Mode of action studies confirm on-target engagement of lysyl-tRNA synthetase inhibitor and lead to new selection marker for Cryptosporidium

• DOI: 10.3389/fcimb.2023.1236814
• Publication Date: 2023-08-04T12:16:06Z
• Authors: Jack C. Hanna; Victor Corpas-Lopez; Simona Seizova; Beatrice L. Colon; Ross Bacchetti; Grant M. J. Hall; Emma M. Sands; Lee Robinson; Beatriz Baragaña; Susan Wyllie; Mattie C. Pawlowic
• Keywords: Cryptosporidium parvum; Mode of Action

Introduction Cryptosporidiosis is a leading cause of diarrheal-associated morbidity and mortality, predominantly affecting children under 5 years old in low-and-middle-income countries. There no effective treatment vaccine. New therapeutics are emerging from drug discovery efforts. It critical that mode action studies performed alongside to ensure the best clinical outcomes. Unfortunately, technology identify validate targets for Cryptosporidium severely lacking. Methods We used C. parvum lysyl-tRNA synthetase ( Cp KRS) DDD01510706 as target-compound pair develop both chemical genetic tools . adapted thermal proteome profiling (TPP) , an unbiased approach target identification. Results Using TPP we identified molecular confirm it KRS. Genetic KRS expressed throughout life cycle this essential parasite survival. Parasites genetically modified over-express or parasites with mutation at compound-binding site resistant DDD01510706. leveraged these mutations generate second selection marker modification R This interchangeable original marker, Neo expands range reverse approaches available study biology. Due sexual nature cycle, parental strains containing different markers can be crossed vivo Discussion Selection produces highly efficient crosses (>99% hybrid progeny), paving way forward genetics