Revealing the rationale behind the differential neutralization of phospholipase A ₂ (PLA ₂ ) enzymes in snake and bee venom by varespladib (LY-315920), a small molecule PLA ₂ inhibitor

Phospholipase A₂ (PLA₂) enzyme found in snakes, bees, and wasps' venoms is responsible for toxicity and pathophysiology of envenomation. Varespladib (VP, LY-315920) is among the extensively researched small molecule inhibitors targeting snake venom (SV) PLA₂ and PLA₂-like proteins. Interestingly, it could not neutralize bee venom (BV) PLA₂. To reveal this puzzle, we compared VP's <i>in silico</i> binding mechanisms with PLA₂s from India's 'Big Four' snake venoms (BFSVs<i>, Naja naja, Daboia russelii, Echis carinatus, and Bungarus caeruleus</i>), nine viper venoms from four continents, and BV. VP binds SV-PLA₂s in the same position but in a different position in the BV-PLA2s due to a clash between its phenyl ring and residues Tyr89 and Ile91, which might be the reason behind its universal activity against SV-PLA₂s but negligible activity against BV-PLA₂. Molecular docking and dynamics simulations identified optimal VP binding conformations with BFSV and BV-PLA₂ proteins. <i>In silico</i> analysis results showed that the BFSV-PLA₂-VP complex exhibited significantly greater binding affinity and stability than other PLA₂-VP complexes, suggesting enhanced molecular interactions. The spectrofluorometric binding data showed a significantly higher binding affinity of VP with BFSV-PLA₂s than BV-PLA₂s, corroborated by differential inhibition of catalytic activity and anticoagulant activity of PLA₂ enzymes from BV and SVs. VP showed significant neutralization of <i>in vivo</i> toxicity, generating reactive oxygen species and altering mitochondrial transmembrane potential induced by PLA₂s from BFSV in the <i>Caenorhabditis elegans</i> model. However, such activities were not shown against BV-PLA₂, indicating the limitations of broad-spectrum inhibitors like VP in neutralizing BV-PLA₂.