Optimal Requirements for High Affinity and Use-Dependent Block of Skeletal Muscle Sodium Channel by N-Benzyl Analogs of Tocainide-Like Compounds ANNAMARIA DE LUCA, SOPHIE TALON, 1 MICHELA DE BELLIS, JEAN-FRANC ¸ OIS DESAPHY, GIOVANNI LENTINI, FILOMENA CORBO, ANTONIO SCILIMATI, CARLO FRANCHINI, VINCENZO TORTORELLA, AND DIANA CONTE CAMERINO Unita ` di Farmacologia, Dipartimento Farmacobiologico (A.D.L., S.T., M.D.B., J.-F.D., D.C.C.) and Dipartimento Farmacochimico (G.L., F.C., A.S., C.F., V.T.), Facolta ` di Farmacia, Universita ` di Bari, Bari, Italy Received December 24, 2002; accepted July 7, 2003 This article is available online at http://molpharm.aspetjournals.org ABSTRACT Newly synthesized tocainide analogs were tested for their state-dependent affinity and use-dependent behavior on so- dium currents (I Na ) of adult skeletal muscle fibers by means of the Vaseline-gap voltage clamp method. The drugs had the pharmacophore amino group constrained in position  [N-(2,6- dimethylphenyl)pyrrolidine-2-carboxamide (To5)] or  [N-(2,6- dimethylphenyl)pyrrolidine-3-carboxamide (To9)] in a proline- like cycle and/or linked to a lipophilic benzyl moiety as in N-benzyl-tocainide (Benzyl-Toc), 1-benzyl-To5 (Benzyl-To5), and 1-benzyl-To9 (Benzyl-To9). I Na were elicited with pulses to -20 mV from different holding potentials (-140, -100, and -70 mV) and stimulation frequencies (2 and 10 Hz). All com- pounds were voltage-dependent and use-dependent channel blockers. The presence of a proline-like cycle increased the potency; i.e., To5 was 3- and 10-fold more effective than Toc in blocking I Na at the holding potential of -140 and -70 mV, respectively. The benzyl group on the amine further enhanced drug effectiveness with the following scale: Benzyl-To9  Ben- zyl-Toc  Benzyl-To5. At a holding potential of -100 mV and 10-Hz stimulation, Benzyl-To9 blocked I Na with a half-maximal concentration of 0.5 M, being 60 and 400 times more potent than To9 and Toc, respectively. The similar effectiveness of Benzyl-Toc and Benzyl-To9 was paralleled by a similar spatial arrangement by equilibrium geometry modeling. In addition, the latter had a higher pK a value that probably contributed to a slow kinetic during its high use-dependent behavior. Benzyl-To5 had its lowest energy level at a more folded conformation that justifies the less favorable profile among the N-benzylated an- alogs. The new compounds are the most potent tocainide-like sodium channel blockers so far described and have high ther- apeutic potentials. The block of voltage-gated Na + channel by local anesthetic- like (LA) drugs has therapeutic value for numerous disorders characterized by abnormal membrane excitability (Clare et al., 2000). Different channel types show different sensitivi- ties to LAs; however, the structural basis for this difference is still poorly understood (Wang et al., 1996; Li et al., 1999; 2002). The selective activity on tissue displaying abnormal excitability pattern is based on the state-dependence of LA effect (i.e., a stronger potency at depolarized membrane po- tential or during high frequency trains of channel activity) (Catterall, 2002). This effect is related to drug interaction with a receptor whose affinity for ligands changes with the state-dependent transitions of the channel, the affinity being the highest when the channels open or inactivate. Mutagen- esis studies identified various amino acid residues, localized in the S6 segments of each homologous domain of the  subunit, as critical for LA binding and activity on Na + chan- nels of various excitable tissues (Ragsdale et al., 1994, 1996; Wright et al., 1998; Li et al., 1999; Nau et al., 1999; Wang et al., 2000; Yarov-Yarovoy et al., 2001, 2002). LAs may interact weakly with the channel at the resting state, presumably through hydrophobic interactions with two aromatic amino acids, Phe and Tyr, on D4-S6 (Ragsdale et al., 1996; Wright et al., 1998; Li et al., 1999; Nau et al., 1999). Voltage-depen- dent gating movements of the channel protein may allow a stronger interaction of the drug at these and other residues, leading to a high-affinity stabilization of the channel in the This work was supported by grant 1208 from Telethon-Italy (to D.C.C.). 1 Present address: Laboratoire de Physiologie Generale, Unite Mixte de Recherche 6018, Centre National de la Recherche Scientifique, Faculte des Sciences et des Techniques, Universite de Nantes, F-44322 Nantes, France. ABBREVIATIONS: LA, local anesthetic; Toc, tocainide; Benzyl-Toc, N-benzyl-tocainide; To5, N-(2,6-dimethylphenyl)pyrrolidine-2-carboxamide; Benzyl-To5, 1-benzyl-N-(2,6-dimethylphenyl)pyrrolidine-2-carboxamide; To9, N-(2,6-dimethylphenyl)pyrrolidine-3-carboxamide; Benzyl-To9, 1-benzyl-N-(2,6-dimethylphenyl)-pyrrolidine-3-carboxamide; MOPS, 3-(N-morpholino)propanesulfonic acid; HP, holding potential; fUDB, frac- tional use-dependent block (percentage reduction of I Na at the steady-state versus the first pulse of 10-Hz train); TB, tonic block. 0026-895X/03/6404-932–945$7.00 MOLECULAR PHARMACOLOGY Vol. 64, No. 4 Copyright © 2003 The American Society for Pharmacology and Experimental Therapeutics 2353/1096785 Mol Pharmacol 64:932–945, 2003 Printed in U.S.A. 932 at ASPET Journals on January 21, 2017 molpharm.aspetjournals.org Downloaded from