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Existing Ca2+ sensitizers have other targets in addition to cardiac troponin (cTn), which can lead to adverse side effects, such as hypotension or arrhythmias. Thus, there is a need to design Ca2+-sensitizing drugs with higher affinity and selectivity for cTn. Previously, we determined that many compounds based on diphenylamine (DPA) were able to bind to a cTnC\u2013cTnI chimera with moderate affinity (Kd \u223c10\u2013120 \u00b5M). Of these compounds, 3-chlorodiphenylamine (3-Cl-DPA) bound most tightly (Kd of 10 \u00b5M). Here, we investigate 3-Cl-DPA further and find that it increases the Ca2+ sensitivity of force development in skinned cardiac muscle. Using NMR, we show that, like the known Ca2+ sensitizers, trifluoperazine (TFP) and bepridil, 3-Cl-DPA is able to bind to the isolated N-terminal domain (N-domain) of cTnC (Kd of 6 \u00b5M). However, while the bulky molecules of TFP and bepridil stabilize the open state of the N-domain of cTnC, the small and flexible 3-Cl-DPA molecule is able to bind without stabilizing this open state. Thus, unlike TFP, which drastically slows the rate of Ca2+ dissociation from the N-domain of isolated cTnC in a dose-dependent manner, 3-Cl-DPA has no effect on the rate of Ca2+ dissociation. On the other hand, the affinity of 3-Cl-DPA for a cTnC\u2013TnI chimera is at least an order of magnitude higher than that of TFP or bepridil, likely because 3-Cl-DPA is less disruptive of cTnI binding to cTnC. Therefore, 3-Cl-DPA has a bigger effect on the rate of Ca2+ dissociation from the entire cTn complex than TFP and bepridil. Our data suggest that 3-Cl-DPA activates the cTn complex via a unique mechanism and could be a suitable scaffold for the development of novel treatments for systolic heart failure.<\/jats:p>","DOI":"10.1085\/jgp.201812131","type":"journal-article","created":{"date-parts":[[2018,11,15]],"date-time":"2018-11-15T10:35:23Z","timestamp":1542278123000},"page":"9-17","update-policy":"https:\/\/doi.org\/10.1085\/jgp.crossmarkpolicy","source":"Crossref","is-referenced-by-count":13,"title":["3-Chlorodiphenylamine activates cardiac troponin by a mechanism distinct from bepridil or TFP"],"prefix":"10.1085","volume":"151","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-2101-9996","authenticated-orcid":true,"given":"Svetlana B.","family":"Tikunova","sequence":"first","affiliation":[{"name":"Department of Physiology and Cell Biology, The Ohio State University, Columbus, OH 1"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Andres","family":"Cuesta","sequence":"additional","affiliation":[{"name":"Department of Physiology and Cell Biology, The Ohio State University, Columbus, OH 1"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Morgan","family":"Price","sequence":"additional","affiliation":[{"name":"Department of Physiology and Cell Biology, The Ohio State University, Columbus, OH 1"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Monica X.","family":"Li","sequence":"additional","affiliation":[{"name":"Departments of Medicine and Biochemistry, University of Alberta, Edmonton, AB, Canada 2"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Natalya","family":"Belevych","sequence":"additional","affiliation":[{"name":"Division of Biosciences, College of Dentistry, The Ohio State University, Columbus, OH 3"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Brandon J.","family":"Biesiadecki","sequence":"additional","affiliation":[{"name":"Department of Physiology and Cell Biology, The Ohio State University, Columbus, OH 1"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Peter J.","family":"Reiser","sequence":"additional","affiliation":[{"name":"Division of Biosciences, College of Dentistry, The Ohio State University, Columbus, OH 3"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Peter M.","family":"Hwang","sequence":"additional","affiliation":[{"name":"Departments of Medicine and Biochemistry, University of Alberta, Edmonton, AB, Canada 2"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jonathan P.","family":"Davis","sequence":"additional","affiliation":[{"name":"Department of Physiology and Cell Biology, The Ohio State University, Columbus, OH 1"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"291","published-online":{"date-parts":[[2018,11,15]]},"reference":[{"key":"2023072705283803900_bib1","doi-asserted-by":"publisher","first-page":"75","DOI":"10.1002\/prot.340170110","article-title":"Primary structure effects on peptide group hydrogen exchange","volume":"17","author":"Bai","year":"1993","journal-title":"Proteins."},{"key":"2023072705283803900_bib2","doi-asserted-by":"publisher","first-page":"e67","DOI":"10.1161\/CIR.0000000000000558","article-title":"Heart Disease and Stroke Statistics-2018 Update: A Report From the American Heart Association","volume":"137","author":"Benjamin","year":"2018","journal-title":"Circulation."},{"key":"2023072705283803900_bib3","doi-asserted-by":"publisher","first-page":"13831","DOI":"10.1021\/bi001106+","article-title":"Acid pairs increase the N-terminal Ca2+ affinity of CaM by increasing the rate of Ca2+ association","volume":"39","author":"Black","year":"2000","journal-title":"Biochemistry."},{"key":"2023072705283803900_bib4","doi-asserted-by":"publisher","first-page":"398","DOI":"10.1111\/j.1476-4431.2012.00768.x","article-title":"A review of the pharmacology and clinical uses of pimobendan","volume":"22","author":"Boyle","year":"2012","journal-title":"J. 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