Effect of structural modification of several groups on the D-ring of grayanotoxin on its depolarization potency in squid giant axon.

The purpose of this investigation was twofold, 1) to clarify the pharmacological significance of various groups on the D-ring of grayanotoxin (GTX) and 2) to determine possible sites on GTX which are available for preparing pharmacological probes. All GTX derivatives were directly applied to the intracellular phase of internally perfused squid giant axons. A dose-response curve for each GTX analog was constructed using depolarization as an index and assuming a simple one-to-one stoichiometry. By comparing EC50 and the maximum depolarization caused by GTX analogs, the main factors affecting potency were found to be: the formation of 1) electrostatic interaction, 2) a hydrogen bond between groups on the D-ring of GTX and part of the Na channel protein and 3) balance between hydrophilicity and hydrophobicity in the GTX molecule. Biologically essential groups like those in the A- and B-rings were not recognized. A suitable site on GTX for synthesizing pharmacological probes is probably position C-17 of grayanotox-15-ene compound, because in chemical modification, bulkier groups can be introduced without a total loss of biological activity of GTX. Another possible site for modification is position C-14R, because this portion is on the molecular surface opposite biologically essential groups. It can be speculated that in GTX binding, possible molecular moiety of the Na channel facing the D-ring of GTX is rich in positively charged amino groups.