This is not evident from experimental structures. inhibitors of BoNT/A targeting the exosites. Hits C1 and C2 showed non-competitive inhibition and likely target the – and -exosites, respectively. The identified exosite inhibitors may provide novel candidates for structure-based development of therapeutics against BoNT/A intoxication. neurotoxins (BoNTs) are classified as Tier 1 Select Agent toxins by the Centers for Disease Control and Prevention [1,2]. Serotype A (BoNT/A) is one of seven known serotypes of botulinum neurotoxins (ACG), and has an estimated human LD50 of only 1 1 ng/kg [3]. The toxin consists of a single 150 kDa polypeptide chain that is post-translationally proteolysed into a ~100 kDa heavy Sebacic acid chain (HC) and a ~50 Sebacic acid kDa light chain (LC) [4]. The toxins mechanism of action is known to involve cleavage of one of the three soluble half-lives [18]. Developing tight binding non-chelating inhibitors of BoNT/A has proven to be a difficult task in part due to the high conformational plasticity of the binding pocket and induced conformational changes in adjacent loops upon substrate or inhibitor binding [19]. The exceptionally large substrate binding surface of BoNT/A poses an extremely challenging problem to design effective small molecule inhibitors that are capable of disrupting the extensive protein-protein interactions within the substrate binding interface. The – and -exosites of BoNT/A, which were first addressed by Breidenbach and Brunger, provide intriguing alternatives for small molecule inhibition of enzyme-substrate interactions [9]. The -exosite is located on the rear surface of the protein (relative to the active site) and consists of four helices, while the -exosite lies in a dynamic loop region adjacent to the active site and forms the hallmark three-stranded antiparallel -sheet interaction involving the substrate SNAP-25 [9]. While studies have indicated that these exosites play an important role in substrate recognition and catalysis, the potential for small molecule binding and structure-based inhibitor design at these sites has been largely unexplored. Compared to the deep pocket of the active site, these regions appear to be relatively shallow and undefined. Therefore, questions still remain as to whether the exosites are amenable to small molecule binding. A single domain antibody was recently shown to inhibit SNAP-25 cleavage and bind to a small crevice in the -exosite with a low-nM Kd, suggesting that low nM inhibition may be possible [20]. Recently, studies from Jandas group showed that the natural products of phenolic caffeoyl derivatives such as D/L-chicoric acid exhibited noncompetitive partial inhibition of BoNT/A [21]. The combination of D-chicoric acid Sebacic acid with an active-site inhibitor, 2,4-dichlorocinnamic hydroxamate, displayed nonmutually exclusive inhibition. More interestingly, another non-competitive inhibitor, lomofungin, was identified which also exhibited synergistic inhibition against BoNT/A when used in combination with 2,4-dichlorocinnamic hydroxamate and chicoric acid [22]. While no structural evidence has been generated, it has been speculated based upon kinetic data that the binding regions of the two small molecules might map to the – and -exosites [22]. The discovery of exosite inhibitors of BoNT/A Mouse monoclonal to AXL inspired us to further investigate the small molecule binding interactions and molecular mechanisms of inhibition at the exosites. The synergy of exosite inhibition provides a valuable approach for designing novel inhibitors against BoNT intoxication. Herein, we applied computational approaches to explore the structural features of the exosites of BoNT/A using chicoric acid and lomofungin as model probes. The potential binding interactions of these small molecules at the exosites were investigated using an unbiased ensemble docking search and stepwise binding mode analysis. To gain insight into the structural basis of synergistic inhibition, we modeled a tripartite inhibitor binding complex of BoNT/A with a hydroxamate inhibitor bound at the active site, D-chicoric acid bound at the -exosite, and lomofungin bound at the -exosite. The tripartite inhibitor binding complex was analyzed in comparison with the substrate SNAP-25 binding complex, and revealed details of the binding site preferences and key residue determinants contributing to synergistic inhibition at the exosites. Finally, based upon the predicted binding models of the two exosite inhibitors, we performed high throughput screening to identify novel inhibitors targeting the exosites of BoNT/A. Materials and methods Structure and inhibitors of BoNT/A The crystallographic coordinates of BoNT/A in complex with the substrate, SNAP-25, (PDB code 1XTG) was used as the initial model [9]. The SNAP-25 was removed and the two mutated residues, E224Q and Y366F, were changed back to the wild type. The resulting structure was energy-minimized prior to MD simulations. Three inhibitors of BoNT/A were used as probes.