The retrieved Pvalues of the statistically significant genes were corrected by using a step-up false breakthrough rate

Alternatively, small-molecule inhibitors of BoNT are sought to antagonize the extracellular or intracellular toxin and can be potentially used to treat pre- and post-exposure. Additionally, if stockpiled in dry, sunlight free, temperature-controlled locations, chemically stable small-molecules would remain viable for many years. In contrast, vaccines possess comparatively shorter self-lives. Moreover, with respect to the development of small-molecule therapeutics, the BoNT/A-LC represents a top priority as it possesses the longest duration of activity in the neuronal cytosol in comparison to other BoNT-LCs known to cause botulism in human. Research efforts to identify antagonists against BoNT intoxication have dramatically increased in recent years. However, the discovery and development of BoNT/A small-molecule inhibitors have been a challenging task for researchers since long. Part of the difficulty in this endeavor can be attributed to the unusually large peptide substrate-enzyme interface that requires a small-molecule with high affinity to effectively block substrate binding. Also, the BoNT and its domains show considerable conformational flexibility, making design of effective inhibitors complicated. Despite these challenges, a number of papers have been published on the initial steps to discover and develop inhibitors of BoNT/A protease activity using different approaches. Using high throughput screening of the NCI Diversity Set as well as a series of 4-aminoquinolines, Burnett et al. identified several small-molecule inhibitors of BoNT/A from which a common pharmacophore was predicted using molecular modeling. Quinolinol derivatives were reported to inhibit BoNT/A as determined by biochemical, cell and tissue based assay. Mechanism of QAQ binding to BoNT/A-LC and mode of inhibition was studied in detail by Lai et al.. Similarly, a high throughput screening of a library of hydroxamates resulted in the selection of 4- dichlorocinnamic hydroxamate as a lead structure for further development. Capkova et al. structurally modified 2, 4- dichlorocinnamic acid hydroxamate to improve its potency. On the other hand, a computational screen of 2.5 million compounds resulted in the identification of an inhibitor with a Ki of 12 mM, but this value was later invalidated. Computer-aided optimization of this inhibitor resulted in an analog that showed a two-fold improvement in inhibitory potency and displayed competitive kinetics by chelating the active site zinc atom. Though the above approaches have resulted in the identification of a number of small-molecules as BoNT/A inhibitors, no compound has yet advanced to pre-clinical development. The majority of such molecules reportedly demonstrated to be effective in enzymatic assays and a few small-molecules have been tested in cell-based assays. But the information shows that small-molecules can significantly protect mammals against BoNT/A is scanty. We screened the ChemBridge and NSC libraries, consisting of millions of compounds of unknown function for similarity search to 8-hyroxy quinolinol lead, NSC 84096. Since some of these compounds were commercially available and their functions are currently undefined, we reasoned that novel inhibitors could be identified. Herein we report the effective small-molecule BoNT/A inhibitors with promising in vivo pharmacokinetics. Our results demonstrate that small-molecule can protect mice against pre and post BoNT/A challenge and support pursuit of small-molecule inhibitor as a cost effective alternative for treating botulism and for biodefence measures. In the wake of the events of September 11, 2001, research efforts aimed at the discovery of potent antagonists for agents of bioterrorism have increased exponentially. However, despite the plethora of new data that has emerged in the past 5 years, an established pharmacophore validated through in vivo models of exposure remains elusive. Indeed, in the case of BoNT, few studies have reported the assessment of any small-molecule antagonist in animal models. Peptidomimetics and hydroxamic acid-based inhibitors have been developed that display inhibitory effects in the high nM range for the light chain of the BoNT serotype A. Compounds that contain zinc-coordinating sulfhydryl moieties might potentially inhibit host zinc proteases thereby making them poor therapeutic leads. The characteristically poor pharmacokinetics of hydroxamates, their instability, and their reported toxicity, which is likely due to their inhibition of an array of metalloproteases, also make them problematic as therapeutic agents. We focused on the 8-hydroxy quinolinol lead NSC 84096 for database search. Selection of this compound was based on: NSC 84096 is reported to be very potent and serotype A selective inhibitors ; it is reported to be non toxic and active in cell based and mouse phrenic nerve hemi-diaphragm assays ; there are quinolinols in clinical trials for Alzheimer’s disease and cancer ; and quinolinol-based drugs such as linolasept and vioform are available in the market.

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