A Cryptosporidium PI(4)K inhibitor is a drug candidate for cryptosporidiosis
Identifieur interne : 000A55 ( Pmc/Corpus ); précédent : 000A54; suivant : 000A56A Cryptosporidium PI(4)K inhibitor is a drug candidate for cryptosporidiosis
Auteurs : Ujjini H. Manjunatha ; Sumiti Vinayak ; Jennifer A. Zambriski ; Alexander T. Chao ; Tracy Sy ; Christian G. Noble ; Ghislain M. C. Bonamy ; Ravinder R. Kondreddi ; Bin Zou ; Peter Gedeck ; Carrie F. Brooks ; Gillian T. Herbert ; Adam Sateriale ; Jayesh Tandel ; Susan Noh ; Suresh B. Lakshminarayana ; Siau H. Lim ; Laura B. Goodman ; Christophe Bodenreider ; Gu Feng ; Lijun Zhang ; Francesca Blasco ; Juergen Wagner ; F. Joel Leong ; Boris Striepen ; Thierry T. DiaganaSource :
- Nature [ 0028-0836 ] ; 2017.
Abstract
Diarrheal disease is responsible for 8.6% of global child mortality. Recent epidemiological studies found the protozoan parasite
Url:
DOI: 10.1038/nature22337
PubMed: 28562588
PubMed Central: 5473467
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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">A <italic>Cryptosporidium</italic> PI(4)K inhibitor is a drug candidate for cryptosporidiosis</title><author><name sortKey="Manjunatha, Ujjini H" sort="Manjunatha, Ujjini H" uniqKey="Manjunatha U" first="Ujjini H." last="Manjunatha">Ujjini H. Manjunatha</name><affiliation><nlm:aff id="A1">Novartis Institute for Tropical Diseases, Singapore 138670</nlm:aff></affiliation></author><author><name sortKey="Vinayak, Sumiti" sort="Vinayak, Sumiti" uniqKey="Vinayak S" first="Sumiti" last="Vinayak">Sumiti Vinayak</name><affiliation><nlm:aff id="A2">Center for Tropical and Emerging Global Diseases, University of Georgia, Athens, GA, USA</nlm:aff></affiliation></author><author><name sortKey="Zambriski, Jennifer A" sort="Zambriski, Jennifer A" uniqKey="Zambriski J" first="Jennifer A." last="Zambriski">Jennifer A. Zambriski</name><affiliation><nlm:aff id="A3">Washington State University, College of Veterinary Medicine, Paul G. Allen School for Global Animal Health, Pullman, WA, USA</nlm:aff></affiliation></author><author><name sortKey="Chao, Alexander T" sort="Chao, Alexander T" uniqKey="Chao A" first="Alexander T." last="Chao">Alexander T. Chao</name><affiliation><nlm:aff id="A1">Novartis Institute for Tropical Diseases, Singapore 138670</nlm:aff></affiliation></author><author><name sortKey="Sy, Tracy" sort="Sy, Tracy" uniqKey="Sy T" first="Tracy" last="Sy">Tracy Sy</name><affiliation><nlm:aff id="A3">Washington State University, College of Veterinary Medicine, Paul G. Allen School for Global Animal Health, Pullman, WA, USA</nlm:aff></affiliation></author><author><name sortKey="Noble, Christian G" sort="Noble, Christian G" uniqKey="Noble C" first="Christian G." last="Noble">Christian G. Noble</name><affiliation><nlm:aff id="A1">Novartis Institute for Tropical Diseases, Singapore 138670</nlm:aff></affiliation></author><author><name sortKey="Bonamy, Ghislain M C" sort="Bonamy, Ghislain M C" uniqKey="Bonamy G" first="Ghislain M. C." last="Bonamy">Ghislain M. C. Bonamy</name><affiliation><nlm:aff id="A1">Novartis Institute for Tropical Diseases, Singapore 138670</nlm:aff></affiliation></author><author><name sortKey="Kondreddi, Ravinder R" sort="Kondreddi, Ravinder R" uniqKey="Kondreddi R" first="Ravinder R." last="Kondreddi">Ravinder R. Kondreddi</name><affiliation><nlm:aff id="A1">Novartis Institute for Tropical Diseases, Singapore 138670</nlm:aff></affiliation></author><author><name sortKey="Zou, Bin" sort="Zou, Bin" uniqKey="Zou B" first="Bin" last="Zou">Bin Zou</name><affiliation><nlm:aff id="A1">Novartis Institute for Tropical Diseases, Singapore 138670</nlm:aff></affiliation></author><author><name sortKey="Gedeck, Peter" sort="Gedeck, Peter" uniqKey="Gedeck P" first="Peter" last="Gedeck">Peter Gedeck</name><affiliation><nlm:aff id="A1">Novartis Institute for Tropical Diseases, Singapore 138670</nlm:aff></affiliation></author><author><name sortKey="Brooks, Carrie F" sort="Brooks, Carrie F" uniqKey="Brooks C" first="Carrie F." last="Brooks">Carrie F. 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Herbert</name><affiliation><nlm:aff id="A2">Center for Tropical and Emerging Global Diseases, University of Georgia, Athens, GA, USA</nlm:aff></affiliation></author><author><name sortKey="Sateriale, Adam" sort="Sateriale, Adam" uniqKey="Sateriale A" first="Adam" last="Sateriale">Adam Sateriale</name><affiliation><nlm:aff id="A2">Center for Tropical and Emerging Global Diseases, University of Georgia, Athens, GA, USA</nlm:aff></affiliation></author><author><name sortKey="Tandel, Jayesh" sort="Tandel, Jayesh" uniqKey="Tandel J" first="Jayesh" last="Tandel">Jayesh Tandel</name><affiliation><nlm:aff id="A4">Department of Cellular Biology, University of Georgia, Athens, GA, USA</nlm:aff></affiliation></author><author><name sortKey="Noh, Susan" sort="Noh, Susan" uniqKey="Noh S" first="Susan" last="Noh">Susan Noh</name><affiliation><nlm:aff id="A3">Washington State University, College of Veterinary Medicine, Paul G. Allen School for Global Animal Health, Pullman, WA, USA</nlm:aff></affiliation><affiliation><nlm:aff id="A5">USDA-Agricultural Research Service, Animal Disease Research Unit and Washington State University, Department of Veterinary Microbiology and Pathology, Washington Animal Disease Diagnostic Laboratory, Pullman, WA, USA</nlm:aff></affiliation></author><author><name sortKey="Lakshminarayana, Suresh B" sort="Lakshminarayana, Suresh B" uniqKey="Lakshminarayana S" first="Suresh B." last="Lakshminarayana">Suresh B. Lakshminarayana</name><affiliation><nlm:aff id="A1">Novartis Institute for Tropical Diseases, Singapore 138670</nlm:aff></affiliation></author><author><name sortKey="Lim, Siau H" sort="Lim, Siau H" uniqKey="Lim S" first="Siau H." last="Lim">Siau H. Lim</name><affiliation><nlm:aff id="A1">Novartis Institute for Tropical Diseases, Singapore 138670</nlm:aff></affiliation></author><author><name sortKey="Goodman, Laura B" sort="Goodman, Laura B" uniqKey="Goodman L" first="Laura B." last="Goodman">Laura B. Goodman</name><affiliation><nlm:aff id="A6">Cornell University, College of Veterinary Medicine, Department of Population Medicine and Diagnostic Sciences, Ithaca, NY, USA</nlm:aff></affiliation></author><author><name sortKey="Bodenreider, Christophe" sort="Bodenreider, Christophe" uniqKey="Bodenreider C" first="Christophe" last="Bodenreider">Christophe Bodenreider</name><affiliation><nlm:aff id="A1">Novartis Institute for Tropical Diseases, Singapore 138670</nlm:aff></affiliation></author><author><name sortKey="Feng, Gu" sort="Feng, Gu" uniqKey="Feng G" first="Gu" last="Feng">Gu Feng</name><affiliation><nlm:aff id="A1">Novartis Institute for Tropical Diseases, Singapore 138670</nlm:aff></affiliation></author><author><name sortKey="Zhang, Lijun" sort="Zhang, Lijun" uniqKey="Zhang L" first="Lijun" last="Zhang">Lijun Zhang</name><affiliation><nlm:aff id="A7">China Novartis Institute for Biomedical Research, Shanghai 201203, China</nlm:aff></affiliation></author><author><name sortKey="Blasco, Francesca" sort="Blasco, Francesca" uniqKey="Blasco F" first="Francesca" last="Blasco">Francesca Blasco</name><affiliation><nlm:aff id="A1">Novartis Institute for Tropical Diseases, Singapore 138670</nlm:aff></affiliation></author><author><name sortKey="Wagner, Juergen" sort="Wagner, Juergen" uniqKey="Wagner J" first="Juergen" last="Wagner">Juergen Wagner</name><affiliation><nlm:aff id="A1">Novartis Institute for Tropical Diseases, Singapore 138670</nlm:aff></affiliation></author><author><name sortKey="Leong, F Joel" sort="Leong, F Joel" uniqKey="Leong F" first="F. Joel" last="Leong">F. Joel Leong</name><affiliation><nlm:aff id="A1">Novartis Institute for Tropical Diseases, Singapore 138670</nlm:aff></affiliation></author><author><name sortKey="Striepen, Boris" sort="Striepen, Boris" uniqKey="Striepen B" first="Boris" last="Striepen">Boris Striepen</name><affiliation><nlm:aff id="A2">Center for Tropical and Emerging Global Diseases, University of Georgia, Athens, GA, USA</nlm:aff></affiliation><affiliation><nlm:aff id="A4">Department of Cellular Biology, University of Georgia, Athens, GA, USA</nlm:aff></affiliation></author><author><name sortKey="Diagana, Thierry T" sort="Diagana, Thierry T" uniqKey="Diagana T" first="Thierry T." last="Diagana">Thierry T. Diagana</name><affiliation><nlm:aff id="A1">Novartis Institute for Tropical Diseases, Singapore 138670</nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">28562588</idno><idno type="pmc">5473467</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5473467</idno><idno type="RBID">PMC:5473467</idno><idno type="doi">10.1038/nature22337</idno><date when="2017">2017</date><idno type="wicri:Area/Pmc/Corpus">000A55</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000A55</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">A <italic>Cryptosporidium</italic> PI(4)K inhibitor is a drug candidate for cryptosporidiosis</title><author><name sortKey="Manjunatha, Ujjini H" sort="Manjunatha, Ujjini H" uniqKey="Manjunatha U" first="Ujjini H." last="Manjunatha">Ujjini H. Manjunatha</name><affiliation><nlm:aff id="A1">Novartis Institute for Tropical Diseases, Singapore 138670</nlm:aff></affiliation></author><author><name sortKey="Vinayak, Sumiti" sort="Vinayak, Sumiti" uniqKey="Vinayak S" first="Sumiti" last="Vinayak">Sumiti Vinayak</name><affiliation><nlm:aff id="A2">Center for Tropical and Emerging Global Diseases, University of Georgia, Athens, GA, USA</nlm:aff></affiliation></author><author><name sortKey="Zambriski, Jennifer A" sort="Zambriski, Jennifer A" uniqKey="Zambriski J" first="Jennifer A." last="Zambriski">Jennifer A. Zambriski</name><affiliation><nlm:aff id="A3">Washington State University, College of Veterinary Medicine, Paul G. Allen School for Global Animal Health, Pullman, WA, USA</nlm:aff></affiliation></author><author><name sortKey="Chao, Alexander T" sort="Chao, Alexander T" uniqKey="Chao A" first="Alexander T." last="Chao">Alexander T. Chao</name><affiliation><nlm:aff id="A1">Novartis Institute for Tropical Diseases, Singapore 138670</nlm:aff></affiliation></author><author><name sortKey="Sy, Tracy" sort="Sy, Tracy" uniqKey="Sy T" first="Tracy" last="Sy">Tracy Sy</name><affiliation><nlm:aff id="A3">Washington State University, College of Veterinary Medicine, Paul G. Allen School for Global Animal Health, Pullman, WA, USA</nlm:aff></affiliation></author><author><name sortKey="Noble, Christian G" sort="Noble, Christian G" uniqKey="Noble C" first="Christian G." last="Noble">Christian G. Noble</name><affiliation><nlm:aff id="A1">Novartis Institute for Tropical Diseases, Singapore 138670</nlm:aff></affiliation></author><author><name sortKey="Bonamy, Ghislain M C" sort="Bonamy, Ghislain M C" uniqKey="Bonamy G" first="Ghislain M. C." last="Bonamy">Ghislain M. C. Bonamy</name><affiliation><nlm:aff id="A1">Novartis Institute for Tropical Diseases, Singapore 138670</nlm:aff></affiliation></author><author><name sortKey="Kondreddi, Ravinder R" sort="Kondreddi, Ravinder R" uniqKey="Kondreddi R" first="Ravinder R." last="Kondreddi">Ravinder R. Kondreddi</name><affiliation><nlm:aff id="A1">Novartis Institute for Tropical Diseases, Singapore 138670</nlm:aff></affiliation></author><author><name sortKey="Zou, Bin" sort="Zou, Bin" uniqKey="Zou B" first="Bin" last="Zou">Bin Zou</name><affiliation><nlm:aff id="A1">Novartis Institute for Tropical Diseases, Singapore 138670</nlm:aff></affiliation></author><author><name sortKey="Gedeck, Peter" sort="Gedeck, Peter" uniqKey="Gedeck P" first="Peter" last="Gedeck">Peter Gedeck</name><affiliation><nlm:aff id="A1">Novartis Institute for Tropical Diseases, Singapore 138670</nlm:aff></affiliation></author><author><name sortKey="Brooks, Carrie F" sort="Brooks, Carrie F" uniqKey="Brooks C" first="Carrie F." last="Brooks">Carrie F. Brooks</name><affiliation><nlm:aff id="A2">Center for Tropical and Emerging Global Diseases, University of Georgia, Athens, GA, USA</nlm:aff></affiliation></author><author><name sortKey="Herbert, Gillian T" sort="Herbert, Gillian T" uniqKey="Herbert G" first="Gillian T." last="Herbert">Gillian T. Herbert</name><affiliation><nlm:aff id="A2">Center for Tropical and Emerging Global Diseases, University of Georgia, Athens, GA, USA</nlm:aff></affiliation></author><author><name sortKey="Sateriale, Adam" sort="Sateriale, Adam" uniqKey="Sateriale A" first="Adam" last="Sateriale">Adam Sateriale</name><affiliation><nlm:aff id="A2">Center for Tropical and Emerging Global Diseases, University of Georgia, Athens, GA, USA</nlm:aff></affiliation></author><author><name sortKey="Tandel, Jayesh" sort="Tandel, Jayesh" uniqKey="Tandel J" first="Jayesh" last="Tandel">Jayesh Tandel</name><affiliation><nlm:aff id="A4">Department of Cellular Biology, University of Georgia, Athens, GA, USA</nlm:aff></affiliation></author><author><name sortKey="Noh, Susan" sort="Noh, Susan" uniqKey="Noh S" first="Susan" last="Noh">Susan Noh</name><affiliation><nlm:aff id="A3">Washington State University, College of Veterinary Medicine, Paul G. Allen School for Global Animal Health, Pullman, WA, USA</nlm:aff></affiliation><affiliation><nlm:aff id="A5">USDA-Agricultural Research Service, Animal Disease Research Unit and Washington State University, Department of Veterinary Microbiology and Pathology, Washington Animal Disease Diagnostic Laboratory, Pullman, WA, USA</nlm:aff></affiliation></author><author><name sortKey="Lakshminarayana, Suresh B" sort="Lakshminarayana, Suresh B" uniqKey="Lakshminarayana S" first="Suresh B." last="Lakshminarayana">Suresh B. Lakshminarayana</name><affiliation><nlm:aff id="A1">Novartis Institute for Tropical Diseases, Singapore 138670</nlm:aff></affiliation></author><author><name sortKey="Lim, Siau H" sort="Lim, Siau H" uniqKey="Lim S" first="Siau H." last="Lim">Siau H. Lim</name><affiliation><nlm:aff id="A1">Novartis Institute for Tropical Diseases, Singapore 138670</nlm:aff></affiliation></author><author><name sortKey="Goodman, Laura B" sort="Goodman, Laura B" uniqKey="Goodman L" first="Laura B." last="Goodman">Laura B. Goodman</name><affiliation><nlm:aff id="A6">Cornell University, College of Veterinary Medicine, Department of Population Medicine and Diagnostic Sciences, Ithaca, NY, USA</nlm:aff></affiliation></author><author><name sortKey="Bodenreider, Christophe" sort="Bodenreider, Christophe" uniqKey="Bodenreider C" first="Christophe" last="Bodenreider">Christophe Bodenreider</name><affiliation><nlm:aff id="A1">Novartis Institute for Tropical Diseases, Singapore 138670</nlm:aff></affiliation></author><author><name sortKey="Feng, Gu" sort="Feng, Gu" uniqKey="Feng G" first="Gu" last="Feng">Gu Feng</name><affiliation><nlm:aff id="A1">Novartis Institute for Tropical Diseases, Singapore 138670</nlm:aff></affiliation></author><author><name sortKey="Zhang, Lijun" sort="Zhang, Lijun" uniqKey="Zhang L" first="Lijun" last="Zhang">Lijun Zhang</name><affiliation><nlm:aff id="A7">China Novartis Institute for Biomedical Research, Shanghai 201203, China</nlm:aff></affiliation></author><author><name sortKey="Blasco, Francesca" sort="Blasco, Francesca" uniqKey="Blasco F" first="Francesca" last="Blasco">Francesca Blasco</name><affiliation><nlm:aff id="A1">Novartis Institute for Tropical Diseases, Singapore 138670</nlm:aff></affiliation></author><author><name sortKey="Wagner, Juergen" sort="Wagner, Juergen" uniqKey="Wagner J" first="Juergen" last="Wagner">Juergen Wagner</name><affiliation><nlm:aff id="A1">Novartis Institute for Tropical Diseases, Singapore 138670</nlm:aff></affiliation></author><author><name sortKey="Leong, F Joel" sort="Leong, F Joel" uniqKey="Leong F" first="F. Joel" last="Leong">F. Joel Leong</name><affiliation><nlm:aff id="A1">Novartis Institute for Tropical Diseases, Singapore 138670</nlm:aff></affiliation></author><author><name sortKey="Striepen, Boris" sort="Striepen, Boris" uniqKey="Striepen B" first="Boris" last="Striepen">Boris Striepen</name><affiliation><nlm:aff id="A2">Center for Tropical and Emerging Global Diseases, University of Georgia, Athens, GA, USA</nlm:aff></affiliation><affiliation><nlm:aff id="A4">Department of Cellular Biology, University of Georgia, Athens, GA, USA</nlm:aff></affiliation></author><author><name sortKey="Diagana, Thierry T" sort="Diagana, Thierry T" uniqKey="Diagana T" first="Thierry T." last="Diagana">Thierry T. Diagana</name><affiliation><nlm:aff id="A1">Novartis Institute for Tropical Diseases, Singapore 138670</nlm:aff></affiliation></author></analytic><series><title level="j">Nature</title><idno type="ISSN">0028-0836</idno><idno type="eISSN">1476-4687</idno><imprint><date when="2017">2017</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p id="P1">Diarrheal disease is responsible for 8.6% of global child mortality. Recent epidemiological studies found the protozoan parasite <italic>Cryptosporidium</italic> to be a leading cause of pediatric diarrhea with particularly grave impact on infants and immunocompromised individuals. There is neither a vaccine nor effective treatment. We establish a drug discovery process built on scalable phenotypic assays and mouse models that takes advantage of transgenic parasites. Screening a library of compounds with anti-parasitic activity we identified pyrazolopyridines as inhibitors of <italic>Cryptosporidium parvum</italic> and <italic>C. hominis</italic>. Oral treatment with the pyrazolopyridine KDU731 results in potent reduction in intestinal infection of immunocompromised mice. Treatment also leads to rapid resolution of diarrhea and dehydration in neonatal calves, a clinical model of cryptosporidiosis that closely resembles human infection. Our results suggest the <italic>Cryptosporidium</italic> lipid kinase PI(4)K as a target for pyrazolopyridines and warrant further preclinical evaluation of KDU731 as a drug candidate for the treatment of cryptosporidiosis.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Liu, L" uniqKey="Liu L">L Liu</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Kotloff, Kl" uniqKey="Kotloff K">KL Kotloff</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Platts Mills, Ja" uniqKey="Platts Mills J">JA Platts-Mills</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Checkley, W" uniqKey="Checkley W">W Checkley</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Amadi, B" uniqKey="Amadi B">B Amadi</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Amadi, B" uniqKey="Amadi B">B 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<front><journal-meta><journal-id journal-id-type="nlm-journal-id">0410462</journal-id><journal-id journal-id-type="pubmed-jr-id">6011</journal-id><journal-id journal-id-type="nlm-ta">Nature</journal-id><journal-id journal-id-type="iso-abbrev">Nature</journal-id><journal-title-group><journal-title>Nature</journal-title></journal-title-group><issn pub-type="ppub">0028-0836</issn><issn pub-type="epub">1476-4687</issn></journal-meta><article-meta><article-id pub-id-type="pmid">28562588</article-id><article-id pub-id-type="pmc">5473467</article-id><article-id pub-id-type="doi">10.1038/nature22337</article-id><article-id pub-id-type="manuscript">EMS72141</article-id><article-categories><subj-group subj-group-type="heading"><subject>Article</subject></subj-group></article-categories><title-group><article-title>A <italic>Cryptosporidium</italic> PI(4)K inhibitor is a drug candidate for cryptosporidiosis</article-title></title-group><contrib-group><contrib contrib-type="author" equal-contrib="yes"><name><surname>Manjunatha</surname><given-names>Ujjini H.</given-names></name><xref ref-type="aff" rid="A1">1</xref></contrib><contrib contrib-type="author" equal-contrib="yes"><name><surname>Vinayak</surname><given-names>Sumiti</given-names></name><xref ref-type="aff" rid="A2">2</xref></contrib><contrib contrib-type="author" equal-contrib="yes"><name><surname>Zambriski</surname><given-names>Jennifer A.</given-names></name><xref ref-type="aff" rid="A3">3</xref><xref ref-type="corresp" rid="CR1">*</xref></contrib><contrib contrib-type="author"><name><surname>Chao</surname><given-names>Alexander T.</given-names></name><xref ref-type="aff" rid="A1">1</xref></contrib><contrib contrib-type="author"><name><surname>Sy</surname><given-names>Tracy</given-names></name><xref ref-type="aff" rid="A3">3</xref></contrib><contrib contrib-type="author"><name><surname>Noble</surname><given-names>Christian G.</given-names></name><xref ref-type="aff" rid="A1">1</xref></contrib><contrib contrib-type="author"><name><surname>Bonamy</surname><given-names>Ghislain M.C.</given-names></name><xref ref-type="aff" rid="A1">1</xref></contrib><contrib contrib-type="author"><name><surname>Kondreddi</surname><given-names>Ravinder R.</given-names></name><xref ref-type="aff" rid="A1">1</xref></contrib><contrib contrib-type="author"><name><surname>Zou</surname><given-names>Bin</given-names></name><xref ref-type="aff" rid="A1">1</xref></contrib><contrib contrib-type="author"><name><surname>Gedeck</surname><given-names>Peter</given-names></name><xref ref-type="aff" rid="A1">1</xref></contrib><contrib contrib-type="author"><name><surname>Brooks</surname><given-names>Carrie F.</given-names></name><xref ref-type="aff" rid="A2">2</xref></contrib><contrib contrib-type="author"><name><surname>Herbert</surname><given-names>Gillian T.</given-names></name><xref ref-type="aff" rid="A2">2</xref></contrib><contrib contrib-type="author"><name><surname>Sateriale</surname><given-names>Adam</given-names></name><xref ref-type="aff" rid="A2">2</xref></contrib><contrib contrib-type="author"><name><surname>Tandel</surname><given-names>Jayesh</given-names></name><xref ref-type="aff" rid="A4">4</xref></contrib><contrib contrib-type="author"><name><surname>Noh</surname><given-names>Susan</given-names></name><xref ref-type="aff" rid="A3">3</xref><xref ref-type="aff" rid="A5">5</xref></contrib><contrib contrib-type="author"><name><surname>Lakshminarayana</surname><given-names>Suresh B.</given-names></name><xref ref-type="aff" rid="A1">1</xref></contrib><contrib contrib-type="author"><name><surname>Lim</surname><given-names>Siau H.</given-names></name><xref ref-type="aff" rid="A1">1</xref></contrib><contrib contrib-type="author"><name><surname>Goodman</surname><given-names>Laura B.</given-names></name><xref ref-type="aff" rid="A6">6</xref></contrib><contrib contrib-type="author"><name><surname>Bodenreider</surname><given-names>Christophe</given-names></name><xref ref-type="aff" rid="A1">1</xref></contrib><contrib contrib-type="author"><name><surname>Feng</surname><given-names>Gu</given-names></name><xref ref-type="aff" rid="A1">1</xref></contrib><contrib contrib-type="author"><name><surname>Zhang</surname><given-names>Lijun</given-names></name><xref ref-type="aff" rid="A7">7</xref></contrib><contrib contrib-type="author"><name><surname>Blasco</surname><given-names>Francesca</given-names></name><xref ref-type="aff" rid="A1">1</xref></contrib><contrib contrib-type="author"><name><surname>Wagner</surname><given-names>Juergen</given-names></name><xref ref-type="aff" rid="A1">1</xref></contrib><contrib contrib-type="author"><name><surname>Leong</surname><given-names>F. Joel</given-names></name><xref ref-type="aff" rid="A1">1</xref></contrib><contrib contrib-type="author"><name><surname>Striepen</surname><given-names>Boris</given-names></name><xref ref-type="aff" rid="A2">2</xref><xref ref-type="aff" rid="A4">4</xref><xref ref-type="corresp" rid="CR1">*</xref></contrib><contrib contrib-type="author"><name><surname>Diagana</surname><given-names>Thierry T.</given-names></name><xref ref-type="aff" rid="A1">1</xref><xref ref-type="corresp" rid="CR1">*</xref></contrib></contrib-group><aff id="A1"><label>1</label>Novartis Institute for Tropical Diseases, Singapore 138670</aff><aff id="A2"><label>2</label>Center for Tropical and Emerging Global Diseases, University of Georgia, Athens, GA, USA</aff><aff id="A3"><label>3</label>Washington State University, College of Veterinary Medicine, Paul G. Allen School for Global Animal Health, Pullman, WA, USA</aff><aff id="A4"><label>4</label>Department of Cellular Biology, University of Georgia, Athens, GA, USA</aff><aff id="A5"><label>5</label>USDA-Agricultural Research Service, Animal Disease Research Unit and Washington State University, Department of Veterinary Microbiology and Pathology, Washington Animal Disease Diagnostic Laboratory, Pullman, WA, USA</aff><aff id="A6"><label>6</label>Cornell University, College of Veterinary Medicine, Department of Population Medicine and Diagnostic Sciences, Ithaca, NY, USA</aff><aff id="A7"><label>7</label>China Novartis Institute for Biomedical Research, Shanghai 201203, China</aff><author-notes><corresp id="CR1"><label>*</label>Correspondence and requests for materials should be addressed to<email>striepen@uga.edu</email>; <email>jzambriski@vetmed.wsu.edu</email>; <email>thierry.diagana@novartis.com</email>.</corresp></author-notes><pub-date pub-type="nihms-submitted"><day>5</day><month>6</month><year>2017</year></pub-date><pub-date pub-type="epub"><day>31</day><month>5</month><year>2017</year></pub-date><pub-date pub-type="ppub"><day>15</day><month>6</month><year>2017</year></pub-date><pub-date pub-type="pmc-release"><day>30</day><month>11</month><year>2017</year></pub-date><volume>546</volume><issue>7658</issue><fpage>376</fpage><lpage>380</lpage><pmc-comment>elocation-id from pubmed: 10.1038/nature22337</pmc-comment>
<permissions><license><license-p>Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:<ext-link ext-link-type="uri" xlink:href="http://www.nature.com/authors/editorial_policies/license.html#terms">http://www.nature.com/authors/editorial_policies/license.html#terms</ext-link></license-p></license></permissions><abstract><p id="P1">Diarrheal disease is responsible for 8.6% of global child mortality. Recent epidemiological studies found the protozoan parasite <italic>Cryptosporidium</italic> to be a leading cause of pediatric diarrhea with particularly grave impact on infants and immunocompromised individuals. There is neither a vaccine nor effective treatment. We establish a drug discovery process built on scalable phenotypic assays and mouse models that takes advantage of transgenic parasites. Screening a library of compounds with anti-parasitic activity we identified pyrazolopyridines as inhibitors of <italic>Cryptosporidium parvum</italic> and <italic>C. hominis</italic>. Oral treatment with the pyrazolopyridine KDU731 results in potent reduction in intestinal infection of immunocompromised mice. Treatment also leads to rapid resolution of diarrhea and dehydration in neonatal calves, a clinical model of cryptosporidiosis that closely resembles human infection. Our results suggest the <italic>Cryptosporidium</italic> lipid kinase PI(4)K as a target for pyrazolopyridines and warrant further preclinical evaluation of KDU731 as a drug candidate for the treatment of cryptosporidiosis.</p></abstract></article-meta></front><body><p id="P2">Infections that cause diarrhea are responsible for nearly 800,000 deaths every year, mostly among young children in resource poor settings<xref rid="R1" ref-type="bibr">1</xref>. Recently, the apicomplexan parasite <italic>Cryptosporidium</italic> was found to be one of the leading causes of infectious diarrhea in children <xref rid="R2" ref-type="bibr">2</xref>,<xref rid="R3" ref-type="bibr">3</xref>and Infection with this parasite is strongly associated with mortality, growth stunting, and developmental deficits<xref rid="R4" ref-type="bibr">4</xref>. The major human pathogens causing cryptosporidiosis, <italic>C. hominis</italic> and <italic>C. parvum</italic>, infect the epithelial cells of the intestine and, through a mechanism that is not fully understood, trigger severe watery diarrheal symptoms. These are particularly long-lasting and often life-threatening in malnourished and immunocompromised children<xref rid="R4" ref-type="bibr">4</xref>. Nitazoxanide, the only approved drug for the treatment of cryptosporidiosis has limited efficacy in those most vulnerable patient populations<xref rid="R5" ref-type="bibr">5</xref>,<xref rid="R6" ref-type="bibr">6</xref>. Cryptosporidiosis is also a well-recognized opportunistic infection in adult AIDS and transplant patients<xref rid="R4" ref-type="bibr">4</xref>. Infection occurs through ingestion of the spore-like oocyst stage which shows remarkable resistance to water chlorination. Therefore, even in countries that apply advanced water treatment infection is common, <italic>Cryptosporidium</italic> is the cause of 50% or disease outbreaks linked to recreational water use in the United States. The search for cryptosporidiosis therapeutics has been hindered by the many technical challenges faced when working with this notoriously intractable parasite<xref rid="R7" ref-type="bibr">7</xref>. Here we establish a cryptosporidiosis drug discovery screening process combining phenotypic in vitro assays with novel animal models that take advantage of transgenic parasites<xref rid="R8" ref-type="bibr">8</xref>.</p><sec id="S1"><title><italic>Cryptosporidium</italic> compound screen</title><p id="P3">To discover new treatments for cryptosporidiosis we assembled a set of 6220 compounds with known activity against various protozoan parasites and screened against <italic>C. parvum</italic> in a high content imaging infection assay in HCT-8 cells (see <xref ref-type="supplementary-material" rid="SM">supplementary materials</xref>). Notably, many anti-malarials (spiroindolones<xref rid="R9" ref-type="bibr">9</xref>, cyclomarins<xref rid="R10" ref-type="bibr">10</xref> and imidazolopiperazines<xref rid="R11" ref-type="bibr">11</xref>) lacked activity against <italic>Cryptosporidium</italic>, however 154 compounds showed >60% growth inhibition at 5 μM. Secondary screening using a novel cytopathic effect (CPE) based <italic>C. parvum</italic> assay confirmed several scaffolds, with imidazopyrazines<xref rid="R12" ref-type="bibr">12</xref>,<xref rid="R13" ref-type="bibr">13</xref> and pyrazolopyridines<xref rid="R14" ref-type="bibr">14</xref> showing sub-micromolar cellular activity (<xref ref-type="fig" rid="F1">Fig. 1a-d</xref> and <xref ref-type="table" rid="T1">Table 1</xref>, structures provided in <xref ref-type="fig" rid="F4">Extended Data Fig.1</xref>). We evaluated ˜200 pyrazolopyridine analogs and found correlation between activity against <italic>C. parvum</italic> and <italic>Plasmodium falciparum</italic> (R<sup>2</sup>=0.702, <xref ref-type="fig" rid="F1">Fig. 1d</xref>) suggesting that the mechanism of action of pyrazolopyridines is conserved between these two parasites. No such correlation was observed with toxicity against HepG2 (R<sup>2</sup>=0.071) (<xref ref-type="fig" rid="F5">Extended Data Fig. 2</xref>). <italic>C. hominis</italic> is responsible for the majority of clinical infections<xref rid="R15" ref-type="bibr">15</xref>. We thus evaluated a subset of pyrazolopyridine analogs against <italic>C. hominis</italic>, and found potency comparable to <italic>C. parvum</italic> (R<sup>2</sup> =0.872, <xref ref-type="fig" rid="F1">Fig. 1e</xref> and <xref ref-type="table" rid="T1">Table 1</xref>).</p></sec><sec id="S2"><title>Pyrazolopyridines inhibit CpPI(4)K</title><p id="P4">Knowing that pyrazolopyridines and imidazopyrazines exert their antimalarial activity through inhibition of the <italic>Plasmodium</italic> lipid kinase PI(4)K <xref rid="R12" ref-type="bibr">12</xref>,<xref rid="R16" ref-type="bibr">16</xref>, we searched for potential <italic>Cryptosporidium</italic> orthologs. The genomes of both <italic>C. parvum</italic> and <italic>C. hominis</italic> encode multiple putative lipid kinases and the PI(4)K catalytic domain of cgd8_4500 and its <italic>C. hominis</italic> homolog Chro.80518 show 71.8% amino acid sequence similarity to <italic>P. falciparum</italic> PI(4)K. We expressed cgd8_4500 in insect cells and purified the protein (CpPI(4)K) which displays phosphatidylinositol (PI) kinase activity with a K<sub>m</sub> for ATP and PI of 3 and 0.4 μM, respectively (<xref ref-type="fig" rid="F6">Extended Data Fig. 3</xref>). Using this assay, we showed that the imidazopyrazine KDU691 and the pyrazolopyridine KDU731 are potent inhibitors of CpPI(4)K enzymatic activity with IC<sub>50</sub> values of 17 and 25 nM, respectively (<xref ref-type="fig" rid="F1">Fig. 1f</xref>). Both compounds have a >50 fold selectivity window against the human PI(4)K IIIβ homologue (<xref ref-type="table" rid="T2">Extended Data Table 1</xref>).</p><p id="P5">When we measured diverse imidazopyrazine and pyrazolopyridine analogs against CpPI(4)K enzymatic activity and <italic>C. parvum</italic> growth in cells we found tight correlation (R<sup>2</sup>=0.902) (<xref ref-type="fig" rid="F1">Fig. 1g</xref>), suggesting that the anti-<italic>Cryptosporidium</italic> activity is directly mediated by CpPI(4)K inhibition. Further genetic and structural insights are needed to unambiguously establish PI4(K) as the target. However, we note that the <italic>Plasmodium</italic> PI(4)K inhibitors diaryl-aminopyridine (MMV390048)<xref rid="R17" ref-type="bibr">17</xref> and quinoxaline (BQR695)<xref rid="R12" ref-type="bibr">12</xref> which are inactive against <italic>C. parvum</italic> and <italic>C. hominis</italic> do not inhibit CpPI(4)K enzymatic activity (<xref ref-type="fig" rid="F1">Fig. 1f</xref>). Finally, mechanistic studies revealed an increased K<sub>m</sub> for ATP in the presence of KDU731 (<xref ref-type="fig" rid="F1">Fig. 1h</xref>), suggesting ATP-competitive inhibition similarly to what was previously observed for the <italic>Plasmodium</italic> enzyme<xref rid="R12" ref-type="bibr">12</xref>. Additional structure-activity relationship analysis of pyrazolopyridines against <italic>Cryptosporidium</italic> is found in the <xref ref-type="supplementary-material" rid="SD6">supplementary information</xref>. Taken together our data suggest that pyrazolopyridines are inhibitors of CpPI(4)K that bind to the ATP-binding site of the enzyme with a favorable selectivity window over human PI(4)K.</p></sec><sec id="S3"><title>Pharmacokinetic properties of KDU731</title><p id="P6">The most urgent need for effective cryptosporidiosis treatment is among children under the age of two. A very safe drug profile is thus a key component of the target product profile<xref rid="R18" ref-type="bibr">18</xref>,<xref rid="R19" ref-type="bibr">19</xref>. KDU731 has a selectivity index of more than 100 (CC<sub>50</sub> HepG2 = 15.6 μM and CpCPE EC<sub>50</sub>= 0.1 μM) (<xref ref-type="table" rid="T2">Extended Data Table 1</xref>). In a battery of safety pharmacology assays, the compound does not show intrinsic risks for cardiotoxicity, mutagenicity, clastogenicity, or phototoxicity and it does not significantly bind to a panel of human anti-targets (<xref ref-type="table" rid="T2">Extended Data Table 1</xref> and <xref ref-type="table" rid="T3">2</xref>). KDU731 safety was further evaluated in a 2-week toxicology study in rats using a solid dispersion formulation to maximize systemic exposure (˜25 fold). In this study, no significant histopathological changes and only minor changes in clinical chemistry and hematology were observed (slight elevation of chloride and phosphate ions and increase in red blood cells, see <xref ref-type="supplementary-material" rid="SM">supplementary materials</xref>). Importantly, no hematopoietic toxicity related to potential inhibition of PI3K or PI4K lipid kinases was observed, suggesting that the <italic>in vitro</italic> selectivity of KDU731 translated to lack of host toxicity <italic>in vivo</italic> (<xref ref-type="table" rid="T2">Extended Data Table 1</xref>). Children with cryptosporidiosis in resource limited settings frequently suffer co-morbidities including HIV, TB, and other infections, anti-cryptosporidial drugs will thus likely be used along with other medications and the risk of drug-drug interactions needs to be minimized. Consistent with the requirement, KDU731 did not inhibit any of the major Cytochrome P450 isoenzymes and did not induce human pregnane X receptor (<xref ref-type="table" rid="T2">Extended Data Table 1</xref>).</p><p id="P7">KDU731 pharmacokinetics, metabolism and distribution was examined in mice, rats and rhesus monkeys. Upon intravenous administration, KDU731 displayed a low to moderate volume of distribution (Vss = 1.12 and 2.15 liter/kg), a low total systemic clearance (CL= 6.8 and 16 ml/min /kg) and a half-life ranging from 1-4 hours (<xref ref-type="table" rid="T4">Extended Data Table 3</xref>). KDU731 orally-administered in suspension formulation displayed a moderate half-life (T<sub>1/2</sub> = 2-4 h) in all three species. KDU731 was selected as a drug candidate because it shows moderate-to-low oral bioavailability (37% in rodents, 9% in non-human primates). As <italic>Cryptosporidium</italic> primarily infects the intestinal epithelium, we reasoned that systemic exposure may not be required for efficacy and that limiting systemic exposure may further enhance the safety margin of a cryptosporidiosis drug.</p></sec><sec id="S4"><title>KDU731 treatment of infected mice</title><p id="P8">Current assessment of cryptosporidiosis treatments relies on laborious methods to quantify the parasite in animals. We used genetically-modified parasites to build more facile models. We established the EC<sub>50</sub> of KDU731 for transgenic parasites<xref rid="R8" ref-type="bibr">8</xref> in HCT-8 using Nanoluciferase (Nluc) as a readout and found it comparable to wildtype parasites (<xref ref-type="fig" rid="F7">Extended Data Fig. 4a</xref>). Next, we inoculated 6-8 week old C57BL/6 IFN-γ-knockout mice with 10,000 oocysts and monitored infection following parasite-derived luciferase activity in the feces (<xref ref-type="fig" rid="F2">Fig. 2a</xref>). Infected mice were treated orally with KDU731 in suspension formulation and parasite load was measured in the pooled feces by Nluc and quantitative PCR assay with a high degree of correlation (Spearman coefficient=0.786; two- tailed <italic>P</italic>=0.048). The Nluc assay has a small sample requirement (20 mg) and thus could be conducted on individual mice as well as pooled feces (unpaired <italic>t</italic>-test comparing means of individual and pooled readings showed no significant difference, two-tailed <italic>P</italic>=0.3862). Importantly, KDU731 dramatically reduced oocyst shedding regardless of assay method (<xref ref-type="fig" rid="F2">Fig. 2b</xref> and <xref ref-type="fig" rid="F7">Extended Data Fig. 4b and c</xref>, non-parametric Kruskal-Wallis test, pooled Nluc two-tailed <italic>P</italic> <0.001; fecal qPCR two-tailed <italic>P</italic>=0.0042). In a modified design, KDU731 (10 mg/kg) treatment was initiated at the peak of infection again resulting in significant reduction of oocyst shedding (<xref ref-type="fig" rid="F2">Fig. 2a and c</xref>). At the end of the treatment period three mice per group were killed and intestines were processed for histology. We did not observe parasites in the intestines of treated animals while those that received vehicle showed substantial infection (<xref ref-type="fig" rid="F2">Fig. 2d</xref>).</p><p id="P9">To dynamically evaluate the impact of treatment on parasite load directly in the intestinal tissue, we engineered a new reporter parasite amenable to detection by whole-animal imaging. Mice were infected with parasites expressing red-shifted firefly luciferase<xref rid="R20" ref-type="bibr">20</xref> (Fluc) and imaged following D-luciferin injection. Intestinal tissue load was quantified by PCR and we found strong correlation with tissue luminescence (R<sup>2</sup>= 0.8, <xref ref-type="fig" rid="F9">Extended Data Fig 6 b,c</xref>). KDU731 treatment of mice infected with Fluc parasites was initiated on day 7 when all mice registered high levels of abdominal luciferase activity and mice were imaged daily. Tissue load dropped markedly within 2 days of treatment, and fell below the limit of detection after five days (<xref ref-type="fig" rid="F2">Fig. 2e and g</xref>). Changes in luminescence imaging were mirrored by fecal oocyst shedding quantified by PCR (Spearman coefficient =0.840, two-tailed P= 0.0006, <xref ref-type="fig" rid="F9">Extended Data Fig 6a</xref>). For comparison we evaluated nitazoxanide at 100 mg/kg and observed no change (<xref ref-type="fig" rid="F2">Fig. 2f</xref> and <xref ref-type="fig" rid="F8">Extended Data Fig. 5</xref>), consistent with previous reports on immunosuppressed hosts<xref rid="R5" ref-type="bibr">5</xref>,<xref rid="R21" ref-type="bibr">21</xref>.</p></sec><sec id="S5"><title>Treatment of neonatal calves</title><p id="P10">Newborn calves are naturally susceptible to <italic>C. parvum</italic> infection resulting in fecal oocyst shedding, profuse watery diarrhea and severe dehydration, which closely mirrors human symptomology<xref rid="R22" ref-type="bibr">22</xref>,<xref rid="R23" ref-type="bibr">23</xref>. We challenged 13 neonatal calves with 5 x 10<sup>7</sup> oocysts. Over the duration of enrollment, a complete physical examination was performed every 12 h and clinical data including appetite, mentation, fecal consistency, and hydration status were recorded (<xref ref-type="fig" rid="F3">Fig. 3</xref>) to assign a clinical a score on a scale of 1 (normal) to 3 (severe) in accordance with previously described methods (see rubric in <xref ref-type="supplementary-material" rid="SD6">supplementary information</xref>)<xref rid="R22" ref-type="bibr">22</xref>–<xref rid="R24" ref-type="bibr">24</xref>. Fecal oocyst shedding was enumerated every 24 h by qPCR. The treatment group (n = 7) was subjected to oral treatment with 5 mg/kg KDU731 in suspension formulation every 12 h for 7 days. Treatment was initiated upon development of fulminant diarrhea and fecal oocyst shedding, between day 2 and day 4 post-infection.</p><p id="P11">All calves tolerated KDU731 treatment without compound related abnormalities and treated calves shed significantly fewer oocysts than vehicle treated calves within 3 days of treatment (p <0.0001 on day 3) (<xref ref-type="fig" rid="F3">Fig. 3b</xref>). Treated calves suffered fewer days of severe diarrhea (two-tailed <italic>P</italic> = 0.006) and were significantly less dehydrated (two-tailed <italic>P</italic> < 0.0001) than controls (<xref ref-type="fig" rid="F3">Fig. 3c and d</xref>). Resolution of clinical signs commenced as early as 24 h after treatment was initiated. Within 48 h of treatment 6 out of 7 calves showed no signs of dehydration and within 72 h of treatment 5 of 6 calves had resolution of severe diarrhea (<xref ref-type="fig" rid="F10">Extended Data Fig. 7</xref>). KDU731 displayed limited systemic exposure in calves with a C<sub>max</sub> and AUC of 0.228 μM and 1.9 μM.h respectively (<xref ref-type="fig" rid="F3">Fig. 3e</xref> and <xref ref-type="table" rid="T4">Extended Data Table 3</xref>) confirming that substantial systemic exposure may not be required for parasite clearance and resolution of clinical illness.</p></sec><sec sec-type="conclusions" id="S6"><title>Conclusions</title><p id="P12">Our studies define the pyrazolopyridine KDU731 as a promising anti-cryptosporidial drug candidate active against both <italic>C. parvum</italic> and <italic>C. hominis.</italic> Unlike nitazoxanide, KDU731 demonstrated <italic>in vivo</italic> efficacy in immunocompromised mice. Additionally, treatment in neonatal calves, which closely matches the pathophysiological and pharmacological challenges faced in the treatment of young malnourished children, led to significant decrease in parasite shedding and rapid resolution of diarrhea and dehydration. Our lead candidate, KDU731, displays good anti-cryptosporidial activity and meets a broad range of safety and pharmacology criteria required for a much needed novel cryptosporidiosis therapeutic intervention. Further safety and pharmacological preclinical evaluation is currently ongoing to support the initiation of human clinical trials.</p></sec><sec sec-type="methods" id="S7" specific-use="web-only"><title>Methods</title><sec id="S8"><title>Compounds</title><p id="P13">KDU691, BQR695 and MMV390048 were synthesized as described earlier<xref rid="R12" ref-type="bibr">12</xref>,<xref rid="R25" ref-type="bibr">25</xref>. Synthesis of KDU731 and other pyrazolopyridine analogs is described in <xref ref-type="supplementary-material" rid="SD6">supplementary information</xref> and a patent application<xref rid="R14" ref-type="bibr">14</xref>.</p></sec><sec id="S9"><title>Cells and Parasites</title><p id="P14">Human ileocecal adenocarcinoma cells (HCT-8) were purchased from ATCC (#CCL-34), tested for mycoplasma (at NITD, not at UGA) and maintained in RPMI-1640 medium supplemented with 10 % heat-inactivated horse serum. <italic>C. parvum</italic> and <italic>C. hominis</italic> oocysts were purchased from the Sterling Laboratory, University of Arizona and Tufts University Cummings School of Veterinary Medicine. Oocysts were used in infection experiments less than three months from the date of shedding. Excystation and infection protocols followed established methods with modifications<xref rid="R26" ref-type="bibr">26</xref>. Oocysts were treated with 10 mM hydrochloric acid (HCl) in 1× Hank’s Balanced Salt Solution (HBSS) 10 minutes with agitation at 37°C, then washed twice with non-acidic 1× HBSS. Oocysts were excysted at a density of 10<sup>6</sup> primed oocysts/μL in parasite infection medium (1:1 Leibovitz’s L-15 medium and UltraCULTURE medium supplemented with 2 mM sodium taurocholate, 10 % heat-inactivated horse serum, and 200 μM L-ascorbic acid). All dilutions for subsequent assays were performed in parasite infection medium without sodium taurocholate.</p></sec><sec id="S10"><title>High Content Imaging (HCI) immunofluorescence <italic>Cryptosporidium</italic> screening assay</title><p id="P15">A previously reported <italic>C. parvum</italic> HCI assay in HCT-8 cells was used with modifications <xref rid="R27" ref-type="bibr">27</xref>,<xref rid="R28" ref-type="bibr">28</xref> and is described in detail in the <xref ref-type="supplementary-material" rid="SD6">Supplementary Information</xref>.</p></sec><sec id="S11"><title><italic>Cryptosporidium</italic> EC<sub>50</sub> determination</title><p id="P16">High Content Imaging HCT-8 cell infection assays with <italic>C. parvum</italic> and <italic>C. hominis</italic> were conducted following established protocols<xref rid="R27" ref-type="bibr">27</xref> in 384-well flat black clear-bottom plates using an Opera QEHS (PerkinElmer™) Imaging system. We also developed a cytopathic effect (CPE) based assay monitoring host cell viability using CTG reagent (Promega) that does not require staining or imaging. Further details on EC<sub>50</sub> determination for <italic>C. parvum</italic> and <italic>C. hominis</italic> using both assays in a 10-point dose-response with 3-fold compound dilution to be described elsewhere<xref rid="R29" ref-type="bibr">29</xref>. The EC<sub>50</sub> of KDU731 was also determined using Nluc expressing parasites<xref rid="R8" ref-type="bibr">8</xref>. Briefly, HCT-8 cells were infected with purified Nluc expressing parasites (1000 oocysts per well) and incubated with different concentrations of KDU731 for 48 hours. Culture supernatant was removed from the wells and 200 ul of NanoGlo lysis buffer containing 1:50 of NanoGlo substrate (Promega Corporation) was added to the wells. Lysate was transferred to 96 well plates and luminescence was measured on a Synergy H4 Hybrid Microplate Reader (BioTek Instruments)</p></sec><sec id="S12"><title>Host cell cytotoxicity assay</title><p id="P17">Cytotoxicity against HepG2 ATCC #CRL-10741 was determined as previously described<xref rid="R30" ref-type="bibr">30</xref>. Cells were seeded into 384 well plates at 400 cells per well, incubated at 37°C for 24 hours and exposed to three-fold serially-diluted compounds for 96 hours. Cell viability was monitored using the Cell Counting Kit-8.</p></sec><sec id="S13"><title>Baculovirus expression and purification of <italic>C. parvum</italic> phosphatidylinositol 4-kinase</title><p id="P18">The full-length coding sequence of <italic>C. parvum</italic> PI(4)K (cgd8_4500) was codon-optimized for baculovirus expression, synthesized and cloned into pFastBac-HTb (Invitrogen) in frame with an amino-terminal polyhistidine tag using the <italic>Bam</italic>HI and <italic>Hind</italic>III restriction sites. Recombinant pFastBacHTb-CpPI(4)K bacmid clones were generated by site-specific transposition in <italic>E. coli</italic> DH10Bac (Invitrogen). The bacmid sequence was confirmed by direct DNA sequencing. Bacmid isolation, transfection and selection of the recombinant viruses was performed according to the manufacturer’s protocol (Bac-to-Bac system, Invitrogen). SF9 cells, cultured in SF-900 III serum-free medium, were transfected with recombinant baculovirus at 1/200 (v/v) and incubated at 27 °C for 72 h. Pellets were collected after centrifugation and re-suspended in cell lysis buffer (20 mM Tris-HCl, pH 7.5, 300 mM NaCl, 1mM DTT, 20mM imidazole, 0.01% Triton X-100 and 1× complete protease inhibitor cocktail without EDTA (Roche Diagnostics). The cell suspension was lysed by sonication and clarified supernatant was loaded onto a 1 ml HisTrap affinity column (GE Healthcare) pre-equilibrated with buffer A (20 mM Tris-HCl, pH 7.5, 300 mM NaCl, 1mM DTT, 20mM imidazole, and 1× complete protease inhibitor cocktail without EDTA). The column was washed with (buffer A containing 45 mM imidazole and the bound protein was eluted with buffer A with 90 mM imidazole. Fractions containing CpPI(4)K were pooled, concentrated using Amicon Ultra-15 and purified by a gel-filtration column (Hi-Load 26/60 Superdex 200, GE Healthcare), equilibrated with 20 mM Tris, pH 7.5, 300 mM NaCl, 1 mM DTT and 1× protease inhibitor cocktail without EDTA. Aliquots were flash frozen in liquid nitrogen and immediately stored at −80 °C. Mutations (CpPI4K-S871L and CpPI4K- H1037Y) were introduced into CpPI(4)K in the pFastBac plasmid using the QuikChange Site-Directed Mutagenesis Kit (Agilent) and confirmed by DNA sequencing. Mutant proteins were expressed, purified and assayed as described for the wildtype protein and did not show significant change in their KDU731 IC<sub>50</sub> (see <xref ref-type="supplementary-material" rid="SM">supplementary materials</xref> for full detail)</p></sec><sec id="S14"><title>PI(4)K enzymatic assay</title><p id="P19">The CpPI(4)K enzyme assay was described earlier<xref rid="R12" ref-type="bibr">12</xref>. Briefly, L-α-phosphatidylinositol (Avanti Polar Lipid), dissolved in 3% n-octylglucoside (Roche Diagnostics), was used as lipid substrate using the Transcreener ADP<sub>2</sub> FP detection kit (BellBrook Labs) in black, solid 384-well plates (Corning 3575). The final assay volume was 10 μl and contained 3 nM CpPI(4)K in 10 mM Tris, pH 7.5, 1 mM DTT, 3 μM ATP, 5 mM Mn<sup>2+</sup>, 0.05% Triton X-100 and 10 μM phosphatidylinositol/octylglucoside. The enzyme reaction was performed for 50 min at room temperature and stopped by adding 10 μl of detection mix containing 50mM HEPES, pH7.5, 400mM NaCl, 20mM EDTA, and 0.02% Brij-35, 2 nM AMP Alexa Fluor 633 tracer, and 20 μg ml<sup>−1</sup> ADP antibody (BellBrook Labs). Fluorescence polarization measurements were performed on an Infinite M1000 plate reader (Tecan) with λex = 635 nm and λem = 680 nm (20-nm bandwidth). IC<sub>50</sub> values were calculated using GraphPad Prism software.</p></sec><sec id="S15"><title>Solubility, permeability, <italic>in vitro</italic> metabolic stability and <italic>in vitro</italic> safety assessment</title><p id="P20">Solubility was measured using a miniaturized shake-flask approach and streamlined HPLC analysis as described earlier<xref rid="R31" ref-type="bibr">31</xref>. Parallel artificial membrane assays were carried out using a standard protocol<xref rid="R32" ref-type="bibr">32</xref>. The metabolic stability in liver microsomes was determined using the compound depletion approach and quantified by LC/MS. The assay measures the rate and extent of metabolism as determined by the disappearance of the parent compound, which allows the determination of <italic>in vitro</italic> half-life (t<sub>1/2</sub>), intrinsic clearance and the prediction of metabolic clearance in various species<xref rid="R31" ref-type="bibr">31</xref>. Cardiotoxicity and mini-ames genotoxicity<xref rid="R33" ref-type="bibr">33</xref> risk was measured as previously described<xref rid="R31" ref-type="bibr">31</xref>. All assays for binding to proteins known to bear potential safety liabilities in humans were high-throughput competitive binding assays using specific radiolabeled ligands<xref rid="R31" ref-type="bibr">31</xref>. The phototoxicity assay was performed following OECD Guideline for Testing of Chemicals 432 (<italic>in vitro</italic> 3T3 NRU phototoxicity test).</p></sec><sec id="S16"><title>Cytochrome P450 analysis</title><p id="P21">KDU731 was subjected to CYP450 inhibition analysis using 3 different isoforms<xref rid="R31" ref-type="bibr">31</xref>. The compounds were assessed for time dependent inhibition using CYP3A4<xref rid="R31" ref-type="bibr">31</xref>. The CYP induction assay was carried out using a PXR receptor binding was assayed using LanthaScreen TR-FRET PXR (SXR) competitive binding assay kit from Invitrogen<xref rid="R31" ref-type="bibr">31</xref>.</p></sec><sec id="S17"><title><italic>In vivo</italic> pharmacokinetic analysis</title><p id="P22">Rodent <italic>in vivo</italic> PK analysis was conducted using non-randomized CD-1 female mice (n = 4, 6-8 weeks old) and Wister rats (n = 4, 6-8 weeks old). PK studies in monkeys (n = 3) were performed in rhesus macaques as described<xref rid="R16" ref-type="bibr">16</xref>. Neonatal calf PK studies were performed as part of the efficacy study on day 1 and day 7 of treatment. All procedures involving animals were reviewed and approved by the respective institutional animal care and use committees. KDU731 was formulated in suspension formulation for p.o. dosing and solution formulation for i.v. dosing as described in <xref ref-type="table" rid="T4">Extended Data Table 3</xref>. For rat toxicology studies KDU731 was formulated in solid dispersion (SD). KDU731 (20%) and the required excipients (37.5%Soluplus, 37.5%Eudragit E PO and 5%SLS) were dissolved in ethanol/dichloromethane (v/v=1:1) at a total solid concentration of 10g/L by sonication and heating to 50°C. The solution was spray dried using a Buchi B290 Mini spray dryer with an inlet temperature of 75°C, aspirator set to 100% and pump to 35% followed by overnight drying under vacuum at 40°C. Amorphous nature was confirmed by X-ray powder diffraction analysis and powder was stored at 4°C. SD powder suspension was prepared freshly in 0.5% HPMC in 50mM acetate buffer (pH4.7) at 10 mg/mL of KDU731 (equivalent to SD powder 50mg/mL) and rats were dosed within one hour of preparation. Blood samples for PK studies were collected between 0 and 24 h post dose. Compound plasma concentrations were determined by LC/MS. Plasma samples from pharmacokinetic studies were extracted with acetonitrile: methanol: acetic acid (90:9.8:0.2) containing warfarin as an internal standard, using an 8.8:1 extractant to plasma ratio. Analyte quantitation was performed by LC/MS/MS. Liquid chromatography was performed using an Agilent 1200 HPLC system, with the Agilent Zorbax Phenyl (3.5μ, 4.6 x75 mm) column at an oven temperature of 45ºC, coupled with a API4000 triple quadruple mass spectrometer (Applied Biosystems). PK parameters were determined using Watson LIMS, by non-compartmental analysis.</p></sec><sec id="S18"><title>Rat toxicology study</title><p id="P23">KDU731 solid dispersion powder suspension was prepared freshly in 0.5% HPMC in 50mM acetate buffer (pH4.7) at 10 mg/mL and was orally administered to 5 male Wistar rats at a daily oral dose of 30 and 100 mg/kg/day for two weeks. Five control animals were treated with vehicle only. Rats were obtained from Shanghai SLAC Animal Ltd. and subjected to 3 days of quarantine and acclimatization prior to study begin. All animals were subjected to daily clinical observation, and body weight and food consumption was determined for all animals enrolled in the study. Clinical laboratory evaluations (hematology and clinical chemistry) were performed at the scheduled necropsy on day 15. Organs were examined for gross pathology and weighed prior to fixation and preparation for histology. Samples from organs and tissues prepared from animals assigned to control and high dose groups were examined microscopically. Specifically, the heart, pancreas, gastrointestinal tract (oesophagus, stomach, duodenum, jejunum, ileum, caecum, colon, rectum), kidney, liver, spleen, lung, testes, epididymis, adrenals, and thymus were examined for histopathological changes.</p></sec><sec id="S19"><title>Engineering of a <italic>C. parvum</italic> transgenic parasite strain expressing red-shifted firefly luciferase</title><p id="P24">The 5’ UTR and 3’UTR of the <italic>C. parvum</italic> actin gene was amplified from parasite genomic DNA and ligated into <italic>Kpn</italic>I/<italic>Cla</italic>I and <italic>Spe</italic>I/<italic>Bam</italic>HI sites of plasmid TK-Eno-Nluc-Neo-TK <xref rid="R8" ref-type="bibr">8</xref> respectively. The coding sequence for red-shifted luciferase<xref rid="R20" ref-type="bibr">20</xref> was amplified from pTubRE9 vector (kind gift from Markus Meissner, University of Glasgow) and cloned into <italic>Sal</italic>I/<italic>Nhe</italic>I restriction sites replacing Nluc. A 404 bp fragment of the 5’TK flank, the <italic>tk</italic> gene and a ribosomal 3’UTR was inserted upstream of the 5’ actin UTR using Gibson Assembly cloning (New England Biolabs). The final vector, along with the Cas9 plasmid containing a TK guide RNA (GAAGAATACAATTTCTAAGG) that targets the 3’ end of the <italic>tk</italic> gene was used to transfect <italic>C. parvum</italic> sporozoites. Sporozoites were delivered via surgery into the small intestine of C57BL/6 IFN-γ knockout mice (B6.129S7-Ifngtm1Ts/J, Jackson Laboratories) using procedures described previously<xref rid="R8" ref-type="bibr">8</xref>. Note that UGA1 Nluc parasites were generated using <italic>C. parvum</italic> IOWA II oocysts purchased from Sterling Parasitology Laboratory, University of Arizona whereas the UGA2 Fluc strain was engineered using <italic>C. parvum</italic> IOWA II oocysts purchased from Bunch Grass Farms, Deary, ID.</p></sec><sec id="S20"><title>Mouse model following fecal oocyst load</title><p id="P25">All mouse studies describe in this section were approved by the Institutional Animal Care and Use Committee of the University of Georgia (animal use protocol no. A2016 01-028-Y1-A4). C57BL/6 IFN-γ-knockout mice aged 6–8 weeks were selected randomly for each group (n=5) and infected with 10,000 <italic>C. parvum</italic> UGA1 Nluc<xref rid="R8" ref-type="bibr">8</xref> oocysts. No statistical tests were used to predetermine sample size. KDU731 was formulated in 0.5 % w/v methylcellulose and 0.5%w/v polysorbate in water and administered to mice daily for 7 days by oral gavage beginning on day 3 or day 11 post-infection. Control mice were given only vehicle. Mice were monitored for weight loss, fur ruffling, hunched posture and inactivity. Mice showing a weight loss of <=15% were euthanized. Fecal samples were collected (not blinded) and luminescence measurements were performed as described<xref rid="R8" ref-type="bibr">8</xref> (parasite Nluc activity is stable in feces when refrigerated). Measurements were performed on feces collected from individual mice as well as from cage-wide (pooled) collections. For Nluc assay, 20 mg of feces was mashed in 1 ml of lysis buffer (50mM TrisHCl (pH7.6), 2mM DTT, 2mM EDTA, 10% glycerol, 1% triton-X 100), ten 3 mm glass beads were added to the tube followed by vigorous agitation using a vortex mixer for 1 minute and a a short spin to pellet fecal material. Three aliquots of 100 μl lysate were dispensed into 96 well white plates and an equal amount of NanoGlo reconstituted buffer containing 1:50 of Nano-Glo substrate (Promega) was added, luminescence was read using a Synergy H4 Hybrid Microplate Reader (BioTek Instruments).</p><p id="P26">To measure oocyst load by quantitative real-time PCR (qRT-PCR), DNA was isolated from 100 mg of feces. Samples were subjected to five rounds of freezing in liquid nitrogen and thawing in 100°C heater block prior to DNA isolation using the ZR Fecal DNA miniprep kit (Zymo Research Corp., Irvine, CA). qRT-PCR was performed using sample DNA along with standards prepared from uninfected feces spiked with oocysts. PCR primers JVAF and JAVR and probe 5’FAM labeled JVAR<xref rid="R34" ref-type="bibr">34</xref> were used along with the following cycling parameters: denaturation at 95°C for 3 min, followed by 40 cycles of denaturation at 95°C for 10 sec, annealing at 60°C for 30 sec. Each 20μl PCR reaction contained 10 μl of SSoAdvanced™ universal probes supermix (Bio-Rad Laboratories, Hercules, CA), 0.4375 μM of each primer, 0.125 μM probe and 2 μl DNA.</p></sec><sec id="S21"><title>Mouse model following tissue parasite load using whole-animal imaging</title><p id="P27">IFN-γKO mice (n=5 per group) were infected with 10,000 <italic>C. parvum</italic> UGA2 Fluc oocysts expressing red-shifted firefly luciferase and given a daily dose of 10 mg/kg KDU731, Nitazoxanide (Sigma) at 100mg/kg, or control formulation for one week by oral gavage starting on day 7 post-infection. Parasite tissue load was measured by <italic>in vivo</italic> imaging. At the beginning of the experiment the abdominal area of mice was shaved with clippers to increase the signal during imaging on an IVIS Lumina II system, (Caliper Life Sciences). Mice were injected subcutaneously with 125 mg/kg D-luciferin (Gold Biotechnology Inc., St Louis MO, USA). 5 minutes after luciferin injection, mice were anaesthetized in an induction chamber using 3% isofluorane and then placed in the camera chamber. Mice were kept under anesthesia by administering isofluorane through individual nose cones. Images were acquired using a setting of F-stop=1/16, binning= medium and an exposure time of 5 minutes. Regions of interest (ROI) were selected for each mouse and total flux (photons/sec) was quantified using IVIS Lumina II Living Image 4.0 Software (Caliper Life Sciences). To validate measurements of parasite tissue burden, mice infected for one week were imaged, killed and the small intestine was removed and cut into 12 (1 cm) segments, and flushed with PBS. Sections were imaged 3 times in PBS with D-luciferin after which genomic DNA was isolated from each segment using the ZR Fecal DNA miniprep kit (Zymo Research Corp., Irvine, CA). qPCR was performed using parameters described above and parasite burden was established against a standard curve of samples with known parasite DNA content.</p></sec><sec id="S22"><title>Histology of intestinal tissue</title><p id="P28">Mice treated with KDU731 or control formulation were killed and intestinal tissue was collected within 15 minutes of death. Sections of the small intestine were taken from the 1-2 cm region anterior to the cecum, flushed with PBS and fixed overnight in 10% buffered formalin. Fixed samples were embedded in paraffin and 4 μm sections were cut (RM225 Microtome, Leica, Buffalo Grove, IL). Sections were deparafinized and stained with hematoxylin and eosin.</p></sec><sec id="S23"><title>Neonatal calf efficacy study</title><p id="P29">All calves used in this study were cared for in compliance with the Washington State University Institutional Animal Care and Use Committee. Sample size was calculated assuming that 85% of treated calves had resolution of clinical illness by the end of the treatment period (48 hrs post-administration of treatment #14) as compared to 15% of control calves. Assuming a type I error risk of 5% and a type II error risk of 80%, 7 calves were needed in the treatment group, plus 2 positive controls. In the event of calf death or removal from the study, an additional 4 calves were added to the control group (n = 6). Sample size was calculated using Epi Info. Fifteen Friesian-Holstein bull calves were enrolled in November 2015 and February 2016. At birth, all calves enrolled in November were randomized to treatment with KDU731 (n = 7), positive infection control (n = 2), and negative infection control (n = 1). The 5 calves enrolled in February were positive controls. The perineum of the dam was cleaned with povidone–iodine scrub and calves were delivered onto single-use plastic sheets to prevent exposure to environmental pathogens. Calves with abnormal physical examination findings and those weighing less than 29.5 kg at birth were excluded. Enrolled calves received 4L ≥ 50g IgG/L commercial colostrum replacer (Land O’Lakes Inc.) and a 3 ml subcutaneous injection of vitamin E and selenium (Merck Animal Health). Calves were then transported from the commercial dairy farm to Washington State University where they were housed in individual box stalls in a BSL-2 facility. Shatter-proof mirrors were provided for enrichment. Within 48 h of birth, blood samples were collected and evaluated for adequate passive transfer of colostral immunity. Calves were offered a commercial 20% protein/20% fat non-medicated milk replacer (Land O’Lakes Inc.) every 12 h via nipple bucket. At each feeding, calves were fed an average of 8.8 g of dry matter per kg of birth weight for the duration of the study. Water was provided <italic>ad libitum</italic>. All calves randomized to treatment or positive control groups were experimentally challenged within the first 48 h of life with 5×10<sup>7</sup><italic>C. parvum</italic> oocysts (Iowa II, Bunch Grass Farm, ID) via the rigid portion of an oroesophageal feeding tube. Oocysts were within 1 month of isolation and were cleaned in 0.6% sodium hypochlorite for 1 minute and then washed four times with PBS. The negative control calf was sham-challenged to maintain blinding of study personnel.</p><p id="P30">To facilitate collection of blood for plasma pharmacokinetic analysis, a long-term intravenous catheter (MILACATH, MILA International) was aseptically placed in the jugular vein within the first 48-72 hours of life. Calves were sedated with Xylazine 0.1 ml IV (20mg/ml) (Akorn Animal Health). Sedation was reversed with Atipamezole 0.1ml IM (5mg/ml) (Zoetis). Beginning at birth, a fecal sample was collected directly from the rectum every 24 h. A complete physical exam was performed every 12 h and clinical data including appetite, mentation, fecal consistency, and hydration status were recorded. Clinical data was evaluated on a scale of 1 (normal) to 3 (severe) in accordance with previously described methods (see rubric in <xref ref-type="supplementary-material" rid="SD6">supplementary information</xref>)<xref rid="R23" ref-type="bibr">23</xref>,<xref rid="R24" ref-type="bibr">24</xref>.</p><p id="P31">KDU731 was prepared as a 5mg/ml suspension formulation in 0.5% w/v Methylcellulose and 0.5% w/v Tween-80 in water and KDU731 treatment was initiated when a calf began shedding oocysts and had a fecal consistency score of 3. Calves were induced to suckle and then KDU731 was given orally via an oral dosing syringe. Calves were treated every 12 h for 7 days at a dose of 5 mg/kg birth weight at least 2 h after feeding. Pharmacokinetic sampling was conducted on day 1 and day 7 of treatment. Blood was drawn prior to and at multiple time-points following administration. A fecal sample was collected 1 h and 12 h following administration. On days 2–6 of treatment, blood was collected prior to KDU731 administration. On day 3 of life, a fecal sample was tested <italic>E. coli</italic> K99, on day 7 for <italic>Salmonella</italic>, rotavirus, and corona virus. Of the 7 treatment calves, two calves (N101 and N107) were randomly selected to be euthanized 24 hours after administration of the final KDU731 treatment. A positive control calf (N104) was also euthanized at the same time. Euthanasia was via captive bolt and induction of bilateral pneumothorax in order to avoid confounding histologic findings. Calves were submitted for necropsy to the Washington State Animal Disease Diagnostic Laboratory. Samples of the liver, spleen, kidney, and small intestine were collected for histologic analysis. The remaining calves continued in the study until the cessation of fecal oocyst shedding as determined by immunofluorescence microscopy (Merifluor, Meridian Diagnostics). Upon cessation of fecal oocyst shedding (2 consecutive negative fecal samples over 48 h), calves were euthanized and submitted to the Washington State Animal Disease Diagnostic Laboratory for necropsy.</p><p id="P32">Oocysts counts were interpolated by qRT-PCR at the Cornell Animal Health Diagnostic Center using serial dilutions of commercially purified <italic>C. parvum</italic> oocysts (Waterborne, Inc., New Orleans, LA). Total nucleic acid was extracted from supernatants of 200 mg of fecal sample, oocyst suspension, or negative control homogenized in 400 μl of PBS using a magnetic bead based automated procedure (AM1840, Applied Biosytems, Foster City, CA). An exogenous control (MS2 phage) was added to the lysis buffer to control for PCR inhibition<xref rid="R35" ref-type="bibr">35</xref>. qPCR for <italic>Cryptosporidium</italic> Spp. 18S rRNA was performed on the Applied Biosystems 7500-FAST platform using commercial master mix (ToughMix, Quantabio) and oligonucleotides previously described<xref rid="R36" ref-type="bibr">36</xref>. This count was standardized by the fecal dry weight percentage. 5-10 g portion of each original fecal sample was dried at 108 °C for a minimum of 24 h (Squaroid Vaccuum Oven, Labline, India) and weighed<xref rid="R24" ref-type="bibr">24</xref>.</p><p id="P33">Data were analyzed using descriptive and inferential methods. The Shapiro–Wilk test was used to determine if data were non-Gaussian. Depending on the distribution of data, continuous variables were evaluated using the student’s t test, analysis of variance, or the Wilcoxon Rank Sum test. Analysis of variance was used to assess differences in fecal oocyst counts and fecal consistency score between the KDU731 treated and untreated calves. The effect of co-morbidity on outcomes of interest was evaluated using the student’s t test. Data were analyzed using JMP Pro 11.0 (SAS Institute Inc).</p></sec></sec><sec sec-type="extended-data" id="S24"><title>Extended Data</title><fig id="F4" orientation="portrait" position="anchor"><label>Extended Data Figure 1</label><caption><title>Structures of the pyrazolopyridines and other compounds described in <xref ref-type="table" rid="T1">Table 1</xref>.</title><p id="P34">Important structural determinants required for anti-<italic>Cryptosporidium</italic> activity in pyrazolopyridines are shown in blue.</p></caption><graphic xlink:href="emss-72141-f004"></graphic></fig><fig id="F5" orientation="portrait" position="anchor"><label>Extended Data Figure 2</label><caption><title>Anti-<italic>Cryptosporidium</italic> activity does not correlate with mammalian cell toxicity.</title><p id="P35">Correlation of <italic>C. parvum</italic> cytopathic effect vs HepG2 cytotoxicity assay for selected pyrazolopyridine and imidazopyrazine analogs along with BQR695 and MMV390048. Data shown here is geometric mean EC<sub>50</sub>, with at least 2 biological replicates.</p></caption><graphic xlink:href="emss-72141-f005"></graphic></fig><fig id="F6" orientation="portrait" position="anchor"><label>Extended Data Figure 3</label><caption><title>Recombinant <italic>C. parvum</italic> cgd8_4500 shows phosphatidylinositide kinase activity.</title><p id="P36">cgd8_4500 was expressed in insect cells using a Baculovirus system and recombinant enzyme was purified. A Michaelis-Menten plot of phosphatidylinositide kinase reaction with 3 nM CpPI(4)K enzyme at varying ATP concentration is shown. Data shown here is a representative graph of two independent biological replicates.</p></caption><graphic xlink:href="emss-72141-f006"></graphic></fig><fig id="F7" orientation="portrait" position="anchor"><label>Extended Data Figure 4</label><caption><title>KDU731 inhibits <italic>C. parvum</italic> Nluc parasites in vitro and in vivo.</title><p id="P37">a, EC50 determination of KDU731 against UGA1 Nluc transgenic parasites grown in HCT-8 cultures using luciferase activity as read out. Representative data is shown, three technical replicates. b, Mice (n=5) were infected with 10,000 UGA1 Nluc oocysts and treated orally three days post-infection with 1, 5 or 10 mg/kg KDU731 for one week. Fecal oocyst load was determined by measuring parasite luciferase activity (b) or parasite DNA by qPCR (c) in feces pooled from entire cage of 5 mice (20 mg feces for Nluc and 100 mg for PCR assay). b,c, Means for three technical replicates are shown. Error bars represent s.d. Pooled Nluc experiments for vehicle and 10mg/kg dose were repeated in three biological replicates and a representative result is shown.</p></caption><graphic xlink:href="emss-72141-f007"></graphic></fig><fig id="F8" orientation="portrait" position="anchor"><label>Extended Data Figure 5</label><caption><title>Nitazoxanide does not reduce intestinal parasite load in IFN-γ KO mice.</title><p id="P38">Mice (n=5) were infected with 10,000 UGA2 FLuc oocysts and 7 days post-infection animals were treated daily for a week with 100 mg/kg Nitazoxanide or vehicle. Mice were monitored by whole-animal-imaging. Radiance scale shows total flux in photon/sec.</p></caption><graphic xlink:href="emss-72141-f008"></graphic></fig><fig id="F9" orientation="portrait" position="anchor"><label>Extended Data Figure 6</label><caption><title>Parasite intestinal load measured by qPCR correlates with fecal shedding and tissue luminescence.</title><p id="P39">a, Mice were infected with 10,000 UGA2 FLuc oocysts and 7 days post-infection animals were treated daily for a week with vehicle or 10 mg/kg KDU731. Whole animal imaging during the treatment period is shown in <xref ref-type="fig" rid="F2">Fig 2g</xref>. Fecal oocyst load was determined by measuring parasite DNA by qPCR in feces pooled from a cage of 5 mice. b, Mice (n=4) were infected with 50,000 UGA2 FLuc oocysts and imaged after one week. Mice were killed and the small intestine was resected and imaged (representative image shown). Infection of the intestine ranges in intensity from heavy in the ileum to more moderate in the jejunum and cecum (see radiance scale bar for comparison). Intestines were cut into 12 segments and luminescence of each segment was recorded. c, Quantitative PCR analysis of intestinal segments was performed in triplicate and plotted against the respective luminescence measurements. Regression analysis found robust correlation of tissue luminescence and PCR for parasite DNA with an R2 of 0.8.</p></caption><graphic xlink:href="emss-72141-f009"></graphic></fig><fig id="F10" orientation="portrait" position="anchor"><label>Extended Data Figure 7</label><caption><title>Effect of KDU731 on severity of diarrhea and dehydration in the neonatal calf model of cryptosporidiosis.</title><p id="P40">Severity of diarrhea and dehydration in individual calves challenged with 5 x 10<sup>7</sup><italic>C. parvum</italic> oocysts. Infected calves were treated with vehicle (n=6) or with KDU731 (n=7); n represents the number of calves. Every 12 h, calves were stimulated to defecate, fecal consistency was evaluated, and hydration status was assessed. Fecal consistency and hydration scores were assigned per the study rubric (see <xref ref-type="supplementary-material" rid="SD6">supplementary information</xref>). The schematic representation shows the fecal consistency (<bold>a</bold>) and hydration scores (<bold>b</bold>) throughout the drug treatment period. Fecal consistency and hydration began to improve within 48 h of initiating treatment with KDU731.</p></caption><graphic xlink:href="emss-72141-f010"></graphic></fig><table-wrap id="T2" position="anchor" orientation="portrait"><label>Extended Data Table 1</label><caption><title>Physicochemical properties and safety profiling data for KDU731</title></caption><table frame="hsides" rules="groups"><thead><tr><th align="left" valign="top" rowspan="1" colspan="1">Properties</th><th align="left" valign="top" rowspan="1" colspan="1">KDU731</th></tr></thead><tbody><tr><td align="left" valign="top" rowspan="1" colspan="1"><bold>Physicochemical</bold></td><td align="left" valign="top" rowspan="1" colspan="1"></td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">Molecular Weight (Da)</td><td align="left" valign="top" rowspan="1" colspan="1">396.41</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">Solubility (pH 6.8) (μM)</td><td align="left" valign="top" rowspan="1" colspan="1">20</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">Lipophilicity (logP)</td><td align="left" valign="top" rowspan="1" colspan="1">1.7</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">PAMPA (% calc fraction absorbed)</td><td align="left" valign="top" rowspan="1" colspan="1">34.7</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">Caco2 permeability (ratio B-A/A-B)</td><td align="left" valign="top" rowspan="1" colspan="1">2.42</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1"><italic>In vitro</italic> clearance (M/R/D/Mk/H)</td><td align="left" valign="top" rowspan="1" colspan="1"></td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">Microsomal CLint [μl/min•mg]</td><td align="left" valign="top" rowspan="1" colspan="1">50 / 29 / 36 / 39 / 13</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">Hepatocytes [μl/min/million cells]</td><td align="left" valign="top" rowspan="1" colspan="1">- / 6 / < 0.6 / <4 /12</td></tr><tr><td colspan="2" align="left" valign="top" rowspan="1"><hr></hr></td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1"><bold>Cellular activity (μM)</bold></td><td align="left" valign="top" rowspan="1" colspan="1"></td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">Cytotoxicity HepG2 (CC<sub>50</sub>)</td><td align="left" valign="top" rowspan="1" colspan="1">15.6</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1"><italic>C. parvum</italic> / <italic>C. hominis</italic> (EC<sub>50</sub>)</td><td align="left" valign="top" rowspan="1" colspan="1">0.10 / 0.13</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">HepG2 CC<sub>50</sub> / <italic>C. parvum</italic> EC<sub>50</sub> ratio (SI)</td><td align="left" valign="top" rowspan="1" colspan="1">> 100</td></tr><tr><td colspan="2" align="left" valign="top" rowspan="1"><hr></hr></td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1"><bold>% Plasma protein binding</bold></td><td align="left" valign="top" rowspan="1" colspan="1"></td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">M/R/D/H</td><td align="left" valign="top" rowspan="1" colspan="1">- / 87.7 / 70.8 / 79.1</td></tr><tr><td colspan="2" align="left" valign="top" rowspan="1"><hr></hr></td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1"><bold>Human lipid and related kinase enzyme IC<sub>50</sub> (μM)</bold></td><td align="left" valign="top" rowspan="1" colspan="1"></td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">Pl(4)β</td><td align="left" valign="top" rowspan="1" colspan="1"></td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">Pl(3)α</td><td align="left" valign="top" rowspan="1" colspan="1">1.4</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">Pl(3)β</td><td align="left" valign="top" rowspan="1" colspan="1">1</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">PIK(3)Cγ</td><td align="left" valign="top" rowspan="1" colspan="1">1.9</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">PIK(3)Cδ</td><td align="left" valign="top" rowspan="1" colspan="1">0.88</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">VPS34</td><td align="left" valign="top" rowspan="1" colspan="1">0.39</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">mTOR</td><td align="left" valign="top" rowspan="1" colspan="1">>9.1</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1"></td><td align="left" valign="top" rowspan="1" colspan="1">4.3</td></tr><tr><td colspan="2" align="left" valign="top" rowspan="1"><hr></hr></td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1"><bold>CYP P450 isoforms inhibition (IC<sub>50</sub> μM)</bold></td><td align="left" valign="top" rowspan="1" colspan="1"></td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">Reversible 3A4</td><td align="left" valign="top" rowspan="1" colspan="1">>50</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">Reversible 2D6</td><td align="left" valign="top" rowspan="1" colspan="1">>50</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">Reversible 2C9</td><td align="left" valign="top" rowspan="1" colspan="1">>4.8</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">Time dependent inhibition 3A4</td><td align="left" valign="top" rowspan="1" colspan="1">Negative</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">CYP induction, PXR functional assay</td><td align="left" valign="top" rowspan="1" colspan="1">>10</td></tr><tr><td colspan="2" align="left" valign="top" rowspan="1"><hr></hr></td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1"><bold>Cardiotoxicity</bold></td><td align="left" valign="top" rowspan="1" colspan="1"></td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">hERG Binding (μM)</td><td align="left" valign="top" rowspan="1" colspan="1">>30</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">Q-Patch IC<sub>50</sub> (μM)</td><td align="left" valign="top" rowspan="1" colspan="1">28</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">Patch Clamp Nav1.5 Quattro IC<sub>50</sub> (μM)</td><td align="left" valign="top" rowspan="1" colspan="1">>50</td></tr><tr><td colspan="2" align="left" valign="top" rowspan="1"><hr></hr></td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1"><bold>Genotoxicity</bold></td><td align="left" valign="top" rowspan="1" colspan="1"></td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">Mini-AMES</td><td align="left" valign="top" rowspan="1" colspan="1">Negative</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">Micronucleus test (MNT)</td><td align="left" valign="top" rowspan="1" colspan="1">Negative</td></tr><tr><td colspan="2" align="left" valign="top" rowspan="1"><hr></hr></td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1"><bold>Phototoxicity</bold> (PIF values)</td><td align="left" valign="top" rowspan="1" colspan="1">Negative (3.1)</td></tr><tr><td colspan="2" align="left" valign="top" rowspan="1"><hr></hr></td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1"><bold>Safety pharmacology profiling<xref ref-type="table-fn" rid="TFN4">*</xref></bold></td><td align="left" valign="top" rowspan="1" colspan="1">No significant</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">(selected receptors, ion-channels, transporters, kinases etc),</td><td align="left" valign="top" rowspan="1" colspan="1">binding/inhibition</td></tr></tbody></table><table-wrap-foot><fn id="TFN4"><label>*</label><p id="P41">Details in <xref ref-type="table" rid="T3">Extended Data Table 2</xref>. Data presented here is from assays repeated at least 2 times</p></fn></table-wrap-foot></table-wrap><table-wrap id="T3" position="anchor" orientation="portrait"><label>Extended Data Table 2</label><caption><title>Effect of KDU731 on radio-ligand binding to a panel of human recombinant receptors and pharmacologically relevant proteases / kinases</title></caption><table frame="hsides" rules="groups"><thead><tr><th align="left" rowspan="1" colspan="1">Binding assay<xref ref-type="table-fn" rid="TFN5">*</xref></th><th align="left" rowspan="1" colspan="1">IC<sub>50</sub> (μM)</th><th align="left" rowspan="1" colspan="1">Kinase assay</th><th align="left" rowspan="1" colspan="1">IC<sub>50</sub> (μM)</th></tr></thead><tbody><tr><td align="left" rowspan="1" colspan="1">Adenosine 1 receptor</td><td align="left" rowspan="1" colspan="1">>30</td><td align="left" rowspan="1" colspan="1">cABLT315</td><td align="left" rowspan="1" colspan="1">>10</td></tr><tr><td align="left" rowspan="1" colspan="1">Adenosine 2A receptor</td><td align="left" rowspan="1" colspan="1">>30</td><td align="left" rowspan="1" colspan="1">ALK</td><td align="left" rowspan="1" colspan="1">>10</td></tr><tr><td align="left" rowspan="1" colspan="1">Adenosine 3 receptor</td><td align="left" rowspan="1" colspan="1">>30</td><td align="left" rowspan="1" colspan="1">Aurora-A K</td><td align="left" rowspan="1" colspan="1">>10</td></tr><tr><td align="left" rowspan="1" colspan="1">Adenosine transporter</td><td align="left" rowspan="1" colspan="1">>30</td><td align="left" rowspan="1" colspan="1">AXL</td><td align="left" rowspan="1" colspan="1">>10</td></tr><tr><td align="left" rowspan="1" colspan="1">Adrenergic ß1</td><td align="left" rowspan="1" colspan="1">>30</td><td align="left" rowspan="1" colspan="1">BTK</td><td align="left" rowspan="1" colspan="1">>10</td></tr><tr><td align="left" rowspan="1" colspan="1">Adrenergic α2B receptor</td><td align="left" rowspan="1" colspan="1">>30</td><td align="left" rowspan="1" colspan="1">CDK2A</td><td align="left" rowspan="1" colspan="1">>10</td></tr><tr><td align="left" rowspan="1" colspan="1">Adrenergic α2C receptor</td><td align="left" rowspan="1" colspan="1">>30</td><td align="left" rowspan="1" colspan="1">CDK4D1</td><td align="left" rowspan="1" colspan="1">>10</td></tr><tr><td align="left" rowspan="1" colspan="1">Angiotensin II AT1 receptor</td><td align="left" rowspan="1" colspan="1">>30</td><td align="left" rowspan="1" colspan="1">EGFR</td><td align="left" rowspan="1" colspan="1">>10</td></tr><tr><td align="left" rowspan="1" colspan="1">Benzodiazepine receptor <xref ref-type="table-fn" rid="TFN5">♦</xref></td><td align="left" rowspan="1" colspan="1">>30</td><td align="left" rowspan="1" colspan="1">EPHA4</td><td align="left" rowspan="1" colspan="1">>10</td></tr><tr><td align="left" rowspan="1" colspan="1">Cholecystokinin A receptor</td><td align="left" rowspan="1" colspan="1">>30</td><td align="left" rowspan="1" colspan="1">EPHB4</td><td align="left" rowspan="1" colspan="1">>10</td></tr><tr><td align="left" rowspan="1" colspan="1">Cholecystokinin B receptor</td><td align="left" rowspan="1" colspan="1">>30</td><td align="left" rowspan="1" colspan="1">FGFR3</td><td align="left" rowspan="1" colspan="1">>10</td></tr><tr><td align="left" rowspan="1" colspan="1">COX-1 assay <xref ref-type="table-fn" rid="TFN5">#</xref></td><td align="left" rowspan="1" colspan="1">>30</td><td align="left" rowspan="1" colspan="1">GSK3B</td><td align="left" rowspan="1" colspan="1">>10</td></tr><tr><td align="left" rowspan="1" colspan="1">COX-2 assay</td><td align="left" rowspan="1" colspan="1">>30</td><td align="left" rowspan="1" colspan="1">IGF1R</td><td align="left" rowspan="1" colspan="1">>10</td></tr><tr><td align="left" rowspan="1" colspan="1">Dopamine D2 receptor</td><td align="left" rowspan="1" colspan="1">>30</td><td align="left" rowspan="1" colspan="1">INSR</td><td align="left" rowspan="1" colspan="1">>10</td></tr><tr><td align="left" rowspan="1" colspan="1">Dopamine D3 receptor</td><td align="left" rowspan="1" colspan="1">>30</td><td align="left" rowspan="1" colspan="1">JAK1</td><td align="left" rowspan="1" colspan="1">>10</td></tr><tr><td align="left" rowspan="1" colspan="1">Dopamine transporter</td><td align="left" rowspan="1" colspan="1">>30</td><td align="left" rowspan="1" colspan="1">JAK2</td><td align="left" rowspan="1" colspan="1">6.7</td></tr><tr><td align="left" rowspan="1" colspan="1">Endothelin A receptor</td><td align="left" rowspan="1" colspan="1">>30</td><td align="left" rowspan="1" colspan="1">JAK3</td><td align="left" rowspan="1" colspan="1">>10</td></tr><tr><td align="left" rowspan="1" colspan="1">Ghrelin receptor</td><td align="left" rowspan="1" colspan="1">>30</td><td align="left" rowspan="1" colspan="1">KDR</td><td align="left" rowspan="1" colspan="1">>10</td></tr><tr><td align="left" rowspan="1" colspan="1">Histamine H1 receptor</td><td align="left" rowspan="1" colspan="1">>30</td><td align="left" rowspan="1" colspan="1">KIT</td><td align="left" rowspan="1" colspan="1">>10</td></tr><tr><td align="left" rowspan="1" colspan="1">Histamine H3 receptor</td><td align="left" rowspan="1" colspan="1">>30</td><td align="left" rowspan="1" colspan="1">LCK</td><td align="left" rowspan="1" colspan="1">>10</td></tr><tr><td align="left" rowspan="1" colspan="1">Melanocortin MC3 receptor</td><td align="left" rowspan="1" colspan="1">>30</td><td align="left" rowspan="1" colspan="1">MAP3K8</td><td align="left" rowspan="1" colspan="1">>10</td></tr><tr><td align="left" rowspan="1" colspan="1">Monoamine oxydase A</td><td align="left" rowspan="1" colspan="1">>30</td><td align="left" rowspan="1" colspan="1">MAPK14</td><td align="left" rowspan="1" colspan="1">>10</td></tr><tr><td align="left" rowspan="1" colspan="1">Motilin receptor</td><td align="left" rowspan="1" colspan="1">>30</td><td align="left" rowspan="1" colspan="1">MAPK1</td><td align="left" rowspan="1" colspan="1">>10</td></tr><tr><td align="left" rowspan="1" colspan="1">Muscarinic M1 receptor</td><td align="left" rowspan="1" colspan="1">>30</td><td align="left" rowspan="1" colspan="1">MET</td><td align="left" rowspan="1" colspan="1">>10</td></tr><tr><td align="left" rowspan="1" colspan="1">Muscarinic M3 receptor</td><td align="left" rowspan="1" colspan="1">>30</td><td align="left" rowspan="1" colspan="1">PDGFRA</td><td align="left" rowspan="1" colspan="1">>10</td></tr><tr><td align="left" rowspan="1" colspan="1">Nicotinic (CNS) receptor</td><td align="left" rowspan="1" colspan="1">>30</td><td align="left" rowspan="1" colspan="1">PDPK1</td><td align="left" rowspan="1" colspan="1">>10</td></tr><tr><td align="left" rowspan="1" colspan="1">NMDA channel site receptor</td><td align="left" rowspan="1" colspan="1">>30</td><td align="left" rowspan="1" colspan="1">PKN1</td><td align="left" rowspan="1" colspan="1">>10</td></tr><tr><td align="left" rowspan="1" colspan="1">Norepinephrine transporter</td><td align="left" rowspan="1" colspan="1">>30</td><td align="left" rowspan="1" colspan="1">PKN2</td><td align="left" rowspan="1" colspan="1">>10</td></tr><tr><td align="left" rowspan="1" colspan="1">Opiate δ receptor</td><td align="left" rowspan="1" colspan="1">>30</td><td align="left" rowspan="1" colspan="1">PRKCA</td><td align="left" rowspan="1" colspan="1">>10</td></tr><tr><td align="left" rowspan="1" colspan="1">Opiate κ receptor</td><td align="left" rowspan="1" colspan="1">>30</td><td align="left" rowspan="1" colspan="1">PRKCQ</td><td align="left" rowspan="1" colspan="1">>10</td></tr><tr><td align="left" rowspan="1" colspan="1">Phosphodiesterase 3</td><td align="left" rowspan="1" colspan="1">>4.5</td><td align="left" rowspan="1" colspan="1">RET</td><td align="left" rowspan="1" colspan="1">>10</td></tr><tr><td align="left" rowspan="1" colspan="1">Phosphodiesterase 4D</td><td align="left" rowspan="1" colspan="1">>30</td><td align="left" rowspan="1" colspan="1">ROCK2</td><td align="left" rowspan="1" colspan="1">>10</td></tr><tr><td align="left" rowspan="1" colspan="1">Serotonin 5-HT 2C receptor</td><td align="left" rowspan="1" colspan="1">>30</td><td align="left" rowspan="1" colspan="1">SYK</td><td align="left" rowspan="1" colspan="1">>10</td></tr><tr><td align="left" rowspan="1" colspan="1">Serotonin 5-HT3 receptor</td><td align="left" rowspan="1" colspan="1">>30</td><td align="left" rowspan="1" colspan="1">TYK2</td><td align="left" rowspan="1" colspan="1">>10</td></tr><tr><td align="left" rowspan="1" colspan="1">Serotonin transporter</td><td align="left" rowspan="1" colspan="1">>30</td><td align="left" rowspan="1" colspan="1">ZAP70</td><td align="left" rowspan="1" colspan="1">>10</td></tr><tr><td align="left" rowspan="1" colspan="1">Vasopressin V1a receptor</td><td align="left" rowspan="1" colspan="1">>30</td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td><td colspan="2" align="left" valign="top" rowspan="1"><hr></hr></td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"><bold>Protease assays</bold></td><td align="left" rowspan="1" colspan="1"></td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">Caspases 3</td><td align="left" rowspan="1" colspan="1">>30</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">Cathepsin D</td><td align="left" rowspan="1" colspan="1">>30</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">Matrix Metalloproteinases MMP08</td><td align="left" rowspan="1" colspan="1">>30</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">Thrombin</td><td align="left" rowspan="1" colspan="1">>30</td></tr></tbody></table><table-wrap-foot><fn id="TFN5"><p id="P42">*all human except <sup>♦</sup>rat <sup>#</sup>Ovine</p></fn><fn id="TFN6"><p id="P43">Assays were repeated at least twice</p></fn></table-wrap-foot></table-wrap><table-wrap id="T4" position="anchor" orientation="portrait"><label>Extended Data Table 3</label><caption><title><italic>In vivo</italic> pharmacokinetic analysis of KDU731 in mice, rats, monkeys and calves.</title></caption><table frame="hsides" rules="groups"><thead><tr><th align="left" rowspan="1" colspan="1">Route</th><th align="left" rowspan="1" colspan="1">Parameter</th><th align="left" rowspan="1" colspan="1">Units</th><th align="left" colspan="2" rowspan="1">Mice</th><th align="left" colspan="2" rowspan="1">Rats</th><th align="left" colspan="2" rowspan="1">Rats TK<xref ref-type="table-fn" rid="TFN11">*</xref></th><th align="left" colspan="2" rowspan="1">Monkeys</th><th align="left" colspan="2" rowspan="1">Calves</th></tr></thead><tbody><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">Dose</td><td align="left" rowspan="1" colspan="1">mg/kg</td><td align="left" rowspan="1" colspan="1">2.3</td><td align="left" rowspan="1" colspan="1">24.7</td><td align="left" rowspan="1" colspan="1">2.3</td><td align="left" rowspan="1" colspan="1">21.3</td><td align="left" rowspan="1" colspan="1">30</td><td align="left" rowspan="1" colspan="1">100</td><td align="left" rowspan="1" colspan="1">3</td><td align="left" rowspan="1" colspan="1">10</td><td align="left" rowspan="1" colspan="1">5<xref ref-type="table-fn" rid="TFN12">a</xref></td><td align="left" rowspan="1" colspan="1">5<xref ref-type="table-fn" rid="TFN13">b</xref></td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">C<sub>max</sub></td><td align="left" rowspan="1" colspan="1">nM</td><td align="left" rowspan="1" colspan="1">406</td><td align="left" rowspan="1" colspan="1">2788</td><td align="left" rowspan="1" colspan="1">161</td><td align="left" rowspan="1" colspan="1">532</td><td align="left" rowspan="1" colspan="1">19168</td><td align="left" rowspan="1" colspan="1">28373</td><td align="left" rowspan="1" colspan="1">72</td><td align="left" rowspan="1" colspan="1">120</td><td align="left" rowspan="1" colspan="1">228<xref ref-type="table-fn" rid="TFN12">a</xref></td><td align="left" rowspan="1" colspan="1">341<xref ref-type="table-fn" rid="TFN13">b</xref></td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">AUC</td><td align="left" rowspan="1" colspan="1">nM*h</td><td align="left" rowspan="1" colspan="1">2306</td><td align="left" rowspan="1" colspan="1">22624</td><td align="left" rowspan="1" colspan="1">1844</td><td align="left" rowspan="1" colspan="1">5235</td><td align="left" rowspan="1" colspan="1">93026 (18x)</td><td align="left" rowspan="1" colspan="1">130979 (25x)</td><td align="left" rowspan="1" colspan="1">1620</td><td align="left" rowspan="1" colspan="1">2580</td><td align="left" rowspan="1" colspan="1">1909<xref ref-type="table-fn" rid="TFN12">a</xref></td><td align="left" rowspan="1" colspan="1">2011<xref ref-type="table-fn" rid="TFN13">b</xref></td></tr><tr><td align="left" rowspan="4" valign="top" colspan="1">p.o.</td><td align="left" rowspan="1" colspan="1">T<sub>1/2</sub></td><td align="left" rowspan="1" colspan="1">hours</td><td align="left" rowspan="1" colspan="1">2.47</td><td align="left" rowspan="1" colspan="1">1.39</td><td align="left" rowspan="1" colspan="1">4.3</td><td align="left" rowspan="1" colspan="1">3.14</td><td align="left" rowspan="1" colspan="1">-</td><td align="left" rowspan="1" colspan="1">-</td><td align="left" rowspan="1" colspan="1">-</td><td align="left" rowspan="1" colspan="1">-</td><td align="left" rowspan="1" colspan="1">-</td><td align="left" rowspan="1" colspan="1">-</td></tr><tr><td align="left" rowspan="1" colspan="1">F</td><td align="left" rowspan="1" colspan="1">%</td><td align="left" rowspan="1" colspan="1">37</td><td align="left" rowspan="1" colspan="1">34</td><td align="left" rowspan="1" colspan="1">23</td><td align="left" rowspan="1" colspan="1">7</td><td align="left" rowspan="1" colspan="1">-</td><td align="left" rowspan="1" colspan="1">-</td><td align="left" rowspan="1" colspan="1">9</td><td align="left" rowspan="1" colspan="1">4</td><td align="left" rowspan="1" colspan="1">-</td><td align="left" rowspan="1" colspan="1">-</td></tr><tr><td align="left" rowspan="1" colspan="1">C<sub>max</sub> d.n</td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">177</td><td align="left" rowspan="1" colspan="1">113</td><td align="left" rowspan="1" colspan="1">70</td><td align="left" rowspan="1" colspan="1">25</td><td align="left" rowspan="1" colspan="1">638.9</td><td align="left" rowspan="1" colspan="1">283.7</td><td align="left" rowspan="1" colspan="1">24</td><td align="left" rowspan="1" colspan="1">12</td><td align="left" rowspan="1" colspan="1">45.6</td><td align="left" rowspan="1" colspan="1">68.2</td></tr><tr><td align="left" rowspan="1" colspan="1">AUCd.n</td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">1003</td><td align="left" rowspan="1" colspan="1">916</td><td align="left" rowspan="1" colspan="1">802</td><td align="left" rowspan="1" colspan="1">245.8</td><td align="left" rowspan="1" colspan="1">3100.9 (13x)</td><td align="left" rowspan="1" colspan="1">1309.8 (5x)</td><td align="left" rowspan="1" colspan="1">540</td><td align="left" rowspan="1" colspan="1">258</td><td align="left" rowspan="1" colspan="1">381.8</td><td align="left" rowspan="1" colspan="1">402.2</td></tr><tr><td colspan="13" align="left" valign="top" rowspan="1"><hr></hr></td></tr><tr><td align="left" rowspan="4" valign="middle" colspan="1">i.v.</td><td align="left" rowspan="1" colspan="1">Dose</td><td align="left" rowspan="1" colspan="1">mg/kg</td><td align="left" colspan="2" rowspan="1">5</td><td align="left" colspan="2" rowspan="1">2.5</td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td><td align="left" colspan="2" rowspan="1">0.3</td><td align="left" colspan="2" rowspan="1">-</td></tr><tr><td align="left" rowspan="1" colspan="1">Vss</td><td align="left" rowspan="1" colspan="1">L/kg</td><td align="left" colspan="2" rowspan="1">1.12</td><td align="left" colspan="2" rowspan="1">2.15</td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td><td align="left" colspan="2" rowspan="1">2.1</td><td align="left" colspan="2" rowspan="1">-</td></tr><tr><td align="left" rowspan="1" colspan="1">CL</td><td align="left" rowspan="1" colspan="1">mL/min/kg</td><td align="left" colspan="2" rowspan="1">16</td><td align="left" colspan="2" rowspan="1">12.4</td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td><td align="left" colspan="2" rowspan="1">6.8</td><td align="left" colspan="2" rowspan="1">-</td></tr><tr><td align="left" rowspan="1" colspan="1">T<sub>1/2</sub></td><td align="left" rowspan="1" colspan="1">hours</td><td align="left" colspan="2" rowspan="1">1.06</td><td align="left" colspan="2" rowspan="1">3.3</td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td><td align="left" colspan="2" rowspan="1">4.7</td><td align="left" colspan="2" rowspan="1">-</td></tr></tbody></table><table-wrap-foot><fn id="TFN7"><p id="P44">Mice, Rats, Monkeys and Calves n = 4, 4, 3 and 7 respectively.</p></fn><fn id="TFN8"><p id="P45">C<sub>max</sub>, maximum concentration achieved; AUC, area under curve (0-24 hours); T<sub>1/2</sub>, half-life; F, percentage oral bioavailability; C<sub>max</sub> d.n, dose-normalized</p><p id="P46">Cmax; AUC d.n, dose-normalized AUC; Vss, steady state volume of distribution; CL, clearance; TK, toxicokinetic study.</p></fn><fn id="TFN9"><p id="P47">p.o. denotes oral gavage formulated in 0.5% w/v methylcellulose and 0.5% w/v Polysorbate80 in water except for rats TK study.</p></fn><fn id="TFN10"><p id="P48">i.v. denotes intravenous injection formulated in PEG300/D5W (3:1, v/v) for mice; Propylene Glycol / Tween 80 / Water (20:20:60, v/v) for rats and Methylpyrrolidone / PEG200 (10:90, v/v) for monkeys.</p></fn><fn id="TFN11"><label>*</label><p id="P49">Rats TK: day 1 TK analysis of KDU731 formulated in solid dispersion (SD) formulation as described in <xref ref-type="sec" rid="S7">Methods</xref> section; numbers in parenthesis indicate the exposure multiples compared 21.3 mg/kg suspension formulation.</p></fn><fn id="TFN12"><p id="P50">Calves<sup>a</sup>, day 1, first dose PK data with KDU731 5 mg/kg BID dosing, AUC is 0-12 h.</p></fn><fn id="TFN13"><p id="P51">Calves<sup>b</sup>, day 7, 13<sup>th</sup> dose PK data with KDU731 5 mg/kg BID dosing, AUC is 0-12 h.</p></fn></table-wrap-foot></table-wrap></sec><sec sec-type="supplementary-material" id="SM"><title>Supplementary Information</title><p id="P52">Further details on assembly and screening of NITD parasite box and calf efficacy study are provided as supplementary information. Supplementary Information is linked to the online version of the paper at <ext-link ext-link-type="uri" xlink:href="http://www.nature.com/nature/index.html">www.nature.com/nature</ext-link>.</p><supplementary-material content-type="local-data" id="SD1"><label>Source Data Fig 2</label><media xlink:href="NIHMS72141-supplement-Source_Data_Fig_2.xlsx" mimetype="application" mime-subtype="octet-stream" orientation="portrait" xlink:type="simple" id="d36e2312" position="anchor"></media></supplementary-material><supplementary-material content-type="local-data" id="SD2"><label>Source Data Fig 3</label><media xlink:href="NIHMS72141-supplement-Source_Data_Fig_3.xlsx" mimetype="application" mime-subtype="octet-stream" orientation="portrait" xlink:type="simple" id="d36e2316" position="anchor"></media></supplementary-material><supplementary-material content-type="local-data" id="SD3"><label>Source Data for ED Figure 4 (b and c)</label><media xlink:href="NIHMS72141-supplement-Source_Data_for_ED_Figure_4__b_and_c_.xlsx" mimetype="application" mime-subtype="octet-stream" orientation="portrait" xlink:type="simple" id="d36e2320" position="anchor"></media></supplementary-material><supplementary-material content-type="local-data" id="SD4"><label>Source Data for ED Figure 6 (a and c)</label><media xlink:href="NIHMS72141-supplement-Source_Data_for_ED_Figure_6__a_and_c_.xlsx" mimetype="application" mime-subtype="octet-stream" orientation="portrait" xlink:type="simple" id="d36e2324" position="anchor"></media></supplementary-material><supplementary-material content-type="local-data" id="SD5"><label>Source Data for ED Figure 7a and b</label><media xlink:href="NIHMS72141-supplement-Source_Data_for_ED_Figure_7a_and_b.xlsx" mimetype="application" mime-subtype="octet-stream" orientation="portrait" xlink:type="simple" id="d36e2328" position="anchor"></media></supplementary-material><supplementary-material content-type="local-data" id="SD6"><label>Supplementary Information</label><media xlink:href="NIHMS72141-supplement-Supplementary_Information.pdf" mimetype="application" mime-subtype="pdf" orientation="portrait" xlink:type="simple" id="d36e2332" position="anchor"></media></supplementary-material><supplementary-material content-type="local-data" id="SD7"><label>Supplementary table</label><media xlink:href="NIHMS72141-supplement-Supplementary_table.pdf" mimetype="application" mime-subtype="pdf" orientation="portrait" xlink:type="simple" id="d36e2336" position="anchor"></media></supplementary-material></sec></body><back><ack id="S25"><title>Acknowledgments</title><p>We thank Saul Tzipori and Donald Girouard (Cummings School of Veterinary Medicine, Tufts University) for <italic>C. hominis</italic> oocysts; Bakela Nare (SCYNEXIS, US) for screening; Boon Heng Lee and Jeremy Selva (NITD) for HCI data; Margaret Weaver (Novartis Institutes for Biomedical Research (NIBR)) for rat toxicology studies; Irene Mueller (NIBR, US) for monkey PK; Bryan Yeung, Oliver Simon (NITD), Jason Roland, Venkatataiah Bollu, Arnab Chatterjee, Advait Nagle, Robert Moreau and Pranab Kumar Mishra (GNF) for compound synthesis; other NIBR colleagues for profiling; Jeremy Burrows (MMV) and Kelly Chibale (UCT) for MMV390048; Markus Meissner (University of Glasgow) for a plasmid carrying the red-shifted firefly luciferase gene. This work was supported in part by the Novartis Institutes for Biomedical Research (NIBR), the Wellcome Trust (Pathfinder #107678/Z/15/Z to BS and UM) and the National Institutes of Health (NIH R01AI112427 to BS). Inhibitors of the <italic>Plasmodium</italic> PI4K were discovered with the support of translational grants (WT078285 and WT096157) from the Wellcome Trust and funding from the Medicines for Malaria Venture (MMV). BS is a Georgia Research Alliance Distinguished Investigator and AS is supported by NIH fellowship F32AI124518. We thank our colleagues from Novartis Institute for Tropical Diseases (NITD), UGA, WSU’s Office of the Campus Veterinarian, Animal Resource Unit, and Office of Research Support and Operations and Renee Anderson, 5D Dairy Farm for their support. We are also grateful to the animal science and veterinary students at WSU for their invaluable participation in data collection and care of the research calves.</p></ack><fn-group><fn fn-type="con" id="FN1"><p id="P53"><bold>Author contributions</bold></p><p id="P54">U.H.M., S.V., J.A.Z., B.S. and T.T.D. conceived and designed study. B.S. wrote grant application with contribution from U.H.M. U.H.M., A.T.C. and G.M.C.B., developed <italic>C. parvum</italic> screening assays; C.G.N. and S.H.L. developed enzyme assay; C.B. analyzed P. falciparum EC50 data; U.H.M., P.G. and T.T.D. assembled screening library; R.R.K., BZ, performed compound synthesis; U.H.M., B.Z. and JW analyzed structure activity relationship; S.B.L. and F.B. analyzed in vivo pharmacokinetics data; L.Z. optimized formulation; U.H.M., G.F., F.J.L. and T.T.D. analyzed in vivo efficacy and toxicology results S.V., A.S., and B.S. designed mouse models based on transgenic parasites. S.V., A.S. and J.T. constructed transgenic parasites. S.V., A.S., C.F.B., G.T.H. validated mouse models and G.T.H., S.V. and C.F.B. tested compound. J.A.Z developed calf model and analyzed calf data, T.L.S. executed calf model, S.N. conducted anatomic pathology review for efficacy and toxicity, L.B.G. developed and executed calf stool analytics. B.S., S.V., U.H.M., and T.T.D. wrote the manuscript with contributions from J.A.Z., A.T.C., C.G.N. and S.B.L.</p></fn><fn id="FN2"><p id="P55"><bold>Author Information</bold></p><p id="P56">Reprints and permissions information is available at <ext-link ext-link-type="uri" xlink:href="http://www.nature.com/reprints/index.html">www.nature.com/reprints</ext-link></p><p id="P57">Requests for compounds (NITD) and transgenic parasite strains (UGA) are subject to a Material Transfer Agreement.</p></fn><fn fn-type="COI-statement" id="FN3"><p id="P58">The authors declare competing financial interests. RRK, BZ and BY are named as inventors on a pyrazolopyridine patent application related to this work (WO20140788002A1). UHM and TTD are named as inventors on a pending cryptosporidiosis patent applications related to this work; all NITD affiliated authors are employees of Novartis and some own shares of Novartis.</p></fn><fn id="FN4"><p id="P59"><bold>Data Availability</bold></p><p id="P60">Source data analyzed for mouse and calf infection experiments shown in <xref ref-type="supplementary-material" rid="SD1">Figure 2</xref> and <xref ref-type="supplementary-material" rid="SD2">3</xref> are included as excel files.</p></fn></fn-group><ref-list><ref id="R1"><label>1</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Liu</surname><given-names>L</given-names></name><etal></etal></person-group><article-title>Global, regional, and national causes of under-5 mortality in 2000-15: an updated systematic analysis with implications for the Sustainable Development Goals</article-title><source>Lancet</source><year>2016</year><pub-id 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Hits with inhibition >3 SD are shown in red <bold>b</bold>, Structure of the pyrazolopyridine lead KU731. <bold>c</bold>, <italic>In vitro</italic> activity of KDU731 (red), MMV3900048 (maroon) and nitazoxanide (black) against <italic>C. parvum</italic> (solid line) and <italic>C. hominis</italic> (dashed line). <bold>d</bold>, Correlation of growth inhibition (EC<sub>50</sub>) of selected compounds between <italic>C. parvum</italic> (Cp) and <italic>P. falciparum</italic> (Pf). Pyrazolopyridine analogs are shown in red, imidazopyrazines in blue, quinoxaline in green, and diaryl-aminopyridine in maroon. <bold>e</bold>, Correlation of growth inhibition (EC<sub>50</sub>) between <italic>C. parvum</italic> and <italic>C. hominis</italic> for selected compounds. <bold>f</bold>, Inhibition of phosphatidylinositide kinase activity of purified enzyme by KDU731, KDU691, MMV390048 and BQR695 (means ± s.e. with at least 3 biological replicates) in presence of 3 μM ATP. <bold>g</bold>, Correlation between inhibition of PI kinase activity of purified CpPI(4)K enzyme (IC<sub>50</sub>) and growth inhibition (EC<sub>50</sub>) of <italic>C. parvum</italic> with selected compounds (colors as in <bold>d</bold>). <bold>h,</bold> CpPI(4)K activity across a range of ATP concentrations in the presence of 1.56 - 50 nM KDU731. Data shown in <bold>c</bold> and <bold>f</bold> represent mean ± s.e., n=3 biological replicates, representative data shown. Experiments shown in <bold>d</bold>, <bold>e</bold>, and <bold>g</bold> were repeated at least twice and geometric mean EC<sub>50</sub> values are plotted, and <bold>h</bold> has been repeated twice (biological replicates) and one representative assay is shown.</p></caption><graphic xlink:href="emss-72141-f001"></graphic></fig><fig id="F2" orientation="portrait" position="float"><label>Figure 2</label><caption><title>KDU731 has potent activity against <italic>Cryptosporidium</italic> in immunocompromised IFN-γ KO mice.</title><p><bold>a</bold>, Mice were infected with 10,000 <italic>C. parvum</italic> UGA1 Nluc or UGA2 Fluc oocysts. Parasite load in the feces was determined by measuring fecal Nluc activity and parasite tissue load was quantified by whole animal imaging of Fluc activity. Different 7 day treatment courses are indicated in red. Fecal luciferase measurements of individual mice with treatment initiated after 3 (<bold>b</bold>) or 11 (<bold>c</bold>) days of infection (red, vehicle control shown in black), n=5 and n=9 mice respectively, representative of two biological replicates for b and c. <bold>d</bold>, Histology of the ileum of infected mice (shown in <bold>c</bold>) after one week of KDU731 treatment compared to vehicle treated control on day 18 (n=3 biological replicates, representative images shown here). Note numerous intracellular parasite stages (white arrowheads) and extracellular oocysts (black arrowhead) in the control absent in the treated mice. Vehicle treated animals showed disorganized columnar epithelial cells and loss of brush border when compared to KDU731 treated mice. White box indicates section shown at higher magnification to the right. Mice (n=5) infected with UGA2 FLuc were treated on day 7 with 10mg/kg of KDU731 (red (<bold>e</bold>) and <bold>g</bold>), 100 mg/kg nitazoxanide (red (<bold>f</bold>), also see <xref ref-type="fig" rid="F8">Extended Data Fig. 5</xref>), or vehicle (black, <bold>e</bold> and <bold>f</bold>) for one week. Animals were monitored by whole-animal-imaging and a radiance scale and quantification of total flux in photon/sec is shown. *Animals shown on the baseline were below the level of detection.</p></caption><graphic xlink:href="emss-72141-f002"></graphic></fig><fig id="F3" orientation="portrait" position="float"><label>Figure 3</label><caption><title>Therapeutic efficacy of KDU731 in neonatal calf clinical model of cryptosporidiosis.</title><p><bold>a</bold>, Within 48 h of birth, calves were challenged with 5x10<sup>7</sup><italic>C. parvum</italic> oocysts. Fecal oocyst shedding was enumerated via qPCR and calves were clinically evaluated every 12 h. Oral treatment with KDU731 (5mg/kg every 12 hours for 7 days) was initiated when calves showed severe diarrhea (Fecal Consistency Score = 3) and oocysts in their feces. KDU731 treated calves shed significantly fewer oocysts in their stool (<bold>b</bold>), had significantly fewer days of severe diarrhea (<bold>c</bold>) and were significantly less dehydrated (<bold>d</bold>) than untreated calves. <bold>e</bold>, Day 1 and day 7 plasma PK profile of KDU731. Data shown here (panels b,c,d, e) are from infected calves that were treated with vehicle (n=6) or KDU731 (n = 7), n represents the number of calves. <italic>In vitro</italic> EC<sub>90</sub> is shown as dotted line. Error bars are SEM. Data shown here (c and d) as a “box and whiskers” plot, the box extends from the 25th to 75th percentiles and whiskers with min to max showing all data points. Data in panel b and e were determined to display non-Gaussian distribution and were log-transformed prior to statistical analysis using <italic>t</italic>-tests with two-tailed *<italic>P</italic><0.05, **0.01, ***0.001, and ****0.0001. Panels c and d were determined to be normally distributed and analyzed using t-tests<italic>.</italic></p></caption><graphic xlink:href="emss-72141-f003"></graphic></fig><table-wrap id="T1" position="float" orientation="portrait"><label>Table 1</label><caption><title>Activity of pyrazolopyridine analogs and other known PI(4) kinase inhibitors</title></caption><table frame="hsides" rules="groups"><thead><tr><th align="left" rowspan="1" colspan="1">Compound</th><th align="left" rowspan="1" colspan="1"><italic>Cp</italic> CPE EC<sub>50</sub></th><th align="left" rowspan="1" colspan="1"><italic>Cp</italic> HCI IC<sub>50</sub></th><th align="left" rowspan="1" colspan="1"><italic>Ch</italic> CPE EC<sub>50</sub></th><th align="left" rowspan="1" colspan="1"><italic>Cp</italic> PI(4)K IC<sub>50</sub></th><th align="left" rowspan="1" colspan="1"><italic>Pf</italic> 3D7 IC<sub>50</sub></th><th align="left" rowspan="1" colspan="1">HepG2 CC<sub>50</sub></th></tr></thead><tbody><tr><td align="left" rowspan="1" colspan="1">KDU731</td><td align="left" rowspan="1" colspan="1">0.107 ± 0.039</td><td align="left" rowspan="1" colspan="1">0.063 ± 0.028</td><td align="left" rowspan="1" colspan="1">0.130 ± 0.074</td><td align="left" rowspan="1" colspan="1">0.025 ± 0.004</td><td align="left" rowspan="1" colspan="1">0.003 ± 0.001</td><td align="left" rowspan="1" colspan="1">15.621 ± 8.621</td></tr><tr><td align="left" rowspan="1" colspan="1">KDU691</td><td align="left" rowspan="1" colspan="1">0.096 ± 0.044</td><td align="left" rowspan="1" colspan="1">0.054 ± 0.029</td><td align="left" rowspan="1" colspan="1">0.082 ± 0.017</td><td align="left" rowspan="1" colspan="1">0.017 ± 0.012</td><td align="left" rowspan="1" colspan="1">0.108 ± 0.061</td><td align="left" rowspan="1" colspan="1">27.291 ± 14.169</td></tr><tr><td align="left" rowspan="1" colspan="1">MMV390048</td><td align="left" rowspan="1" colspan="1">12.792 ± 6.264</td><td align="left" rowspan="1" colspan="1">13.422</td><td align="left" rowspan="1" colspan="1">11.85 ± 2.758</td><td align="left" rowspan="1" colspan="1">8.335 ± 2.355</td><td align="left" rowspan="1" colspan="1">0.042 ± 0.022</td><td align="left" rowspan="1" colspan="1">26.156 ± 16.063</td></tr><tr><td align="left" rowspan="1" colspan="1">BQR695</td><td align="left" rowspan="1" colspan="1">11.837 ± 1.889</td><td align="left" rowspan="1" colspan="1">8.344</td><td align="left" rowspan="1" colspan="1">8.565</td><td align="left" rowspan="1" colspan="1">7.305 ± 0.841</td><td align="left" rowspan="1" colspan="1">0.118 ± 0.055</td><td align="left" rowspan="1" colspan="1">26.711 ± 13.870</td></tr><tr><td align="left" rowspan="1" colspan="1">Nitazoxanide</td><td align="left" rowspan="1" colspan="1">14.286 ± 7.127</td><td align="left" rowspan="1" colspan="1">2.927 ± 0.808</td><td align="left" rowspan="1" colspan="1">> 20.000</td><td align="left" rowspan="1" colspan="1">> 10.000</td><td align="left" rowspan="1" colspan="1">> 10.000</td><td align="left" rowspan="1" colspan="1">12.749 ± 2.378</td></tr><tr><td align="left" rowspan="1" colspan="1">KDU370</td><td align="left" rowspan="1" colspan="1">1.540</td><td align="left" rowspan="1" colspan="1">nd</td><td align="left" rowspan="1" colspan="1">2.543</td><td align="left" rowspan="1" colspan="1">0.212</td><td align="left" rowspan="1" colspan="1">0.074 ± 0.032</td><td align="left" rowspan="1" colspan="1">> 50.000</td></tr><tr><td align="left" rowspan="1" colspan="1">KDZ464</td><td align="left" rowspan="1" colspan="1">0.113 ± 0.072</td><td align="left" rowspan="1" colspan="1">0.232</td><td align="left" rowspan="1" colspan="1">0.119</td><td align="left" rowspan="1" colspan="1">0.003</td><td align="left" rowspan="1" colspan="1">0.005 ± 0.001</td><td align="left" rowspan="1" colspan="1">21.222 ± 10.594</td></tr><tr><td align="left" rowspan="1" colspan="1">LNN134</td><td align="left" rowspan="1" colspan="1">1.951 ± 0.340</td><td align="left" rowspan="1" colspan="1">1.031</td><td align="left" rowspan="1" colspan="1">1.937</td><td align="left" rowspan="1" colspan="1">0.488</td><td align="left" rowspan="1" colspan="1">0.126 ± 0.115</td><td align="left" rowspan="1" colspan="1">40.025 ± 17.278</td></tr><tr><td align="left" rowspan="1" colspan="1">LMW740</td><td align="left" rowspan="1" colspan="1">> 20.000</td><td align="left" rowspan="1" colspan="1">> 20.000</td><td align="left" rowspan="1" colspan="1">> 20.000</td><td align="left" rowspan="1" colspan="1">>10</td><td align="left" rowspan="1" colspan="1">0.521</td><td align="left" rowspan="1" colspan="1">> 50.000</td></tr><tr><td align="left" rowspan="1" colspan="1">KEL204</td><td align="left" rowspan="1" colspan="1">> 20.000</td><td align="left" rowspan="1" colspan="1">> 20.000</td><td align="left" rowspan="1" colspan="1">> 20.000</td><td align="left" rowspan="1" colspan="1">3.701</td><td align="left" rowspan="1" colspan="1">1.432</td><td align="left" rowspan="1" colspan="1">> 50.000</td></tr></tbody></table><table-wrap-foot><fn id="TFN1"><p id="P61"><italic>Cp</italic>, <italic>C. parvum</italic>; <italic>Ch</italic>, <italic>C. hominis</italic>; <italic>Pf</italic>, <italic>P. falciparum</italic>; CPE, Cytopathic Effect assay; HCI; High Content Imaging assay</p></fn><fn id="TFN2"><p id="P62">EC<sub>50</sub>, IC<sub>50</sub> and CC<sub>50</sub><italic>values</italic> are in μM nd, not determined</p></fn><fn id="TFN3"><p id="P63">Data shown here are means ± s.e. , n=3 biological replicates</p></fn></table-wrap-foot></table-wrap></floats-group></pmc></record>Pour manipuler ce document sous Unix (Dilib)
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