Molecular insights into the local anesthetic receptor within voltage-gated sodium channels using hydroxylated analogs of mexiletine

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.

Molecular Dissection of Lubeluzole Use–Dependent Block of Voltage-Gated Sodium Channels Discloses New Therapeutic Potentials

“…For instance, we observed a significant ∼20% reduction of Y1593C sodium currents at 10 Hz in the absence of drug (dashed line in Fig. 5B), which suggests the slowing of recovery from inactivation (O'Reilly et al, 2000;Xiao et al, 2001;Desaphy et al, 2012). Note, however, that the corresponding Y1767C in the cardiac hNav1.5 isoform accelerates the recovery from inactivation, which potentiates mutant channel inhibition by the open-channel blocker ranolazine (Huang et al, 2011).…”

“…Structurally, lubeluzole contains an aryloxy moiety and a protonable amine linked by a short carbon chain, conferring hydrophobicity (log P 5 4.08 6 0.01) and basicity (pKa 5 7.14 6 0.02), respectively. The pKa of lubeluzole is close to that of lidocaine (pKa 5 7.9; Hille, 1977b), but is less basic than that of mexiletine (pKa 5 9.3; Desaphy et al, 2012). Thus, the partition of lubeluzole molecules between neutral and charged forms at pH 7.4 is quite similar to lidocaine, close to a 1-to-1 ratio.…”

“…To take into account use-dependent inhibition occurring at 10 Hz in the absence of drug, an additional variable, namely, I max , was introduced in eq. 1 to fit the concentration-response relationship (Desaphy et al, 2012). The calculated I max value 6 the S.E.…”

“…binding to the inactivated channel is a phenylalanine side chain that may interact electrostatically with the charged amine of LA drugs (p-cation interaction) (Ragsdale et al, 1994;Pless et al, 2011;Desaphy et al, 2012). Nonconservative mutations of this residue disrupt binding of LAs to inactivated sodium channels, resulting in a complete abolition of usedependent block.…”

Recent advances in voltage-gated sodium channels, their pharmacology and related diseases

“…Several ongoing studies are aimed at understanding the intimate drug-channel molecular interactions to design more efficacious and safer drugs (Fozzard et al, 2011;Desaphy et al, 2012;Morris et al, 2012). Gating properties of these channels are also affected during trauma injury (Morris et al, 2012).…”