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 goal. 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-cTnI chimera with moderate affinity (K_d ∼10-120 µM). Of these compounds, 3-chlorodiphenylamine (3-Cl-DPA) bound most tightly (K_d of 10 µM). 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 (K_d of 6 µM). 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-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.