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 [(3)H]arginine to [(3)H]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 microg of this venom extract, and over 95% inhibition of the three isoforms, nNOS, eNOS, and iNOS, was found with doses of 60 microg in 60 microl 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 microM of isosolenopsin A, whereas the activity of iNOS was inhibited by only about 20% at the same concentration. The IC(50) for each of three NOS isoforms was approximately 18 +/- 3.9 microM for nNOS, 156 +/- 10 microM for eNOS, and >1000 microM for iNOS, respectively. Kinetic studies showed isosolenopsin A inhibition to be noncompetitive with L-arginine (K(i) = 19 +/- 2 microM). 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.

Diversity of peptide toxins from stinging ant venoms

“…Ant venom is composed of a complex mixture of chemicals such as proteins, enzymes, biogenic amines, peptides, hydrocarbons, formic acid and alkaloids (Davies et al, 2004;Kem et al, 2004;Yi et al, 2003). All these compounds are produced by the venom gland, which consists of two free cylindrical elongated and convoluted tubes, linked to a venom reservoir (Ortiz and Mathias, 2006).…”

Venom alkaloids of monogyne and polygyne forms of the red imported fire ant, Solenopsis invicta, in Taiwan

“…Consequently, stings of red imported fire ants represent a significant human health hazard (Stafford, 1996;Vinson, 1997;Kemp et al, 2000). Yi et al (2003) indicated that three major NOS (nitric oxide synthase) isoforms (nNOS, eNOS and iNOS) should be inhibited by S. invicta venom alkaloids, and isosolenopsin A (cis-2-methyl-6-n-undecylpiperidine), an alkaloid component of fire ant venom, was synthesized and tested for inhibitory activity against three NOS isoforms. The results showed that activities of nNOS and eNOS isoforms were over 95 % inhibited with 1000 mM of isosolenopsin A, whereas the enzyme activity for iNOS was inhibited by only about 20 % with the same concentration of isosolenopsin A (Yi et al, 2003).…”

“…Yi et al (2003) indicated that three major NOS (nitric oxide synthase) isoforms (nNOS, eNOS and iNOS) should be inhibited by S. invicta venom alkaloids, and isosolenopsin A (cis-2-methyl-6-n-undecylpiperidine), an alkaloid component of fire ant venom, was synthesized and tested for inhibitory activity against three NOS isoforms. The results showed that activities of nNOS and eNOS isoforms were over 95 % inhibited with 1000 mM of isosolenopsin A, whereas the enzyme activity for iNOS was inhibited by only about 20 % with the same concentration of isosolenopsin A (Yi et al, 2003). Both solenopsin A (trans-2-methyl-6-n-undecylpiperidine) and isosolenopsin A cause seizures and depress cardiovascular functions in rats (Howell et al, 2005).…”

CHAPTER 4. Venoms-Based Drug Discovery: Bioassays, Electrophysiology, High-Throughput Screens and Target Identification

“…neuronal nitric oxide synthase) involved in cellular signalling and neuronal excitability. 22,37,[89][90][91] Electrophysiological recordings have been instrumental in determining the varied pharmacological effects of toxins and venom components on cellular function, and have provided fundamental insight into ion channel structure and function. For example, nAChRs were isolated by virtue of high affinity binding of a-bungarotoxin from snake venom, [92][93][94] whereas the pore region of the K + channel was rst dened through electrophysiological characterisation of mutants with altered binding of charybdotoxin from scorpion venom.…”