RT Journal Article SR Electronic T1 Characterization of the Substituted N-Triazole Oxindole TROX-1, a Small-Molecule, State-Dependent Inhibitor of Cav2 Calcium Channels JF Molecular Pharmacology JO Mol Pharmacol FD American Society for Pharmacology and Experimental Therapeutics SP 488 OP 497 DO 10.1124/mol.111.075226 VO 81 IS 3 A1 Andrew M. Swensen A1 James Herrington A1 Randal M. Bugianesi A1 Ge Dai A1 Rodolfo J. Haedo A1 Kevin S. Ratliff A1 McHardy M. Smith A1 Vivien A. Warren A1 Stephen P. Arneric A1 Cyrus Eduljee A1 David Parker A1 Terrance P. Snutch A1 Scott B. Hoyt A1 Clare London A1 Joseph L. Duffy A1 Gregory J. Kaczorowski A1 Owen B. McManus YR 2012 UL http://molpharm.aspetjournals.org/content/81/3/488.abstract AB Biological, genetic, and clinical evidence provide validation for N-type calcium channels (CaV2.2) as therapeutic targets for chronic pain. A state-dependent CaV2.2 inhibitor may provide an improved therapeutic window over ziconotide, the peptidyl CaV2.2 inhibitor used clinically. Supporting this notion, we recently reported that in preclinical models, the state-dependent CaV2 inhibitor (3R)-5-(3-chloro-4-fluorophenyl)-3-methyl-3-(pyrimidin-5-ylmethyl)-1-(1H-1,2,4-triazol-3-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 Cav2.2 channels in more detail. When channels are biased toward open/inactivated states by depolarizing the membrane potential under voltage-clamp electrophysiology, TROX-1 inhibits CaV2.2 channels with an IC50 of 0.11 μM. The voltage dependence of CaV2.2 inhibition was examined using automated electrophysiology. TROX-1 IC50 values were 4.2, 0.90, and 0.36 μM at −110, −90, and −70 mV, respectively. TROX-1 displayed use-dependent inhibition of CaV2.2 with a 10-fold IC50 separation between first (27 μM) and last (2.7 μM) pulses in a train. In a fluorescence-based calcium influx assay, TROX-1 inhibited CaV2.2 channels with an IC50 of 9.5 μM under hyperpolarized conditions and 0.69 μM under depolarized conditions. Finally, TROX-1 potency was examined across the CaV2 subfamily. Depolarized IC50 values were 0.29, 0.19, and 0.28 μM by manual electrophysiology using matched conditions and 1.8, 0.69, and 1.1 μM by calcium influx for CaV2.1, CaV2.2, and CaV2.3, respectively. Together, these in vitro data support the idea that a state-dependent, non–subtype-selective CaV2 channel inhibitor can achieve an improved therapeutic window over the relatively state-independent CaV2.2-selective inhibitor ziconotide in preclinical models of chronic pain.