Botulinum neurotoxin inhibitor binding dynamics and kinetics relevant for drug design.

Background: 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).

Methods: 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.

Results: 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.

Conclusion: 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.

General Significance: 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.