Fire ant venom alkaloid, isosolenopsin A, a potent and selective inhibitor of neuronal nitric oxide synthase

Abstract

Massive, multiple fire ant, Solenopsis invicta, stings are often treated aggressively, particularly in the elderly, despite limited evidence of systemic toxicity due to the venom. Over 95% of the S. invicta venom is composed of piperidine alkaloid components, whose toxicity, if any, is unknown. To assess a possible pharmacological basis for systemic toxicity, an alkaloid-rich, protein-free methanol extract of the venom from whole ants was assayed for inhibitory activity on the following nitric oxide synthase (NOS) isoforms, rat cerebellar neuronal (nNOS), bovine recombinant endothelial (eNOS), and murine recombinant immunologic (iNOS). Cytosolic NOS activity was determined by measuring the conversion of [3H]arginine to [3H]citrulline in vitro. Rat nNOS activity was inhibited significantly and in a concentration-dependent manner by the alkaloid-rich venom extract. For nNOS, enzyme activity was inhibited by approximately 50% with 0.33 ± 0.06 μg of this venom extract, and over 95% inhibition of the three isoforms, nNOS, eNOS, and iNOS, was found with doses of 60 μg in 60-μ1 reaction mixture. These results indicate that the alkaloid components of S. invicta venom can produce potent inhibition of all three major NOS isoforms. Isosolenopsin A (cis-2-methyl-6-undecylpiperidine), a naturally occurring fire ant piperidine alkaloid, was synthesized and tested for inhibitory activity against the three NOS isoforms. Enzyme activities for nNOS and eNOS were over 95% inhibited with 1000 μM of isosolenopsin A, whereas the activity of iNOS was inhibited by only about 20% at the same concentration. The IC50 for each of three NOS isoforms was approximately 18 ± 3.9 μM for nNOS, 156 ± 10 μM for eNOS, and > 1000 μM for iNOS, respectively. Kinetic studies showed isosolenopsin A inhibition to be noncompetitive with L-arginine (Ki = 19 ± 2 μM). The potency of isosolenopsin A as an inhibitor of nNOS compares favorably with the inhibitory potency of widely used nNOS inhibitors. Inhibition of NOS isoforms by isosolenopsin A and structurally similar compounds may have toxicological significance with respect to adverse reactions to fire ant stings.