{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,1,20]],"date-time":"2026-01-20T07:50:26Z","timestamp":1768895426761,"version":"3.49.0"},"reference-count":37,"publisher":"American Society for Microbiology","issue":"6","license":[{"start":{"date-parts":[[2018,6,1]],"date-time":"2018-06-01T00:00:00Z","timestamp":1527811200000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/journals.asm.org\/non-commercial-tdm-license"}],"funder":[{"DOI":"10.13039\/100000060","name":"HHS | NIH | National Institute of Allergy and Infectious Diseases","doi-asserted-by":"publisher","award":["R01 AI111341"],"award-info":[{"award-number":["R01 AI111341"]}],"id":[{"id":"10.13039\/100000060","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000060","name":"HHS | NIH | National Institute of Allergy and Infectious Diseases","doi-asserted-by":"publisher","award":["R01 AI089441"],"award-info":[{"award-number":["R01 AI089441"]}],"id":[{"id":"10.13039\/100000060","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000060","name":"HHS | NIH | National Institute of Allergy and Infectious Diseases","doi-asserted-by":"publisher","award":["T32AI007509"],"award-info":[{"award-number":["T32AI007509"]}],"id":[{"id":"10.13039\/100000060","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000199","name":"U.S. Department of Agriculture","doi-asserted-by":"publisher","award":["2014-67015-22106"],"award-info":[{"award-number":["2014-67015-22106"]}],"id":[{"id":"10.13039\/100000199","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100009633","name":"HHS | NIH | Eunice Kennedy Shriver National Institute of Child Health and Human Development","doi-asserted-by":"publisher","award":["R01 HD080670"],"award-info":[{"award-number":["R01 HD080670"]}],"id":[{"id":"10.13039\/100009633","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["journals.asm.org"],"crossmark-restriction":true},"short-container-title":["Antimicrob Agents Chemother"],"published-print":{"date-parts":[[2018,6]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n          <jats:p>\n            In\n            <jats:named-content content-type=\"genus-species\">Toxoplasma gondii<\/jats:named-content>\n            , calcium-dependent protein kinase 1 (CDPK1) is an essential protein kinase required for invasion of host cells. We have developed several hundred CDPK1 inhibitors, many of which block invasion. Inhibitors with similar 50% inhibitory concentrations (IC\n            <jats:sub>50<\/jats:sub>\n            s) were tested in thermal shift assays for their ability to stabilize CDPK1 in cell lysates, in intact cells, or in purified form. Compounds that inhibited parasite growth stabilized CDPK1 in all assays. In contrast, two compounds that showed poor growth inhibition stabilized CDPK1 in lysates but not in cells. Thus, cellular exclusion could explain exceptions in the correlation between the action on the target and cellular activity. We used thermal shift assays to examine CDPK1 in two clones that were independently selected by growth in the CDPK1 inhibitor RM-1-132 and that had increased 50% effective concentrations (EC\n            <jats:sub>50<\/jats:sub>\n            s) for the compound. The A and C clones had distinct point mutations in the CDPK1 kinase domain, H201Q and L96P, respectively, residues that lie near one another in the inactive isoform. Purified mutant proteins showed RM-1-132 IC\n            <jats:sub>50<\/jats:sub>\n            s and thermal shifts similar to those shown by wild-type CDPK1. Reduced inhibitor stabilization (and a presumed reduced interaction) was observed only in cellular thermal shift assays. This highlights the utility of cellular thermal shift assays in demonstrating that resistance involves reduced on-target engagement (even if biochemical assays suggest otherwise). Indeed, similar EC\n            <jats:sub>50<\/jats:sub>\n            s were observed upon overexpression of the mutant proteins, as in the corresponding drug-selected parasites, although high levels of CDPK1(H201Q) only modestly increased resistance compared to that achieved with high levels of wild-type enzyme.\n          <\/jats:p>","DOI":"10.1128\/aac.00051-18","type":"journal-article","created":{"date-parts":[[2018,3,16]],"date-time":"2018-03-16T13:44:45Z","timestamp":1521207885000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":16,"title":["Toxoplasma Calcium-Dependent Protein Kinase 1 Inhibitors: Probing Activity and Resistance Using Cellular Thermal Shift Assays"],"prefix":"10.1128","volume":"62","author":[{"given":"Suzanne","family":"Scheele","sequence":"first","affiliation":[{"name":"Center for Infectious Disease Research, Seattle, Washington, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jennifer A.","family":"Geiger","sequence":"additional","affiliation":[{"name":"Center for Infectious Disease Research, Seattle, Washington, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Amy E.","family":"DeRocher","sequence":"additional","affiliation":[{"name":"Center for Infectious Disease Research, Seattle, Washington, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ryan","family":"Choi","sequence":"additional","affiliation":[{"name":"Department of Medicine, Division of Allergy and Infectious Disease, Center for Emerging and Re-emerging Infectious Diseases (CERID), University of Washington, Seattle, Washington, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Tess R.","family":"Smith","sequence":"additional","affiliation":[{"name":"Department of Medicine, Division of Allergy and Infectious Disease, Center for Emerging and Re-emerging Infectious Diseases (CERID), University of Washington, Seattle, Washington, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Matthew A.","family":"Hulverson","sequence":"additional","affiliation":[{"name":"Department of Medicine, Division of Allergy and Infectious Disease, Center for Emerging and Re-emerging Infectious Diseases (CERID), University of Washington, Seattle, Washington, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Rama Subba Rao","family":"Vidadala","sequence":"additional","affiliation":[{"name":"Department of Chemistry, University of Washington, Seattle, Washington, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Lynn K.","family":"Barrett","sequence":"additional","affiliation":[{"name":"Department of Medicine, Division of Allergy and Infectious Disease, Center for Emerging and Re-emerging Infectious Diseases (CERID), University of Washington, Seattle, Washington, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Dustin J.","family":"Maly","sequence":"additional","affiliation":[{"name":"Department of Chemistry, University of Washington, Seattle, Washington, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ethan A.","family":"Merritt","sequence":"additional","affiliation":[{"name":"Department of Biochemistry, University of Washington, Seattle, Washington, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Kayode K.","family":"Ojo","sequence":"additional","affiliation":[{"name":"Department of Medicine, Division of Allergy and Infectious Disease, Center for Emerging and Re-emerging Infectious Diseases (CERID), University of Washington, Seattle, Washington, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Wesley C.","family":"Van Voorhis","sequence":"additional","affiliation":[{"name":"Department of Medicine, Division of Allergy and Infectious Disease, Center for Emerging 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