3-Chlorodiphenylamine activates cardiac troponin by a mechanism distinct from bepridil or TFP.

Despite extensive efforts spanning multiple decades, the development of highly effective Ca2+ sensitizers for the heart remains an elusive goat. 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 Cfn. Previously, we determined that many compounds based on diphenylamine (DPA) were able to bind to a cTnC-cTnl chimera with moderate affinity (K-d similar to 10-120 mu M). Of these compounds, 3-chlorodiphenytamine (3-Cl-DPA) bound most tightly (K-d of 10 mu M). 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 bepridit, 3-Cl-DPA is able to bind to the isolated N-terminal domain (N-domain) of cTnC (K-d of 6 mu M). 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 stows the rate of Ca(2+ )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-TnI 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.