Abstract
Malaria and cryptosporidiosis, caused by apicomplexan parasites, remain major drivers of global child mortality. New drugs for the treatment of malaria and cryptosporidiosis, in particular, are of high priority; however, there are few chemically validated targets. The natural product cladosporin is active against blood- and liver-stage Plasmodium falciparum and Cryptosporidium parvum in cell-culture studies. Target deconvolution in P. falciparum has shown that cladosporin inhibits lysyl-tRNA synthetase (PfKRS1). Here, we report the identification of a series of selective inhibitors of apicomplexan KRSs. Following a biochemical screen, a small-molecule hit was identified and then optimized by using a structure-based approach, supported by structures of both PfKRS1 and C. parvum KRS (CpKRS). In vivo proof of concept was established in an SCID mouse model of malaria, after oral administration (ED 90 = 1.5 mg/kg, once a day for 4 d). Furthermore, we successfully identified an opportunity for pathogen hopping based on the structural homology between PfKRS1 and CpKRS. This series of compounds inhibit CpKRS and C. parvum and Cryptosporidium hominis in culture, and our lead compound shows oral efficacy in two cryptosporidiosis mouse models. X-ray crystallography and molecular dynamics simulations have provided a model to rationalize the selectivity of our compounds for PfKRS1 and CpKRS vs. (human) HsKRS. Our work validates apicomplexan KRSs as promising targets for the development of drugs for malaria and cryptosporidiosis.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 7015-7020 |
| Number of pages | 6 |
| Journal | Proceedings of the National Academy of Sciences of the United States of America |
| Volume | 116 |
| Issue number | 14 |
| DOIs | |
| State | Published - Apr 2 2019 |
Keywords
- Cryptosporidiosis
- Malaria
- TRNA synthetase
ASJC Scopus subject areas
- General
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Lysyl-tRNA synthetase as a drug target in malaria and cryptosporidiosis. / Baragaña, Beatriz; Forte, Barbara; Choi, Ryan; Hewitt, Stephen Nakazawa; Bueren-Calabuig, Juan A.; Pisco, João Pedro; Peet, Caroline; Dranow, David M.; Robinson, David A.; Jansen, Chimed; Norcross, Neil R.; Vinayak, Sumiti; Anderson, Mark; Brooks, Carrie F.; Cooper, Caitlin A.; Damerow, Sebastian; Delves, Michael; Dowers, Karen; Duffy, James; Edwards, Thomas E.; Hallyburton, Irene; Horst, Benjamin G.; Hulverson, Matthew A.; Ferguson, Liam; Jiménez-Díaz, María Belén; Jumani, Rajiv S.; Lorimer, Donald D.; Love, Melissa S.; Maher, Steven; Matthews, Holly; McNamara, Case W.; Miller, Peter; O’Neill, Sandra; Ojo, Kayode K.; Osuna-Cabello, Maria; Pinto, Erika; Post, John; Riley, Jennifer; Rottmann, Matthias; Sanz, Laura M.; Scullion, Paul; Sharma, Arvind; Shepherd, Sharon M.; Shishikura, Yoko; Simeons, Frederick R.C.; Stebbins, Erin E.; Stojanovski, Laste; Straschil, Ursula; Tamaki, Fabio K.; Tamjar, Jevgenia; Torrie, Leah S.; Vantaux, Amélie; Witkowski, Benoît; Wittlin, Sergio; Yogavel, Manickam; Zuccotto, Fabio; Angulo-Barturen, Iñigo; Sinden, Robert; Baum, Jake; Gamo, Francisco Javier; Mäser, Pascal; Kyle, Dennis E.; Winzeler, Elizabeth A.; Myler, Peter J.; Wyatt, Paul G.; Floyd, David; Matthews, David; Sharma, Amit; Striepen, Boris; Huston, Christopher D.; Gray, David W.; Fairlamb, Alan H.; Pisliakov, Andrei V.; Walpole, Chris; Read, Kevin D.; Van Voorhis, Wesley C.; Gilbert, Ian H.
In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 116, No. 14, 02.04.2019, p. 7015-7020.Research output: Contribution to journal › Article › peer-review
}
TY - JOUR
T1 - Lysyl-tRNA synthetase as a drug target in malaria and cryptosporidiosis
AU - Baragaña, Beatriz
AU - Forte, Barbara
AU - Choi, Ryan
AU - Hewitt, Stephen Nakazawa
AU - Bueren-Calabuig, Juan A.
AU - Pisco, João Pedro
AU - Peet, Caroline
AU - Dranow, David M.
AU - Robinson, David A.
AU - Jansen, Chimed
AU - Norcross, Neil R.
AU - Vinayak, Sumiti
AU - Anderson, Mark
AU - Brooks, Carrie F.
AU - Cooper, Caitlin A.
AU - Damerow, Sebastian
AU - Delves, Michael
AU - Dowers, Karen
AU - Duffy, James
AU - Edwards, Thomas E.
AU - Hallyburton, Irene
AU - Horst, Benjamin G.
AU - Hulverson, Matthew A.
AU - Ferguson, Liam
AU - Jiménez-Díaz, María Belén
AU - Jumani, Rajiv S.
AU - Lorimer, Donald D.
AU - Love, Melissa S.
AU - Maher, Steven
AU - Matthews, Holly
AU - McNamara, Case W.
AU - Miller, Peter
AU - O’Neill, Sandra
AU - Ojo, Kayode K.
AU - Osuna-Cabello, Maria
AU - Pinto, Erika
AU - Post, John
AU - Riley, Jennifer
AU - Rottmann, Matthias
AU - Sanz, Laura M.
AU - Scullion, Paul
AU - Sharma, Arvind
AU - Shepherd, Sharon M.
AU - Shishikura, Yoko
AU - Simeons, Frederick R.C.
AU - Stebbins, Erin E.
AU - Stojanovski, Laste
AU - Straschil, Ursula
AU - Tamaki, Fabio K.
AU - Tamjar, Jevgenia
AU - Torrie, Leah S.
AU - Vantaux, Amélie
AU - Witkowski, Benoît
AU - Wittlin, Sergio
AU - Yogavel, Manickam
AU - Zuccotto, Fabio
AU - Angulo-Barturen, Iñigo
AU - Sinden, Robert
AU - Baum, Jake
AU - Gamo, Francisco Javier
AU - Mäser, Pascal
AU - Kyle, Dennis E.
AU - Winzeler, Elizabeth A.
AU - Myler, Peter J.
AU - Wyatt, Paul G.
AU - Floyd, David
AU - Matthews, David
AU - Sharma, Amit
AU - Striepen, Boris
AU - Huston, Christopher D.
AU - Gray, David W.
AU - Fairlamb, Alan H.
AU - Pisliakov, Andrei V.
AU - Walpole, Chris
AU - Read, Kevin D.
AU - Van Voorhis, Wesley C.
AU - Gilbert, Ian H.
N1 - Funding Information: 16. Cali JJ, et al. (2008) Bioluminescent assays for ADMET. Expert Opin Drug Metab Toxicol 4:103–120. Materials and Methods Full details are in SI Appendix. This includes the following information: (i) the chemical synthesis of compounds described in the paper; (ii) the methods for protein expression and purification, kinetic characterization of enzymes, screening of library, and mode of inhibition studies; (iii) the methods for parasite assays using the different life-cycle stages of P. falciparum and different species of Cryptosporidium; (iv) methods for in vitro drug metabolism and pharmacokinetic assay; (v) methods for in vivo pharmacokinetics and efficacy studies; (vi) details for molecular modeling and dynamics simulations; (vii) details of X-ray crystallography; (viii) ethical use of animals; and (ix) detailed author contributions. Ethical approval for rodent experiments was given by the University of Vermont Institutional Animal Care and Use Committee, The Art of Discovery Institutional Animal Care and Use Committee (TAD-IACUC), Veterinäramt Basel Stadt, the University of Dundee “Welfare and Ethical Use of Animals Committee,” and the University of Georgia Animal Care and Use Committee. Human biological samples were sourced ethically and used under informed consent. ACKNOWLEDGMENTS. We thank the European Synchrotron Radiation Facility for beamtime, highlighting the staff of beamlines BM14 and ID29; Diamond Light Source for beamtime (proposal mx10071); the staff of beamline I24 for assistance with crystal testing and data collection; the entire Seattle Structural Genomics Center for Infectious Disease team; the Division of Biological Chemistry and Drug Discovery Protein Production Team; GlaxoSmithKline for the Tres Campos Antimalarial screening set; the Scottish Blood Transfusion Centre (Ninewells Hospital, Dundee) for providing human erythrocytes; Christoph Fischli and Sibylle Sax at the SwissTPH for technical assistance with the SCID mouse model; and Anja Schäfer for technical assistance with the in vitro antimalarial activity testing. The Art of Discovery thanks Dr. Cristina Eguizabal and the Basque Center of Transfusion and Human Tissues (Galdakao, Spain) and the Bank of Blood and Tissues (Barcelona, Spain) for providing human blood. The University of California, San Diego thanks Jenya Antonova-Koch for help. This work was supported by the Bill and Melinda Gates Foundation through Grant OPP1032548 to the Structure-Guided Drug Discovery Coalition and OPP1134302 (to B.S.). This work was also supported in part from federal funds, from the NIH/National Institute of Allergy and Infectious Diseases Grant R21AI123690 (to K.K.O.) and Contracts HHSN272201200025C and HHSN272201700059C (to P.J.M.); Medicines for Malaria Venture (through access to assays to I.H.G. and through RD/08/2800 to J.B.); Wellcome Trust for support of the X-ray Crystallography Facility 094090, IT support Grant 105021 (to I.H.G.), and Institutional Strategic Support Fund 204816 (to A.V.P.), all at the University of Dundee and for Investigator Award 100993 (to J.B.). 17. Webb MR (1992) A continuous spectrophotometric assay for inorganic phosphate and for measuring phosphate release kinetics in biological systems. Proc Natl Acad Sci USA 89:4884–4887. 18. Placzek S, et al. (2017) BRENDA in 2017: New perspectives and new tools in BRENDA. Nucleic Acids Res 45:D380–D388. 19. Gamo FJ, et al. (2010) Thousands of chemical starting points for antimalarial lead identification. Nature 465:305–310. 20. Khan S, Sharma A, Belrhali H, Yogavel M, Sharma A (2014) Structural basis of malaria parasite lysyl-tRNA synthetase inhibition by cladosporin. J Struct Funct Genomics 15:63–71. 21. Fang P, et al. (2015) Structural basis for specific inhibition of tRNA synthetase by an ATP competitive inhibitor. Chem Biol 22:734–744. 22. Saliba KJ, Kirk K (1999) pH regulation in the intracellular malaria parasite, Plasmodium falciparum. H(+) extrusion via a V-type H(+)-ATPase. J Biol Chem 274:33213–33219. 23. Sanz LM, et al. (2012) P. falciparum in vitro killing rates allow to discriminate between different antimalarial mode-of-action. PLoS One 7:e30949. 24. Baragaña B, et al. (2015) A novel multiple-stage antimalarial agent that inhibits protein synthesis. Nature 522:315–320. 25. Jiménez-Díaz MB, et al. (2009) Improved murine model of malaria using Plasmodium falciparum competent strains and non-myelodepleted NOD-scid IL2Rgammanull mice engrafted with human erythrocytes. Antimicrob Agents Chemother 53:4533–4536. 26. Jumani RS, et al. (2018) A novel piperazine-based drug lead for cryptosporidiosis from the medicines for malaria venture open-access malaria box. Antimicrob Agents Chemother 62:e01505-17. 27. Arnold SLM, et al. (2017) Necessity of bumped kinase inhibitor gastrointestinal ex-posure in treating Cryptosporidium infection. J Infect Dis 216:55–63. 28. Vinayak S, et al. (2015) Genetic modification of the diarrhoeal pathogen Crypto-sporidium parvum. Nature 523:477–480. 29. Manjunatha UH, et al. (2017) A Cryptosporidium PI(4)K inhibitor is a drug candidate for cryptosporidiosis. Nature 546:376–380. 30. Bilokapic S, et al. (2006) Structure of the unusual seryl-tRNA synthetase reveals a distinct zinc-dependent mode of substrate recognition. EMBO J 25:2498–2509. 31. Yaremchuk A, Tukalo M, Grøtli M, Cusack S (2001) A succession of substrate induced conformational changes ensures the amino acid specificity of Thermus thermophilus prolyl-tRNA synthetase: Comparison with histidyl-tRNA synthetase. J Mol Biol 309:989–1002. 32. Zheng H, et al. (2012) Asymmetric total synthesis of cladosporin and isocladosporin. J Org Chem 77:5656–5663. 33. Jain V, Sharma A, Singh G, Yogavel M, Sharma A (2017) Structure-based targeting of orthologous pathogen proteins accelerates antiparasitic drug discovery. ACS Infect Dis 3:281–292. Funding Information: ACKNOWLEDGMENTS. We thank the European Synchrotron Radiation Facility for beamtime, highlighting the staff of beamlines BM14 and ID29; Diamond Light Source for beamtime (proposal mx10071); the staff of beamline I24 for assistance with crystal testing and data collection; the entire Seattle Structural Genomics Center for Infectious Disease team; the Division of Biological Chemistry and Drug Discovery Protein Production Team; GlaxoSmithKline for the Tres Campos Antimalarial screening set; the Scottish Blood Transfusion Centre (Ninewells Hospital, Dundee) for providing human erythrocytes; Christoph Fischli and Sibylle Sax at the SwissTPH for technical assistance with the SCID mouse model; and Anja Schäfer for technical assistance with the in vitro antimalarial activity testing. The Art of Discovery thanks Dr. Cristina Eguizabal and the Basque Center of Transfusion and Human Tissues (Galdakao, Spain) and the Bank of Blood and Tissues (Barcelona, Spain) for providing human blood. The University of California, San Diego thanks Jenya Antonova-Koch for help. This work was supported by the Bill and Melinda Gates Foundation through Grant OPP1032548 to the Structure-Guided Drug Discovery Coalition and OPP1134302 (to B.S.). This work was also supported in part from federal funds, from the NIH/National Institute of Allergy and Infectious Diseases Grant R21AI123690 (to K.K.O.) and Contracts HHSN272201200025C and HHSN272201700059C (to P.J.M.); Medicines for Malaria Venture (through access to assays to I.H.G. and through RD/08/2800 to J.B.); Wellcome Trust for support of the X-ray Crystallography Facility 094090, IT support Grant 105021 (to I.H.G.), and Institutional Strategic Support Fund 204816 (to A.V.P.), all at the University of Dundee and for Investigator Award 100993 (to J.B.).
PY - 2019/4/2
Y1 - 2019/4/2
N2 - Malaria and cryptosporidiosis, caused by apicomplexan parasites, remain major drivers of global child mortality. New drugs for the treatment of malaria and cryptosporidiosis, in particular, are of high priority; however, there are few chemically validated targets. The natural product cladosporin is active against blood- and liver-stage Plasmodium falciparum and Cryptosporidium parvum in cell-culture studies. Target deconvolution in P. falciparum has shown that cladosporin inhibits lysyl-tRNA synthetase (PfKRS1). Here, we report the identification of a series of selective inhibitors of apicomplexan KRSs. Following a biochemical screen, a small-molecule hit was identified and then optimized by using a structure-based approach, supported by structures of both PfKRS1 and C. parvum KRS (CpKRS). In vivo proof of concept was established in an SCID mouse model of malaria, after oral administration (ED 90 = 1.5 mg/kg, once a day for 4 d). Furthermore, we successfully identified an opportunity for pathogen hopping based on the structural homology between PfKRS1 and CpKRS. This series of compounds inhibit CpKRS and C. parvum and Cryptosporidium hominis in culture, and our lead compound shows oral efficacy in two cryptosporidiosis mouse models. X-ray crystallography and molecular dynamics simulations have provided a model to rationalize the selectivity of our compounds for PfKRS1 and CpKRS vs. (human) HsKRS. Our work validates apicomplexan KRSs as promising targets for the development of drugs for malaria and cryptosporidiosis.
AB - Malaria and cryptosporidiosis, caused by apicomplexan parasites, remain major drivers of global child mortality. New drugs for the treatment of malaria and cryptosporidiosis, in particular, are of high priority; however, there are few chemically validated targets. The natural product cladosporin is active against blood- and liver-stage Plasmodium falciparum and Cryptosporidium parvum in cell-culture studies. Target deconvolution in P. falciparum has shown that cladosporin inhibits lysyl-tRNA synthetase (PfKRS1). Here, we report the identification of a series of selective inhibitors of apicomplexan KRSs. Following a biochemical screen, a small-molecule hit was identified and then optimized by using a structure-based approach, supported by structures of both PfKRS1 and C. parvum KRS (CpKRS). In vivo proof of concept was established in an SCID mouse model of malaria, after oral administration (ED 90 = 1.5 mg/kg, once a day for 4 d). Furthermore, we successfully identified an opportunity for pathogen hopping based on the structural homology between PfKRS1 and CpKRS. This series of compounds inhibit CpKRS and C. parvum and Cryptosporidium hominis in culture, and our lead compound shows oral efficacy in two cryptosporidiosis mouse models. X-ray crystallography and molecular dynamics simulations have provided a model to rationalize the selectivity of our compounds for PfKRS1 and CpKRS vs. (human) HsKRS. Our work validates apicomplexan KRSs as promising targets for the development of drugs for malaria and cryptosporidiosis.
KW - Cryptosporidiosis
KW - Malaria
KW - TRNA synthetase
UR - http://www.scopus.com/inward/record.url?scp=85064061317&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85064061317&partnerID=8YFLogxK
U2 - 10.1073/pnas.1814685116
DO - 10.1073/pnas.1814685116
M3 - Article
C2 - 30894487
AN - SCOPUS:85064061317
VL - 116
SP - 7015
EP - 7020
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
SN - 0027-8424
IS - 14
ER -