Botulinum neurotoxins (BoNTs) are the etiological agents responsible for botulism, a disease characterized by peripheral neuromuscular blockade and a characteristic flaccid paralysis of humans. BoNTs are the most lethal known poisons affecting humans and has been recognized as a potential bioterrorist threat. Current treatments for botulinum poisoning are predominately prophylactic in nature relying on passive immunization with antitoxins. Inhibition of the BoNT light chain metalloprotease (LC) has emerged as a new therapeutic strategy for the treatment of botulism that may provide an effective post-exposure remedy. A high-throughput screening effort against the light chain of BoNT serotype A (LC/A) was conducted with the John Hopkins Clinical Compound Library comprised of over 1,500 existing drugs. Lomofungin, a natural product first isolated in the late 1960’s, was identified as an inhibitor of LC/A, displaying classical noncompetitive inhibition kinetics with a Ki of 6.7 ± 0.7 µM. Inhibitor combination studies reveal that lomofungin binding is nonmutually exclusive (synergistic). The inhibition profile of lomofungin has been delineated by the use of both an active site inhibitor, 2,4-dichlorocinnamic hydroxamate, and a noncompetitive inhibitor d-chicoric acid; the mechanistic implications of these observations are discussed. Lastly, cellular efficacy was investigated using a rat primary cell model which demonstrated that lomofungin can protect against SNAP-25 cleavage, the intracellular protein target of LC/A.
A new mechanistic class of BoNT/A zinc metalloprotease inhibitors, from Echinacea, exemplified by the natural product Dchicoric acid (I1) is disclosed. A detailed evaluation of chicoric acid's mechanism of inhibition reveals that the inhibitor binds to an exosite, displays noncompetitive partial mark.hixon@takedasd.com kdjanda@scripps.edu. Supporting Information Available: Full experimental procedures, derivations and characterization for all compounds. This material is available free of charge via the Internet at http://pubs.acs.org Currently, there are no approved pharmacological treatments for BoNT intoxication. Although an effective vaccine is available for immuno-prophylaxis, 3 vaccine approaches cannot reverse the effects after the toxin has reached its target inside the cell. A small molecule pharmacological intervention, especially one that would be effective against the etiological agent responsible for BoNT intoxication, the light chain protease would be highly desirable and obviate vaccine deficiencies. NIH Public AccessThe substrate for BoNT/A is SNAP-25, (synaptosomal-associated protein, 25 kDa). The Michaelis complex involves an extensive network of binding interactions ranging from the active site to the opposite surface of the BoNT/A. In the complex, the N-terminal residues of SNAP-25 (147-167) form an α-helix, imbedded in the rear surface of BoNT/A while the Cterminal residues (201-204) form a distorted β-strand, and the spanning residues are mostly extended.4 Both mutagenesis and kinetics have conclusively shown that the N-terminal α-helix and the C-terminal β-sheet are critical for an efficient substrate binding and cleavage, and have been termed α-and β-exosites, respectively.5 Also, substrate truncation experiments reveal that BoNT/A protease requires a long stretch of SNAP-25, (66-amino acids) to have optimal catalytic activity. Likely, it is the extensive enzyme-substrate binding interactions that make the proteases of BoNTs among the most selective known. This multi-site binding strategy incorporating an exceptionally large substrate-enzyme interface area4 probably accounts for the extreme difficulty in producing potent small molecule inhibitors of the enzyme. In effect, the small molecule must be capable of disrupting these protein-protein interactions.6 While considerable efforts have gone into identifying active site inhibitors of BoNT/A, no report of a small molecule exosite inhibitor has been communicated.7 Herein, we provide strong evidence demonstrating that components from the plant Echinacea are potent exosite inhibitor with unexpected synergistic effect when combined with an active site inhibitor.One of the most popular herbs in the US today is the Native American medicinal plant called Echinacea. It has been used for over 400 years to treat infections and wounds and as a general "cure-all". Main components of Echinacea showing biological and pharmacological activity are the phenolic caffeoyl derivatives 8 including I1, I3, and I4 , Figure 1. We were intrigued by the str...
Botulinum neurotoxins (BoNTs), proteins secreted by the bacteria genus Clostridium, represent a group of extremely lethal toxins and a potential bioterrorism threat. As the current therapeutic options are of a predominantly prophylactic nature and cannot be used en masse, new strategies and ultimately potential treatments are desperately needed to combat any widespread release of these neurotoxins. In these regards, our laboratory has been working on developing new alternatives to treat botulinum intoxication through the development of inhibitors of the light chain proteases, the etiological agent which causes BoNT intoxication. Such a strategy has required the construction of two high throughput screens and small molecule non-peptidic libraries; excitingly, inhibitors of the BoNT/A protease have been uncovered and are being optimized via structure activity relationship studies. Keywordsbotulism; botulinum neurotoxin A; botulinum neurotoxin B; light chain metalloprotease; protease inhibitors
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.