Development and Testing of an In Vitro Assay for Screening of Potential Therapeutic Agents against Na Channel Neurotoxins
Annual rept. 1 Jan-31 Dec 1986
ALABAMA UNIV IN BIRMINGHAM SCHOOL OF MEDICINE
Pagination or Media Count:
The objective was to develop a rapid, reliable and simple screening assay of broad scope and sensitivity for applications in the identification of compounds with potential therapeutic value in the treatment of intoxication induced by a variety of sodium channel neurotoxins. The assay should also be able to accommodate the screening of large numbers of samples. These requirements have been met in the form of an in vitro radioligand binding assay in which the binding of a tritiated analog of bactrochotoxin serves as a sensitive indicator of test compound interactions at any of at least five different binding domains on the sodium channel. A vesicular preparation from rat cerebral cortex, termed synaptoneurosomes, was selected as a source of mammalian voltage-sensitive sodium channels because of the ease of preparation and the suitability of its biophysical characteristics for the binding assay. Experiments are described which delineate conditions and demonstrate the stability of synpatoneurosomes to extended storage periods at -70 C. HM-197, a hexahydropy-rimidine derivative, was further investigated by electrophysiological techniques and was found to antagonize the veratridine- induced depolarization of rat phrenic nerve at concentrations that had no effect on sodium channel function when administered alone. Results support the applicability of the screening assay as described and suggest that HM-197 and similar derivatives should be further investigated for their potential therapeutic value. Keywords tetrodotoxin scorpion toxin pyrethroids action potential.