Botulinum neurotoxin inhibitor binding dynamics and kinetics relevant for drug design

Kruti B Patel; Olga Kononova; Shuowei Cai; Valeri Barsegov; Virinder S Parmar; Raj Kumar; Bal Ram Singh

doi:10.1016/j.bbagen.2021.129933

Botulinum neurotoxin inhibitor binding dynamics and kinetics relevant for drug design

Biochim Biophys Acta Gen Subj. 2021 Sep;1865(9):129933. doi: 10.1016/j.bbagen.2021.129933. Epub 2021 May 21.

Authors

Kruti B Patel¹, Olga Kononova², Shuowei Cai³, Valeri Barsegov², Virinder S Parmar⁴, Raj Kumar⁵, Bal Ram Singh⁶

Affiliations

¹ Botulinum Research Center, Institute of Advanced Sciences, Dartmouth, MA, USA; Department of Chemistry and Biochemistry, University of Massachusetts Dartmouth, North Dartmouth, MA, USA.
² Department of Chemistry and Biochemistry, University of Massachusetts Lowell, Lowell, MA, USA.
³ Department of Chemistry and Biochemistry, University of Massachusetts Dartmouth, North Dartmouth, MA, USA.
⁴ Botulinum Research Center, Institute of Advanced Sciences, Dartmouth, MA, USA; Bioorganic Laboratory, Department of Chemistry, University of Delhi, Delhi, India.
⁵ Botulinum Research Center, Institute of Advanced Sciences, Dartmouth, MA, USA.
⁶ Botulinum Research Center, Institute of Advanced Sciences, Dartmouth, MA, USA; Department of Chemistry and Biochemistry, University of Massachusetts Dartmouth, North Dartmouth, MA, USA; Prime Bio Inc., Dartmouth, MA, USA. Electronic address: bsingh@inads.org.

PMID: 34023445
DOI: 10.1016/j.bbagen.2021.129933

Abstract

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.

Keywords: Botulinum neurotoxin Type A; Drug discovery; Enzyme kinetics; Graphics processing units; Isothermal calorimeter; Ligand binding interactions; Molecular dynamic simulation; Nitrophenyl Psoralen.

Publication types

Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

Botulinum Toxins, Type A / antagonists & inhibitors*
Botulinum Toxins, Type A / metabolism
Drug Design*
Enzyme Inhibitors / chemical synthesis
Enzyme Inhibitors / chemistry
Enzyme Inhibitors / pharmacology*
Ficusin / chemical synthesis
Ficusin / chemistry
Ficusin / pharmacology*
Kinetics
Molecular Dynamics Simulation

Substances

Enzyme Inhibitors
Botulinum Toxins, Type A
Ficusin