TY - JOUR T1 - Characterization of the Substituted N-triazole oxindole, TROX-1, a Small Molecule, State-dependent Inhibitor of Ca<sub>V</sub>2 Calcium Channels JF - Molecular Pharmacology JO - Mol Pharmacol DO - 10.1124/mol.111.075226 SP - mol.111.075226 AU - Andrew M. Swensen AU - James Herrington AU - Randal M. Bugianesi AU - Ge Dai AU - Rodolfo J. Haedo AU - Kevin S. Ratliff AU - McHardy M. Smith AU - Vivien A. Warren AU - Stephen P. Arneric AU - Cyrus Eduljee AU - David Parker AU - Terrance P. Snutch AU - Scott B. Hoyt AU - Clare London AU - Joseph L. Duffy AU - Gregory J. Kaczorowski AU - Owen B. McManus Y1 - 2011/12/21 UR - http://molpharm.aspetjournals.org/content/early/2011/12/21/mol.111.075226.abstract N2 - 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 the state-dependent CaV2 inhibitor TROX-1 has an improved therapeutic window compared to ziconotide in preclinical models. Here we characterize TROX-1 inhibition of CaV2.2 channels in more detail. When channels are biased towards 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 μM at -110 mV, 0.90 μM at -90 mV, and 0.36 μM at -70 mV. 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. Lastly, TROX-1 potency was examined across the CaV2 subfamily. Depolarized IC50 values were 0.29 μM, 0.19 μM and 0.28 μM by manual electrophysiology using matched conditions and 1.8 μM, 0.69 μM 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. ER -