3. Molecular Docking Studies
3.1. Preparation of Ligands and Receptor. The NCI and ChemBridge database libraries were chosen for virtual screening of small-molecule inhibitors on the basis of structure similarity searches. The structures of selected molecules were drawn by Chemsketch (ChemDraw) software (http://www.acdlabs.com) and saved as MDL mol files. The energy minimized pdb files were generated using ArgusLab 4.0.1 (http://www.arguslab.com). Ligand files in the pdb format were opened in AutoDock (4.1) for preparation. Once opened, ligand files were edited and saved in pbdqt format. The three-dimensional structure of BoNT/A-LC (PDB code 3BON) was obtained from the RCSB Protein Data Bank. All water molecules except those which participate in catalysis were removed. The rigid and flexible residues of the protein were selected, and two additional files were created; a file 3BONrigid.pbdqt and file 3BONflex.pbdqt. 3.2. Grid Generation and Running AutoGrid. AutoDock requires pre-calculated grid maps, one for each atom type present in the ligand being docked. AutoGrid 4.1 was used to create autogrid .gpf, .glg, .fld and map files of atoms for protein. The Grid box was constructed around the active site residue Glu262 which plays a pivotal role in the catalytic activity of BoNT/A endopeptidase [40,41]. The active site residues that surrounded by docking box were Phe163, Gln162, Glu164, Cys165, Lys166, Phe194, Glu224, His227, Arg231, Ala236, Ile237, Pro239, Val258, Ser259, Glu261, Arg363, Tyr366, and Zn(II).3.3. Preparing the Docking Parameter File and Running AutoDock. The final step in submitting the docking was to runusing GS800 densitometer and Quantity One software (BioRad, USA).the AutoDock function. To prepare this, the protein’s rigid pbdqt file, the flexible pdbqt file and ligand’s pdbqt file were specified. At the end of a docking process, the output file `.dlg’ showed the docked conformations. These conformations were compared one to another to determine similarities and they were clustered accordingly. The root mean square deviation (RMSD) was used to determine whether two docked conformations are similar enough to be in the same cluster. After that these clusters were ranked from the lowest energy to highest.
7. Evaluation of Small-molecule Inhibitors in Mouse Model
Four inhibitor molecules were selected for in vivo experiment based on in vitro results. Female Balb/c mice (20?5 g weight) were used for in vivo experiments and they were divided into seventeen groups having five mice each (four groups for each molecule and one group for BoNT/A control). Mice from one group received 5LD50 of BoNT/A (in 200 ml PBS) intraperitoneally (ip). All four compounds were tested using four groups of mice as described below. To determine the toxicity of compounds, each of the molecule (1 mM) solubilized in PBS/DMSO (9:1) was injected per mouse ip in second group. In third group, 100 ml (1 mM) of smallmolecule inhibitor mixed with 5 LD50 of BoNT/A holotoxin was injected ip. Fourth group received 5 LD50 of BoNT/A, 30 min before the inhibitor injection. In last group of animal (fifth group) 100 ml each (1 mM) of small-molecules were injected 30 min prior to challenge with 5 LD50 of BoNT/A. All mice were examined for 4 days at hourly interval for survival, behavior, breath, and extraocular symptoms of botulism. All the animal experiments were approved by the Laboratory Ethical Committee on Animal Experimentation of Defence Research and Development Establishment, Gwalior, India via permission no. BT/01//DRDE/ 2009 and all efforts were made to minimize suffering.
4. Screening of Inhibitors using Rat Brain Synaptosome
Test compounds were obtained from the Drug Synthesis and Chemistry Branch, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, NCI (Bethesda, MD) and ChemBridge Corporation (San Diego, CA). BoNT/A peptide inhibitor (RRGC) was purchased from Bio Basic (Ontario, Canada). Compounds selected after virtual screening were tested in cleavage-based rBoNT/A-LC enzymatic assay. The inhibitors were dissolved in absolute DMSO and stock solutions were made up to 20 mM. Initial screening was performed in 40 ml reaction mixture containing 2.05 mg synaptosomes and 6 mM rBoNT/A-LC in cleavage buffer along with the 1 mM small-molecules. For determination of inhibitory molar ratios of small-molecules to rBoNT/A-LC, different concentrations of the small-molecules (100, 200 and 500 mM) and rBoNT/A-LC (25 pM, 200 nM and 6 mM) were used. In control assays, positive control was accomplished without test compound and rBoNT/A-LC and negative control test compound was replaced by DMSO. The incubation was carried out at 37uC for 15 min. The reactions were stopped by adding 46 SDS-PAGE sample buffer followed by heating at 100uC for 5 min.
Results and Discussion
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 [31,36]. The development of small-molecule inhibitors as intraneuronal therapeutics is a crucial unmet need. Our initial impetus for producing recombinant LC as a reagent to be utilized in synaptosome based assay for the screening of potential inhibitors of the BoNT/A-LC protease activity. A 1401 bp DNA fragment encoding BoNT/A-LC along with 14 amino acids of translocation domain and 15 amino acids of Nterminal heavy chain was selected to produce rBoNT/A-LC as already reported in our previous study [38].
5. Determination of the IC 50
Fifty-percent inhibitory concentration (IC50) values for rBoNT/ A-LC were calculated from nine concentrations of compound via dose-response measurements. The reactions were set up in duplicate as described earlier with the decreasing concentrations of inhibitors, keeping the concentration of rBoNT/A-LC constant at 10 nM. Each value is the average of two independent determinations. In all the cases, standard deviations were less than 620%.
6. Western Blot Analysis
Proteins from catalytic reactions were separated on a 13% SDSPAGE before transfer to a 0.2 m nitrocellulose membrane for 60 min at 100 V. After blocking in 5% skim milk/PBS for 2 h at room temperature (RT), the membrane was washed three times for 5 min at room temperature with PBST [2 mM Phosphate buffer (pH 7.4), 137 mM NaCl, 2.7 mM KCl, and 0.1% Tween 20(v/v)]. Rabbit anti-SNAP-25 antibody against N-terminal SNAP-25 protein (Sigma, USA) was added at the dilution of 1:5000 in PBS and the blot was incubated for 1 h at RT and washed four times with PBST. Goat anti-rabbit HRP conjugate antibody at a dilution of 1:30000 into PBS was added and further incubated for 1 h at RT. Membrane was again washed with PBST four times, each of 10 min. Bound antibodies were detected by chemiluminescence using an ECL western blot kit (Biological Industry, Israel) as per manufacturer’s instructions. Film was exposed for 15 s before development.