ABSTRACT
A comprehensive comparative molecular field analysis (CoMFA) model for the binding of ligands to the neuronal voltage-gated sodium channel was generated based on 67 diverse compounds. Earlier published CoMFA models for this target provided µM ligands, but the improved model described here provided structurally novel compounds with low nM IC50. For example, new compounds 94 and 95 had IC50 values of 129 and 119 nM, respectively.
Subject(s)
Quantitative Structure-Activity Relationship , Voltage-Gated Sodium Channels/chemistry , Ligands , Models, MolecularABSTRACT
We report the results on the expression in Escherichia coli of a functional neurotoxin LqqV from the scorpion Leiurus quinquestriatus quinquestriatus. The gene for LqqV was synthesized using recursive PCR and expressed as a poly-histidine-tagged fusion protein in thioredoxin mutant E. coli strain [AD494(DE3)pLysS], thus permitting disulfide-bond formation. When cultured at 37 degrees C, about 50% of the expressed protein is contained as a monomer in the soluble fraction of the E. coli extract. The fusion protein from the soluble fraction was purified and the His-tag was cleaved by thrombin, resulting in a yield of about 1.5 mg/liter. The globular structure of the purified protein was confirmed by NMR and CD spectroscopy. Patch-clamp measurements using native sodium channels in guinea pig ventricular myocytes reveal (1) a slowing of inactivation and (2) a decrease in peak current upon application of toxin, thus confirming the alpha-toxin activity of the purified recombinant protein.
Subject(s)
Cloning, Molecular , Escherichia coli/genetics , Neurotoxins/biosynthesis , Neurotoxins/genetics , Scorpion Venoms/biosynthesis , Scorpion Venoms/genetics , Amino Acid Sequence , Animals , Base Sequence , Guinea Pigs , Magnetic Resonance Spectroscopy , Male , Molecular Sequence Data , Neurotoxins/chemistry , Scorpion Venoms/chemistry , ScorpionsABSTRACT
We previously developed a preliminary 3-D QSAR model for the binding of 14 hydantoins to the neuronal voltage-gated sodium channel; this model was successful in designing an effective non-hydantoin ligand. To further understand structural features that result in optimum binding, here we synthesized a variety of compound classes and evaluated their binding affinities to the neuronal voltage-gated sodium channel using the [3H]-batrachotoxinin A 20-alpha-benzoate ([3H]BTX-B) binding assay. In order to understand the importance of the hydantoin ring for good sodium channel binding, related non-hydantoins such as hydroxy amides, oxazolidinediones, hydroxy acids, and amino acids were included. Two major conclusions were drawn: (1) The hydantoin ring is not critical for compounds with long alkyl side chains, but it is important for compounds with shorter side chains. (2) Relative to Khodorov's pharmacophore, which contains two hydrophobic regions, a third hydrophobic region may enhance binding to provide nanomolar inhibitors.
