Journal Article

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

Baragaña, Beatriz, Seizova, Simona, Colon, Beatrice L., Hanna, Jack C., Corpas-Lopez, Victor, Sands, Emma M., Hall, Grant M. J., Bacchetti, Ross, Robinson, Lee, Wyllie, Susan, Pawlowic, Mattie C.

2023

Overview

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

• Publisher: Frontiers Media SA,Frontiers Media S.A
• Subject: aminoacyl-tRNA synthtase / Binding Sites / Cellular and Infection Microbiology / Child / Child, Preschool / Cryptosporidiosis / Cryptosporidiosis - drug therapy / Cryptosporidium / Cryptosporidium - genetics / Diarrhea / Dihydrofolate reductase / Drug development / Drug discovery / genetic cross / Genetic crosses / Humans / Life cycles / Lysine-tRNA Ligase - genetics / Malnutrition / Metabolism / Metabolites / mode of action / Morbidity / Mutation / Parasites / Propionibacterium acnes / Proteins / Proteomes / Protozoa / selection marker / Therapeutic targets / Transfer RNA / tRNA / tRNA synthetase inhibitor