title: Characterization of the substituted N-triazole oxindole TROX-1, a small-molecule, state-dependent inhibitor of Ca(V)2 calcium channels.
abstract: 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-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 Ca(V)2.2 channels with an IC(50) of 0.11 μM. The voltage dependence of Ca(V)2.2 inhibition was examined using automated electrophysiology. TROX-1 IC(50) values were 4.2, 0.90, and 0.36 μM at -110, -90, and -70 mV, respectively. TROX-1 displayed use-dependent inhibition of Ca(V)2.2 with a 10-fold IC(50) separation between first (27 μM) and last (2.7 μM) 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 μM under hyperpolarized conditions and 0.69 μM under depolarized conditions. Finally, TROX-1 potency was examined across the Ca(V)2 subfamily. Depolarized IC(50) 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 Ca(V)2.1, 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.
authors.affiliation: Department of Ion Channels, Merck Research Laboratories, Rahway, New Jersey, USA.
authors.forename: Andrew M
authors.initials: AM
authors.lastname: Swensen
authors.forename: James
authors.initials: J
authors.lastname: Herrington
authors.forename: Randal M
authors.initials: RM
authors.lastname: Bugianesi
authors.forename: Ge
authors.initials: G
authors.lastname: Dai
authors.forename: Rodolfo J
authors.initials: RJ
authors.lastname: Haedo
authors.forename: Kevin S
authors.initials: KS
authors.lastname: Ratliff
authors.forename: McHardy M
authors.initials: MM
authors.lastname: Smith
authors.forename: Vivien A
authors.initials: VA
authors.lastname: Warren
authors.forename: Stephen P
authors.initials: SP
authors.lastname: Arneric
authors.forename: Cyrus
authors.initials: C
authors.lastname: Eduljee
authors.forename: David
authors.initials: D
authors.lastname: Parker
authors.forename: Terrance P
authors.initials: TP
authors.lastname: Snutch
authors.forename: Scott B
authors.initials: SB
authors.lastname: Hoyt
authors.forename: Clare
authors.initials: C
authors.lastname: London
authors.forename: Joseph L
authors.initials: JL
authors.lastname: Duffy
authors.forename: Gregory J
authors.initials: GJ
authors.lastname: Kaczorowski
authors.forename: Owen B
authors.initials: OB
authors.lastname: McManus
mesh_terms: D002121:Calcium Channel Blockers / Q000737:chemistry* / Q000494:pharmacology; D020864:Calcium Channels, N-Type / Q000187:drug effects*; D002460:Cell Line; D006801:Humans; D007211:Indoles / Q000737:chemistry* / Q000494:pharmacology; D020128:Inhibitory Concentration 50; D008564:Membrane Potentials / Q000187:drug effects; D018408:Patch-Clamp Techniques; D014230:Triazoles / Q000737:chemistry* / Q000494:pharmacology
chemical_list: C551468:5-(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; D002121:Calcium Channel Blockers; D020864:Calcium Channels, N-Type; D007211:Indoles; D014230:Triazoles