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    METAL ION-MEDIATED INACTIVATION AND SMALL MOLECULE INHIBITION OF THE METALLOPROTEASE IN BOTULINUM NEUROTOXIN A

    Cover for METAL ION-MEDIATED INACTIVATION AND SMALL MOLECULE INHIBITION OF THE METALLOPROTEASE IN BOTULINUM NEUROTOXIN A
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    View/Open: Feng_georgetown_0076D_12008.pdf (7.2MB) Bookview

    Creator
    Feng, Minghao
    Advisor
    Yang, David C.H.
    Abstract
    Botulinum neurotoxins are the causative agents of botulism and serotype A (BoNT/A) is the most persistent one among seven Clostridium botulinum serotypes. Current treatments for BoNT/A toxicity do not meet the demand of a potential mass outbreak of botulism. The catalytic domain of BoNT/A light chain (LC) is the main target of the inhibitor designs.
     
    The inhibition and binding data of quinolinol inhibitors suggest that the inhibition of NSC 84096 is likely due to the binding around the active site pocket rather than removing the catalytic Zn2+ of LC/A. NSC 84096 was successfully docked to the active site of LC/A with and without Zn2+. Based on the structural information of known LC/A inhibitors, about 400,000 compounds in the ZINC database were screened by eHiTS-LASSO. everal new scaffolds and potential leads were obtained.
     
    Other than small organic compounds, Zn2+ and Cu2 showed strong inhibition of LC/A activity at micromolar concentrations, implicating that the inhibition is likely associated with specific binding to LC/A. The inhibition of LC/A cysteine and histidine mutants by Zn2+ and the inactivation of LC/A by thiol-selective reagent N-ethylmaleimide suggest that C165 is the likely transition metal binding site.
     
    More than only inhibiting LC/A, Cu2+ was able to inactivate LC/A through Fenton-like reaction in the presence of ascorbate or hydrogen peroxide. Cu2+ effectively inactivated LC/A protease activity at submicromolar concentrations in the presence of both hydrogen peroxide and ascorbate. Cu2+ mediated inactivation of LC/A was time-dependent and irreversible. The second order rate constant of the inactivation of LC/A by Cu2+ was found to be 4.24 ± 0.26 µM-1 min-1.
     
    The Cu2+-mediated inactivation of LC/A was not affected by the addition of the known LC/A inhibitor NSC 84096, which is a quinolinol derivative. NSC 84096 and similar quinolinol inhibitors in combination with Cu2+ could potentially deliver the inhibitors across cell membrane through chelation and subsequently inactivate BoNT/A.
     
    Due to the several-month long persistence of the BoNT/A toxicity, irreversible inactivation may offer a more effective therapy than conventional inhibitors. The Cu2+ mediated inactivation of LC/A developed in the present studies is applicable to similar Cu2+ binding proteins and nucleic acids.
     
    Description
    Ph.D.
    Permanent Link
    http://hdl.handle.net/10822/558231
    Date Published
    2013
    Subject
    botulinum neurotoxin; enzymology; metal binding; metalloprotease; Chemistry; Chemistry;
    Type
    thesis
    Publisher
    Georgetown University
    Extent
    209 leaves
    Collections
    • Graduate Theses and Dissertations - Chemistry
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    Georgetown University Seal
    ©2009 - 2022 Georgetown University Library
    37th & O Streets NW
    Washington DC 20057-1174
    202.687.7385
    digitalscholarship@georgetown.edu
    Accessibility