Abstract
The botulinum neurotoxin (BoNT) family consists of seven antigenically distinct serotypes BoNT/ A-G, acting on peripheral nervous system. BoNT/A is recognized as by far the most toxic serotype. BoNT/A which targets synaptosomal -associated protein of 25 kDa (SNAP-25), is particularly long lived within neurons and requires a longer time for recovery of neuromuscular function. Due to their extreme toxicity and easy production BoNTs have become potential bio-terror agents. Diagnosis of botulism based on the flaccid paralysis symptoms, and takes up to 96 hours to confirm the intoxication. Furthermore effective chemical countermeasures to treat victims after sign and symptoms of botulism have presented are limited. BoNTs are made of tripartite functional domains: 1) a receptor binding domain 2) a translocation domain 3) a zinc protease domain. A zinc protease domain which is also termed as light chain (LC) is highly specific to soluble N-ethylmaleimide- sensitive factor attachment protein receptor (SNARE), which mediates cellular and vesicular membrane fusion during neurotransmitter secretion. Cleavage of SNARE proteins by the LC blocks the release of acetylcholine from the presynapsis, resulting in flaccid paralysis or botulism, and possibly death. So, LCs have been a primary target for the development of therapeutics against botulism. Quinolinol derivatives were found to be effective small molecule inhibitors of botulinum neurotoxin serotype A (BoNT/A). In the current work, we have synthesized 5 Quinolinol derivatives and named the SP1 to SP5. These 5 derivatives were screened using high-throughput FRET-based assay and we observed the effects of electron withdrawing and donating groups on their inhibition activities. Further work was done on potential inhibitor SP3 to determine its mode of action. Enzyme kinetic data and molecular modeling study of potential inhibitor SP3 reveal that it binds to hydrophobic pocket near the active site and exerts it competitive inhibitory effect by blocking the Zn²⁺ but not by chelating it.