SCREENING AND IDENTIFICATION OF POTENTIAL CYANOBACTERIAL CRUDE EXTRACTS/COMPOUNDS AGAINST HIGHLY INFECTIOUS POULTRY VIRUSES AND BACTERIA

<b>Project Methods</b><br> Collection of cyanobioactives and selection of target receptors: Cyanobacterial compounds collected from CyanoMetDB (n = 2124) will be filtered based on molecular weight greater than 700 g/moland hydrogen acceptor or donator capacity (< 2). Then the SMILEs strings of selected compounds will be used to identify and download 3D conformer structural data files (SDF) via the PubChem database search engine. Conservative viral (proteases (IBV), RNA polymerases (IBV and H5N1), neuraminidases (H5N1), M2 ion channel (H5N1), and DNA gyrases (Campylobacter,Salmonella)target receptors which may show more broad-spectrum activity will be selected and crystal structures will be collected from RCSB protein database or generated (ifPDB structures unavailable).In silicoapproaches:Open Babel program will be used for format transformation or 3-D coordinate generation for the ligand cyanobioactives. The MGL Tools will be implemented to process (delete other chains, and heteroatoms (including water), adding missing atoms, hydrogens, and charges) and convert receptor PDB files to PDBQT format. Autodock Vina will be used as a docking engine and docked protein-ligand complex structures will be visualized using Pymol.To validate the stability of the protein-ligand complex, MD simulations will be performed using NAMD. The parameters, structure, and topology files for the ligand will be generated using the CHARMM-GUI Web server. Visual molecular dynamics (VMD) will be used to generate protein structure (PSF) files. We will predict pharmacophore properties; ADME of the potent compounds by submitting canonical simplified molecular input line entry system (SMILES) of the compounds to an online server; ADMETlab 2.0.Preparation of crude extracts and purified top hit cyanobioactives:Selected cyanobacteriawill be purchased from the UTEX culture collection (Austin, TX) and cultivated using suggested growth medium and conditions in bench scale photo-bioreactors.The crude extractsCrude extracts of cyanobacteriawill be prepared as follows: harvested cyanobacterial wet paste will be rapidly frozen in a refrigerator below −20 °C, and subsequently thawed at 0-4 °C until completely de-frozen, and insoluble substances will be removed by centrifugation. The supernatant will be then lyophilized with freeze driers to derive powdered cyanobacterial extract. Cyanobioactiveswill bepurified using solvent extraction and/or chromatography methodsor purchased (if available).Heterologous expression and purification of selected viral and bacterial enzymes: The genes of target viral receptors/enzymeswill be amplified and cloned into the pET21a expression plasmid. The proteins will be expressed inE.coliBL21 and purified using an ÄKTAprime Plus liquid-chromatography system (GE Healthcare) by affinity chromatography.Inhibition kinetics against target receptors:Enzyme inhibition or biochemical assays against viral targets and antiviral activity assays will be conducted. Dose response curves with varying concentrations of crude extract/purified cyanobacterial compound versus enzyme/receptor activity will be plotted and the inhibitor concentration at 50% of enzyme/receptor activity will be determined.CoVs (Mpro and PLpro Assays. Fluorescence resonance energy transfer (FRET)-based cleavage assay will be used for in vitro enzyme inhibition study. Initially, 15 μL of the Mpro/PLpro in reaction buffer at the final concentration of 10 ng/μL and 5 μL of inhibitor control /test inhibitor (at varying concentrations)/inhibitor solvent (positive control) will be pipetted into a 384-well plate. Afterward, the plate will be preincubated for 30 min at room temperature with slow shaking. The enzymatic reaction will be then initiated by adding 5 μL of substrate dissolved in reaction buffer to 25 μL final volume (final concentration 50 μM) and incubated at room temperature for 4 h. The fluorescence signal will be monitored at excitation at 360 nm with an emission wavelength of 460 nm.RNA dependent RNA polymerase (RdRp) assay. The CoVs RdRp Homogeneous Assay Kit will be designed in a convenient AlphaLISA® format, with Digoxigenin- labeled RNA duplex, biotinylated ATP, RdRp assay buffer (2 components plus DTT), and purified RdRp. RdRp Homogeneous Assay Kit will measure the direct incorporation of ATP in the double-stranded RNA chain. The increase in Alpha-counts is proportional to the amount of ATP incorporated into RNA.IAVs. (Neuraminidase inhibition (NI) Assay): The NI assay will be performed using an NA- Fluor™ Influenza Neuraminidase Assay Kit (Applied Biosystems, Foster City, CA, USA) as per the manufacturer's instructions with slight modifications. For the NA-Fluor™ influenza neuraminidase assay, crude extracts/purified compounds will be added to the assay buffer in 96- well plates at concentrations of 0-500 μg/mL. Then the viruses suspended in the assay buffer will be added and incubated at 37 °C.After 30 min, NA-Fluor Substrate will be added to each well and incubated for an additional 2 h, followed by recording the fluorescence (excitation: 365 nm; emission: 415-445 nm)M2 activity inhibition using liposome dye release assays: Unilammellar liposomes containing self-quenching concentrations of carboxyfluorescein (CF) will be prepared as described. M2- mediated CF release will be assessed by incubating up to 50 nM peptide with 50 μM liposomes (determined by rhodamine absorbance).DNA gyrase B inhibitor assay: The GyrB ATPase assay will be carried out in a buffer containing 12.8 nM Gyrase enzyme, 0.08 mg/mL ssDNA, 35 mM Tris, pH 7.5, 24 mM KCl, 2 mM MgCl2, 6.5% glycerol, 2 mM DDT, 1.8 mM spermidine, 0.1 mg/mL BSA, and 5% DMSO solution containing the inhibitor in a total volume of 25 µl. The reaction will be started by adding ATP to a final concentration of 1 mM and allowed to incubate at 30 °C for 60 min. The reaction will be stopped by adding 200 µl of malachite green solution. Color is allowed to develop for 5 min and the absorbance at 600 nm.Cultivation of viruses:Madin-Darby canine kidney (MDCK) cells will be seeded in a T175 flask and grown until 80% confluence before viral propagation. MDCK cells will be infected with virus at a multiplicity of infection (MOI) of 0.001 and incubated for 1 h at 37 °C for adsorption. Then the cells will be washed with PBS and replenished with DMEM containing 1 µg/mL TPCK-treated trypsin (Sigma-Aldrich, Burlington, VT, USA) without FBS. The culture supernatant Will be harvested on day three post-infection, with 50-70% of the cells showing a cytopathic effect, by centrifugation at 4000 rpm, 4 °C for 10 min. The supernatant will be harvested, filtered,and stored in aliquots at −80 °C. Viral titer will be determined by plaque assay.Kinetics of antiviral activity:Host cell lines will be incubated with purified compounds/crude extracts at varying concentrations for 24 h. Then CC50 values will be determined by using an MTT assay. For EC50, host cell lines will be infected with target CoV (IBV) and IAVs (H5N1, H3N2) in presence of a range of purified cyanobioactives and incubated at 37 °C with 5% CO2for 1-10 days. Then EC50values will be measured by monitoring the cytopathic effect or virus yield reduction assays.Evaluation of antibacterial activity:To evaluate the antibacterial activity againstCampylobacterandSalmonella,minimum inhibitory concentration (MIC) will be determined by the microtiter broth dilution method.Statistical Analysis: Each experiment will be performed in triplicate and repeated three times. The results will be expressed as means ± SD. Statistical comparisons will be made by one-way analysis of variance (ANOVA), followed by a Duncan multiple-comparison test. Differences will be considered significant when the p values will be p<0.05. All statistical analyses of data will be performed using SAS 9.2 software (SAS Institute, Inc., Cary, NC).