Characterization of the triazine, T4, a representative from a novel series of CaV2 inhibitors with strong state-dependence, poor use-dependence, and distinctively fast kinetics.

Abstract There is strong pharmacological, biological, and genetic evidence supporting the role of N-type calcium channels (Ca V 2.2) in nociception. There is also human validation data from ziconotide, the Ca V 2.2-selective peptidyl inhibitor used clinically to treat refractory pain. Unfortunately, ziconotide utility is limited by its narrow therapeutic window and required intrathecal route of administration. A major focus has been placed on identifying state-dependent Ca V 2.2 inhibitors to improve safety margins. Much less attention, however, has been given to characterizing the kinetics of Ca V 2.2 inhibitors as a means to further differentiate compounds and maximize therapeutic potential. Here we provide a detailed characterization of the Ca V 2.2 inhibitor T4 in terms of its state-dependence, use-dependence, kinetics, and mechanism of inhibition. Compound T4 displayed a >20-fold difference in potency when measured under inactivating conditions (IC 50 =1.1 μM) as compared to closed-state conditions (IC 50 =25 μM). At 3 μM, T4 produced a 15-fold hyperpolarizing shift in the inactivation curve for Ca V 2.2 while having no effect on channel activation. To assess the kinetic properties of T4 in a more physiological manner, its inhibition kinetics were assessed at 32 °C using 2 mM Ca 2+ as the charge carrier. Surprisingly, the repriming rate for Ca V 2.2 channels at hyperpolarized potentials was similar in both the presence and absence of T4. This was in contrast to other compounds which markedly delayed repriming. Furthermore, T4 inhibited Ca V 2.2 channels more potently when channel inactivation was driven through a tonic sub-threshold depolarization rather than through a use-dependent protocol, despite similar levels of inactivation.