Characterization of the substituted N-triazole oxindole TROX-1, a small-molecule, state-dependent inhibitor of Ca(V)2 calcium channels.

Biological, genetic, and clinical evidence provide validation for N-type calcium channels (Ca(V)2.2) as therapeutic targets for chronic pain. A state-dependent Ca(V)2.2 inhibitor may provide an improved therapeutic window over ziconotide, the peptidyl Ca(V)2.2 inhibitor used clinically. Supporting this notion, we recently reported that in preclinical models, the state-dependent Ca(V)2 inhibitor (3R)-5-(3-chloro-4fluorophenyl)- 3-methyl-3-(pyrimidin-5-ylmethyl)-1-(1H-1,2,4-triazol3- yl)-1,3-dihydro-2H-indol-2-one (TROX-1) has an improved therapeutic window compared with ziconotide. Here we characterize TROX-1 inhibition of Ca(V)2.2 channels in more detail. When channels are biased toward open/ inactivated states by depolarizing the membrane potential under voltage-clamp electrophysiology, TROX-1 inhibits Ca(V)2.2 channels with an IC50 of 0.11 mu M. The voltage dependence of Ca(V)2.2 inhibition was examined using automated electrophysiology. TROX-1 IC50 values were 4.2, 0.90, and 0.36 mu M at -110, -90, and -70 mV, respectively. TROX-1 displayed usedependent inhibition of Ca V 2.2 with a 10-fold IC50 separation between first (27 mu M) and last (2.7 mu M) pulses in a train. In a fluorescence- based calcium influx assay, TROX-1 inhibited Ca(V)2.2 channels with an IC 50 of 9.5 mu M under hyperpolarized conditions and 0.69 mu M under depolarized conditions. Finally, TROX-1 potency was examined across the Ca(V)2 subfamily. Depolarized IC50 values were 0.29, 0.19, and 0.28 mu M by manual electrophysiology using matched conditions and 1.8, 0.69, and 1.1 mu M by calcium influx for Ca(V)2, Ca(V)2.2, and Ca(V)2.3, respectively. Together, these in vitro data support the idea that a state-dependent, non-subtype-selective Ca(V)2 channel inhibitor can achieve an improved therapeutic window over the relatively state-independent Ca(V)2.2-selective inhibitor ziconotide in preclinical models of chronic pain.