Botulinum neurotoxin inhibitor binding dynamics and kinetics relevant for drug design

• Published in: Biochimica et biophysica acta. General subjects Vol. 1865; no. 9; p. 129933
• Main Authors: Patel, Kruti B., Kononova, Olga, Cai, Shuowei, Barsegov, Valeri, Parmar, Virinder S., Kumar, Raj, Singh, Bal Ram
• Format: Journal Article
• Language: English
• Published: Elsevier B.V 01.09.2021
• Subjects: Botulinum neurotoxin Type A; botulinum toxin; botulism; calorimetry; drug design; Drug discovery; enzyme inhibition; Enzyme kinetics; fluorescence emission spectroscopy; Graphics processing units; Isothermal calorimeter; Ligand binding interactions; Molecular dynamic simulation; molecular dynamics; neurotoxicity; Nitrophenyl Psoralen; psoralen; zinc; Ligand binding interactions; SMIs; DMSO; Graphics processing units; HTS; GPUs; Nitrophenyl Psoralen; Molecular dynamic simulation; Botulinum neurotoxin Type A; SNARE; NPP; MD; KM; Vmax; BoNT; SDS-PAGE; Isothermal calorimeter; ΔH; LC; ITC; SASA; Drug discovery; HC; Enzyme kinetics; ΔS
• ISSN: 0304-4165; 1872-8006; 1872-8006
• DOI: 10.1016/j.bbagen.2021.129933

A natural product analog, 3-(4-nitrophenyl)-7H-furo[3,2-g]chromen-7-one, which is a nitrophenyl psoralen (NPP) was found to be an effective inhibitor of botulinum neurotoxin type A (BoNT/A). In this work, we performed enzyme inhibition kinetics and employed biochemical techniques such as isothermal calorimetry (ITC) and fluorescence spectroscopy as well as molecular modeling to examine the kinetics and binding mechanism of NPP inhibitor with BoNT/A LC. Studies of inhibition mechanism and binding dynamics of NPP to BoNT/A light chain (BoNT/A LC) showed that NPP is a mixed type inhibitor for the zinc endopeptidase activity, implying that at least part of the inhibitor-enzyme binding site may be different from the substrate-enzyme binding site. By using biochemical techniques, we demonstrated NPP forms a stable complex with BoNT/A LC. These observations were confirmed by Molecular Dynamics (MD) simulation, which demonstrates that NPP binds to the site near the active site. The NPP binding interferes with BoNT/A LC binding to the SNAP-25, hence, inhibits its cleavage. Based on these results, we propose a modified strategy for designing a molecule to enhance the efficiency of the inhibition against the neurotoxic effect of BoNT. Insights into the interactions of NPP with BoNT/A LC using biochemical and computational approaches will aid in the future development of effective countermeasures and better pharmacological strategies against botulism.