Abstract
We previously showed that the β-adrenoceptor modulators, clenbuterol and propranolol, directly blocked voltage-gated sodium channels, whereas salbutamol and nadolol did not (Desaphy et al., 2003), suggesting the presence of two hydroxyl groups on the aromatic moiety of the drugs as a molecular requisite for impeding sodium channel block. To verify such an hypothesis, we synthesized five new mexiletine analogs by adding one or two hydroxyl groups to the aryloxy moiety of the sodium channel blocker and tested these compounds on hNav1.4 channels expressed in HEK293 cells. Concentration–response relationships were constructed using 25-ms-long depolarizing pulses at −30 mV applied from an holding potential of −120 mV at 0.1 Hz (tonic block) and 10 Hz (use-dependent block) stimulation frequencies. The half-maximum inhibitory concentrations (IC50) were linearly correlated to drug lipophilicity: the less lipophilic the drug, minor was the block. The same compounds were also tested on F1586C and Y1593C hNav1.4 channel mutants, to gain further information on the molecular interactions of mexiletine with its receptor within the sodium channel pore. In particular, replacement of Phe1586 and Tyr1593 by non-aromatic cysteine residues may help in the understanding of the role of π–π or π–cation interactions in mexiletine binding. Alteration of tonic block suggests that the aryloxy moiety of mexiletine may interact either directly or indirectly with Phe1586 in the closed sodium channel to produce low-affinity binding block, and that this interaction depends on the electrostatic potential of the drug aromatic tail. Alteration of use-dependent block suggests that addition of hydroxyl groups to the aryloxy moiety may modify high-affinity binding of the drug amine terminal to Phe1586 through cooperativity between the two pharmacophores, this effect being mainly related to drug lipophilicity. Mutation of Tyr1593 further impaired such cooperativity. In conclusion, these results confirm our former hypothesis by showing that the presence of hydroxyl groups to the aryloxy moiety of mexiletine greatly reduced sodium channel block, and provide molecular insights into the intimate interaction of local anesthetics with their receptor.
Highlights
Mexiletine is a class Ib antiarrhythmic drug, considered as the first choice drug for treating myotonic syndromes (Conte Camerino et al, 2007)
EFFECTS OF MEXILETINE AND ANALOGS ON WILD-TYPE hNav1.4 CHANNELS To compare the effects of mexiletine and its hydroxylated analogs, block of sodium channels was evaluated by measuring the druginduced reduction of I Na elicited at −30 mV for 25 ms every 10 (0.1 Hz) or 0.1 (10 Hz) s from the HP of −120 mV
Increasing the stimulation to 10 Hz further induced the reduction of I Na to a lower level, determining the so-called use-dependent block, which depends www.frontiersin.org on the high-affinity binding to inactivated channels
Summary
Mexiletine is a class Ib antiarrhythmic drug, considered as the first choice drug for treating myotonic syndromes (Conte Camerino et al, 2007). Mexiletine exerts its pharmacological action through blockade of voltage-gated sodium channels, reducing cell excitability. Preferential binding to inactivated channels and usedependent block are the basis of the selective action of mexiletine on pathologic hyperactive tissues. We have shown that little modification of these three components can substantially affect sodium channel blockade in vitro and antimyotonic effects in vivo (Desaphy et al, 1999, 2001; De Luca et al, 2000, 2003, 2004; De Bellis et al, 2006). The use of mexiletine analogs has allowed to get new important information on the molecular dynamic interaction between the drug and its receptor within the sodium channel pore (De Luca et al, 2000, 2003). Sodium channel mutagenesis experiments have defined the amino acids most probably involved in the interaction with LAs (Ragsdale et al, 1994, 1996; Wright et al, 1998; Nau et al, 1999; Wang et al, 2000; Sunami et al, 2001; Yarov-Yarovoy et al, 2001, 2002; O’Leary and Chahine, 2002; McNulty et al, 2007; Ahern et al, 2008)
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