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

Jack C . Hanna, Victoriano Corpas Lopez, Simona Seizova, Beatrice L. Colon, Ross Bacchetti, Grant M. J. Hall, Emma M. Sands, Lee Robinson, Beatriz Baragaña, Susan Wyllie (Lead / Corresponding author), Mattie Pawlowic (Lead / Corresponding author)

Research output: Contribution to journalArticlepeer-review

3 Citations (Scopus)
59 Downloads (Pure)

Abstract

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 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 Cryptosporidium is severely lacking. Methods: We used C. parvum lysyl-tRNA synthetase (CpKRS) and DDD01510706 as a target-compound pair to develop both chemical and genetic tools for mode of action studies for Cryptosporidium. We adapted thermal proteome profiling (TPP) for Cryptosporidium, an unbiased approach for target identification. Results: Using TPP we identified the molecular target of DDD01510706 and confirm that it is CpKRS. Genetic tools confirm that CpKRS is expressed throughout the life cycle and that this target is essential for parasite survival. Parasites genetically modified to over-express CpKRS 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 Cryptosporidium, KRS R. This second selection marker is interchangeable with the original selection marker, Neo R, and expands the range of reverse genetic approaches available to study parasite biology. Due to the sexual nature of the Cryptosporidium life cycle, parental strains containing different drug selection markers can be crossed in vivo. Discussion: Selection with both drug markers produces highly efficient genetic crosses (>99% hybrid progeny), paving the way for forward genetics approaches in Cryptosporidium.

Original languageEnglish
Article number1236814
Number of pages14
JournalFrontiers in Cellular and Infection Microbiology
Volume13
DOIs
Publication statusPublished - 4 Aug 2023

Keywords

  • Cryptosporidiosis
  • mode of action
  • tRNA synthetase inhibitor
  • drug marker
  • Genetic cross
  • Thermal proteome profiling (TPP)
  • selection marker
  • thermal proteome profiling (TPP)
  • aminoacyl-tRNA synthtase
  • cryptosporidiosis
  • genetic cross

ASJC Scopus subject areas

  • Microbiology (medical)
  • Infectious Diseases
  • Microbiology
  • Immunology

Fingerprint

Dive into the research topics of 'Mode of action studies confirm on-target engagement of lysyl-tRNA synthetase inhibitor and lead to new selection marker for Cryptosporidium'. Together they form a unique fingerprint.

Cite this