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

TY - JOUR

T1 - Mode of action studies confirm on-target engagement of lysyl-tRNA synthetase inhibitor and lead to new selection marker for Cryptosporidium

AU - Hanna, Jack C .

AU - Corpas Lopez, Victoriano

AU - Seizova, Simona

AU - Colon, Beatrice L.

AU - Bacchetti, Ross

AU - Hall, Grant M. J.

AU - Sands, Emma M.

AU - Robinson, Lee

AU - Baragaña, Beatriz

AU - Wyllie, Susan

AU - Pawlowic, Mattie

N1 - Funding Information: JCH is supported by the Medical Research Council (MR/N013735/1). This work was funded in part from the Wellcome Trust (Innovations Award to SW and MCP [218448/Z/19/Z], a Centre Award [203134/Z/16/Z and 223608/Z/21/Z], an Institutional Strategic Support Fund [204816/Z/16/Z] and a PhD studentship to EMS (102132/B/13/Z)]). This work was funded in part by a Sir Henry Dale Fellowship from the Wellcome Trust and the Royal Society to MCP (213469/Z/18/Z), and from the Carnegie Trust (Research Incentive Grant to MCP [RIG008676]). GMJH is supported by the Medical Research Council (MR/R015791/1). We thank the chemistry teams within the Drug Discovery Unit (University of Dundee) for providing CpKRS-targeting compounds used in this study and the In Vivo Team in the Drug Discovery Unit for advice and support establishing the mouse efficacy models. Thanks to Flora Caldwell for laboratory support, and Prof David Horn and members of the Mode of Action group for helpful discussion. We would like to acknowledge the University of Dundee School of Life Sciences Resource Unit, Dundee FingerPrints Proteomics Facility, Dundee Imaging Facility, and Dundee Flow Cytometry and Cell Sorting Facility (supported by the Wellcome Trust, grant 097418/Z/11/Z). Thanks to Sumiti Vinayak Alam (University of Illinois) for kindly providing the “CplicHA3-DDD-Eno-Nluc-Neo-Eno” plasmid, to Martin Taylor (London School of Hygiene and Tropical Medicine) for kindly providing the original plasmid containing red-shifted Firefly Luciferase-mNeonGreen fusion (PpyRE9h-mNeon), to Wes Van Voorhis (University of Washington) for kindly sharing compound BKI-1294, and to Boris Striepen (University of Pennsylvania) for kindly providing the CpTrpB antibody. Copyright: © 2023 Hanna, Corpas-Lopez, Seizova, Colon, Bacchetti, Hall, Sands, Robinson, Baragaña, Wyllie and Pawlowic.

PY - 2023/8/4

Y1 - 2023/8/4

N2 - 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.

AB - 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.

KW - Cryptosporidiosis

KW - mode of action

KW - tRNA synthetase inhibitor

KW - drug marker

KW - Genetic cross

KW - Thermal proteome profiling (TPP)

KW - selection marker

KW - thermal proteome profiling (TPP)

KW - aminoacyl-tRNA synthtase

KW - cryptosporidiosis

KW - genetic cross

UR - https://www.scopus.com/pages/publications/85168348211

U2 - 10.3389/fcimb.2023.1236814

DO - 10.3389/fcimb.2023.1236814

M3 - Article

C2 - 37600947

SN - 2235-2988

VL - 13

JO - Frontiers in Cellular and Infection Microbiology

JF - Frontiers in Cellular and Infection Microbiology

M1 - 1236814

ER -