Drug Discovery; Infectious Diseases; Life Sciences; Medical Microbiology; Microbiology; Parasitology; Protein Processing, Post-Translational; Toxoplasma - genetics; Toxoplasma - metabolism; Toxoplasma gondii; Virology
• ISSN: 2058-5276; 2058-5276
• DOI: 10.1038/s41564-022-01249-y

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. A CRISPR-based oligo recombineering (CORe) approach functionally prioritizes electrophile-sensitive cysteines in Toxoplasma gondii and promotes anti-parasitic drug discovery.