All the sensorgrams were processed using the double referencing method to eliminate the nonspecific binding from background contribution and buffer artifacts by subtracting signals from the reference flow cell (i.e., FC1) and from buffer blank injections (in this case, 200 nM GAG) [35]. FC2, 3 and 4 served as repeats. The response was monitored as a function of time (sensorgram) and analyzed using Biacore T200 Evaluation software.multiple paired comparisons were significant at a p value of , 0.01.
Abstract
Botulinum neurotoxins (BoNTs), etiological agents of the life threatening neuroparalytic disease botulism, are the most toxic substances currently known. The potential for the use as bioweapon makes the development of small-molecule inhibitor against these deadly toxins is a top priority. Currently, there are no approved pharmacological treatments for BoNT intoxication. Although an effective vaccine/immunotherapy is available for immuno-prophylaxis but this cannot reverse the effects of toxin inside neurons. A small-molecule pharmacological intervention, especially one that would be effective against the light chain protease, would be highly desirable. Similarity search was carried out from ChemBridge and NSC libraries to the hit (7-(phenyl(8-quinolinylamino)methyl)-8-quinolinol; NSC 84096) to mine its analogs. Several hits obtained were screened for in silico inhibition using AutoDock 4.1 and 19 new molecules selected based on binding energy and Ki. Among these, eleven quinolinol derivatives potently inhibited in vitro endopeptidase activity of botulinum neurotoxin type A light chain (rBoNT/A-LC) on synaptosomes isolated from rat brain which simulate the in vivo system. Five of these inhibitor molecules exhibited IC50 values ranging from 3.0 nM to 10.0 mM. NSC 84087 is the most potent inhibitor reported so far, found to be a promising lead for therapeutic development, as it exhibits no toxicity, and is able to protect animals from pre and post challenge of botulinum neurotoxin type A (BoNT/A).
Citation: Singh P, Singh MK, Chaudhary D, Chauhan V, Bharadwaj P, et al. (2012) Small-Molecule Quinolinol Inhibitor Identified Provides Protection against BoNT/ A in Mice. PLoS ONE 7(10): e47110. doi:10.1371/journal.pone.0047110 Editor: Kamyar Afarinkia, Univ of Bradford, United Kingdom Received May 9, 2012; Accepted September 10, 2012; Published October 11, 2012 Copyright: ?2012 Singh et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: National Cancer Institute, National Institute of Health, USA acknowledged for providing chemicals bearing NSC prefix. Ms. Padma Singh is a JRF working in ICMR (Indian Council of Medical Research) scheme (Ref No: 3/1/3/JRF-2008/MPD-78, 31500). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist.
Introduction
Botulinum neurotoxins (BoNTs), produced by Clostridium botulinum, C. baratii and C. butyricum, consist of seven immunologically distinct serotypes (A) are the causative agents of a lifethreatening neuroparalytic disease known as botulism. The potency, longevity of BoNT intoxication has facilitated use of BoNTs as therapeutic agents [1,2,3,4,5] and the ease with which these toxins can be produced make them potential bioweapons and bioterrorism agents [6,7]. Overdose with BoNT therapeutics can also result in systemic botulism [8]. BoNTs are the only toxin group in the six most dangerous biothreat agents (Category A agents) listed by Centers for Disease Control and Prevention (CDC) (http://www3.niaid.nih.gov/Biodefense/bandc_priority. htm). BoNTs are synthesized as ,150-kDa single-chain protoxins that are post translationally processed by proteolytic cleavage to form a disulfide-linked dimer, composed of a 100-kDa heavy chain (HC) and a 50-kDa light chain (LC) [9]. The HC comprises a 50kDa C-terminal domain (Hc) that participates in the binding of toxin to productive ectoacceptors on the cell surface of peripheral cholinergic nerve cells [10] and the 50-kDa N-terminal domain (Hn) of the HC facilitates the translocation of the LC across an endosomal membrane into the cytosol of the nerve cell [11]. The LCs are zinc metalloendoproteases [3.4.24] that exhibit extraordinary specificities for SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins: SNAP-25, VAMP/synaptobrevin and syntaxin [12,13,14,15]. SNARE proteins are essential for exocytosis of neurotransmitter and cleavage of these protein(s) by BoNT inhibits the release of acetylcholine from synaptic terminals leading to neuromuscular paralysis or botulism [16]. The most effective immunotherapy for protection against BoNTs relies on vaccination with pentavalent toxoid species, although supplies are reserved for high-risk individuals [17]. Moreover vaccination of general public also restricts subsequent BoNT’s therapeutic applications, if needed. There are no therapies available for BoNT mediated post neuronal intoxication. The current treatments for BoNT poisoning are limited to: (i) the administration of antitoxin(s) to neutralize and clear toxin from the circulation which is not effective in post neuronal intoxication [6] and, therefore, would be of limited use following an act of bioterror (as it is likely that victims would seek medical attention only after enervation); and (ii) mechanical ventilation which is necessary once BoNT-induced paralysis compromises thoracic muscle contraction.