Mode of action studies confirm on-target engagement of lysyl-tRNA synthetase inhibitor and lead to new selection marker for Cryptosporidium
IntroductionCryptosporidiosis 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 cr...
| Main Authors: | , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2023-08-01
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| Series: | Frontiers in Cellular and Infection Microbiology |
| Subjects: | |
| Online Access: | https://www.frontiersin.org/articles/10.3389/fcimb.2023.1236814/full |
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| author | Jack C. Hanna Victor Corpas-Lopez Simona Seizova Beatrice L. Colon Ross Bacchetti Grant M. J. Hall Emma M. Sands Lee Robinson Beatriz Baragaña Beatriz Baragaña Susan Wyllie Mattie C. Pawlowic |
| author_facet | Jack C. Hanna Victor Corpas-Lopez Simona Seizova Beatrice L. Colon Ross Bacchetti Grant M. J. Hall Emma M. Sands Lee Robinson Beatriz Baragaña Beatriz Baragaña Susan Wyllie Mattie C. Pawlowic |
| author_sort | Jack C. Hanna |
| collection | DOAJ |
| description | IntroductionCryptosporidiosis 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.MethodsWe 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.ResultsUsing 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, KRSR. This second selection marker is interchangeable with the original selection marker, NeoR, 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.DiscussionSelection with both drug markers produces highly efficient genetic crosses (>99% hybrid progeny), paving the way for forward genetics approaches in Cryptosporidium. |
| first_indexed | 2024-03-12T17:35:00Z |
| format | Article |
| id | doaj.art-d00f990a5eec45dd880bc3d721f16c09 |
| institution | Directory Open Access Journal |
| issn | 2235-2988 |
| language | English |
| last_indexed | 2024-03-12T17:35:00Z |
| publishDate | 2023-08-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Cellular and Infection Microbiology |
| spelling | doaj.art-d00f990a5eec45dd880bc3d721f16c092023-08-04T12:15:41ZengFrontiers Media S.A.Frontiers in Cellular and Infection Microbiology2235-29882023-08-011310.3389/fcimb.2023.12368141236814Mode of action studies confirm on-target engagement of lysyl-tRNA synthetase inhibitor and lead to new selection marker for CryptosporidiumJack C. Hanna0Victor Corpas-Lopez1Simona Seizova2Beatrice L. Colon3Ross Bacchetti4Grant M. J. Hall5Emma M. Sands6Lee Robinson7Beatriz Baragaña8Beatriz Baragaña9Susan Wyllie10Mattie C. Pawlowic11Wellcome Centre for Anti-Infectives Research, School of Life Sciences, University of Dundee, Dundee, United KingdomWellcome Centre for Anti-Infectives Research, School of Life Sciences, University of Dundee, Dundee, United KingdomWellcome Centre for Anti-Infectives Research, School of Life Sciences, University of Dundee, Dundee, United KingdomWellcome Centre for Anti-Infectives Research, School of Life Sciences, University of Dundee, Dundee, United KingdomWellcome Centre for Anti-Infectives Research, School of Life Sciences, University of Dundee, Dundee, United KingdomWellcome Centre for Anti-Infectives Research, School of Life Sciences, University of Dundee, Dundee, United KingdomWellcome Centre for Anti-Infectives Research, School of Life Sciences, University of Dundee, Dundee, United KingdomWellcome Centre for Anti-Infectives Research, School of Life Sciences, University of Dundee, Dundee, United KingdomWellcome Centre for Anti-Infectives Research, School of Life Sciences, University of Dundee, Dundee, United KingdomDrug Discovery Unit, School of Life Sciences, University of Dundee, Dundee, United KingdomWellcome Centre for Anti-Infectives Research, School of Life Sciences, University of Dundee, Dundee, United KingdomWellcome Centre for Anti-Infectives Research, School of Life Sciences, University of Dundee, Dundee, United KingdomIntroductionCryptosporidiosis 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.MethodsWe 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.ResultsUsing 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, KRSR. This second selection marker is interchangeable with the original selection marker, NeoR, 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.DiscussionSelection with both drug markers produces highly efficient genetic crosses (>99% hybrid progeny), paving the way for forward genetics approaches in Cryptosporidium.https://www.frontiersin.org/articles/10.3389/fcimb.2023.1236814/fullcryptosporidiosismode of actionaminoacyl-tRNA synthtasetRNA synthetase inhibitorselection markergenetic cross |
| spellingShingle | Jack C. Hanna Victor Corpas-Lopez Simona Seizova Beatrice L. Colon Ross Bacchetti Grant M. J. Hall Emma M. Sands Lee Robinson Beatriz Baragaña Beatriz Baragaña Susan Wyllie Mattie C. Pawlowic Mode of action studies confirm on-target engagement of lysyl-tRNA synthetase inhibitor and lead to new selection marker for Cryptosporidium Frontiers in Cellular and Infection Microbiology cryptosporidiosis mode of action aminoacyl-tRNA synthtase tRNA synthetase inhibitor selection marker genetic cross |
| title | Mode of action studies confirm on-target engagement of lysyl-tRNA synthetase inhibitor and lead to new selection marker for Cryptosporidium |
| title_full | Mode of action studies confirm on-target engagement of lysyl-tRNA synthetase inhibitor and lead to new selection marker for Cryptosporidium |
| title_fullStr | Mode of action studies confirm on-target engagement of lysyl-tRNA synthetase inhibitor and lead to new selection marker for Cryptosporidium |
| title_full_unstemmed | Mode of action studies confirm on-target engagement of lysyl-tRNA synthetase inhibitor and lead to new selection marker for Cryptosporidium |
| title_short | Mode of action studies confirm on-target engagement of lysyl-tRNA synthetase inhibitor and lead to new selection marker for Cryptosporidium |
| title_sort | mode of action studies confirm on target engagement of lysyl trna synthetase inhibitor and lead to new selection marker for cryptosporidium |
| topic | cryptosporidiosis mode of action aminoacyl-tRNA synthtase tRNA synthetase inhibitor selection marker genetic cross |
| url | https://www.frontiersin.org/articles/10.3389/fcimb.2023.1236814/full |
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