UK‐78,282, a novel piperidine compound that potently blocks the Kv1.3 voltage‐gated potassium channel and inhibits human T cell activation

Abstract

1 UK-78,282, a novel piperidine blocker of the T lymphocyte voltage-gated K + channel, Kv1.3, was discovered by screening a large compound ®le using a high-throughput 86 Rb e ux assay. This compound blocks Kv1.3 with a IC 50 of *200 nM and 1 : 1 stoichiometry. A closely related compound, 325, containing a benzyl moiety in place of the benzhydryl in UK-78,282, is signi®cantly less potent. 2 Three lines of evidence indicate that UK-78,282 inhibits Kv1.3 in a use-dependent manner by preferentially blocking and binding to the C-type inactivated state of the channel. Increasing the fraction of inactivated channels by holding the membrane potential at 750 mV enhances the channel's sensitivity to 282. Decreasing the number of inactivated channels by exposure to *160 mM external K + decreases the sensitivity to UK-78,282. Mutations that alter the rate of Ctype inactivation also change the channel's sensitivity to UK-78,282 and there is a direct correlation between t h and IC 50 values.3 Competition experiments suggest that UK-78,282 binds to residues at the inner surface of the channel overlapping the site of action of verapamil. Internal tetraethylammonium and external charybdotoxin do not compete UK-78,282's action on the channel. 4 UK-78,282 displays marked selectivity for Kv1.3 over several other closely related K + channels, the only exception being the rapidly inactivating voltage-gated K + channel, Kv1.4. 5 UK-78,282 e ectively suppresses human T-lymphocyte activation.

Verapamil Inhibits Proliferation of LNCaP Human Prostate Cancer Cells Influencing K+ Channel Gating

“…Second, the verapamil-produced inhibition of K ϩ channels was not use-dependent: the initial I K evoked after Ϸ30 s preincubation with verapamil solution was inhibited to its steady-state level, indicating that verapamil interacted with the resting form of the channel. Verapamil and other phenylalkylamines were previously considered to be mainly state-dependent open-channel blockers (DeCoursey, 1995;Trequattrini et al, 1998;Rauer and Grissmer, 1999) and K ϩ channel-inactivated state blockers (Tatsuta et al, 1994;Hanson et al, 1999;Zhang et al, 1999). Third, K ϩ channel inhibition by externally applied verapamil in LNCaP cells was rapid and rapidly reversible (Ϸ30 s), suggesting an easy association/dissociation with its binding site.…”

“…We applied two different approaches to evaluate the type of competition between TEA and verapamil during their simultaneous inhibition of K ϩ channels. The first approach relied on the multiplication of the inhibitory effects of two drugs on I K if their inhibition is produced via independent mechanisms Grissmer, 1996, 1999;Hanson et al, 1999). We compared the normalized amplitudes of resting currents recorded in the presence of 1 mM TEA, 10 M verapamil, and both drugs applied together (1 mM TEA ϩ 10 M verapamil).…”

“…The localization of verapamil binding sites has been addressed in several types of voltage-gated K ϩ channels. In most earlier reports, verapamil seemed to cross the membrane in neutral form to block the channel through binding to its internal residue (DeCoursey, 1995;Rauer and Grissmer, 1996;Catacuzzeno et al, 1999;Hanson et al, 1999;Rauer and Grissmer, 1999;Zhang et al, 1999). It is remarkable that, in nearly all previously examined cell types, verapamil-sensitive voltage-activated K ϩ channels possessed C-type inactivation, and one of the more prominent effects of verapamil was a dramatic acceleration of K ϩ -current inactivation.…”

“…It is remarkable that, in nearly all previously examined cell types, verapamil-sensitive voltage-activated K ϩ channels possessed C-type inactivation, and one of the more prominent effects of verapamil was a dramatic acceleration of K ϩ -current inactivation. After experiments on villus enterocytes (Tatsuta et al, 1994), human T-lymphocytes (Hanson et al, 1999), and transfected human ether à go-go-related gene (HERG) channels (Zhang et al, 1999), verapamil was assumed to have either direct or allosteric effects on the molecular structures responsible for C-type inactivation. In contrast, a comparison of the effects of verapamil on mutant and wild-type Kv1.3 channels (Rauer and Grissmer, 1999) did not indicate any direct connection between C-type inactivation and the cumulative blocking effect of phenylalkylamines.…”

Identification of Novel Kv1.3 Blockers Using a Fluorescent Cell-Based Ion Channel Assay

“…10 There are currently various peptide species available, showing binding specificity for Kv1.3 over other shaker-related ion channels, which have contributed enormously to our understanding of this target. The relative restricted expression of Kv1.3 in tissue and its apparent pharmacological importance have led to the development and discovery of more appropriate small-molecule inhibitors, including correolide, 11 WIN 17317-3, 12 UK-78282, 13 and, more recently, a series of compounds based on disubstituted cyclohexyls 14 and benzofuran derivates from Ammi visnaga and 5-methoxypsoralen isolated from the plant Ruta graveolens.…”