Source: TENNESSEE STATE UNIVERSITY submitted to NRP
SCREENING AND IDENTIFICATION OF POTENTIAL CYANOBACTERIAL CRUDE EXTRACTS/COMPOUNDS AGAINST HIGHLY INFECTIOUS POULTRY VIRUSES AND BACTERIA
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
OTHER GRANTS
Reporting Frequency
Annual
Accession No.
1030305
Grant No.
2023-38821-39582
Cumulative Award Amt.
$595,249.00
Proposal No.
2022-09565
Multistate No.
(N/A)
Project Start Date
Apr 1, 2023
Project End Date
Mar 31, 2026
Grant Year
2023
Program Code
[EQ]- Research Project
Recipient Organization
TENNESSEE STATE UNIVERSITY
3500 JOHN A. MERRITT BLVD
NASHVILLE,TN 37209
Performing Department
(N/A)
Non Technical Summary
Highly infectious diseases in poultry caused by influenza and coronaviruses, andCampylobacterandSalmonellaresult in significant economic losses for farmers and can pose a risk to human health. No antiviral drugs are specifically approved for treating influenza or coronavirus infections. Also, the widespread use of antibiotics against bacterial infections has led to increased problems of antibiotic resistance, and the presence of their residues in the environment compromises human and animal health. One potential alternative strategy is exploring natural products with antiviral/antibacterial to reduce reliance on antibiotics in the poultry industry. Cyanobacteria are relatively easy to cultivate in open and closed systems, do not need arable land, and better atmosphere carbon capture and solar energy conversion efficiency make them a potentially sustainable, cost-effective, and environment-friendly source of natural products. Antibacterial and antiviral activities of some cyanobacterial extracts/metabolites against some microbes have been reported in the literature studies. However, the proposed work aims at entire small molecule (> 700 g/mol) cyanobacterial metabolites from CyanoMetDB to identify potential inhibitors against prominent drug target receptors and evaluation of antiviral/antibacterial activities of identified compounds and their source cyanobacterial crude extracts against specific highly infectious pathogens in poultry. In essence, we will explore potential crude cyanobacterial extracts/purified compounds for prophylactic and therapeutic control of highly infectious poultry viruses and bacteria while gaining a fundamental understanding of their mechanisms of action and their broad-spectrum activity to enhance the ongoing research and education programs at Tennessee State University (TSU). The scientific knowledge gained through the proposed work helps to develop broad-spectrum antimicrobial feed supplements and their efficiency evaluation by animal challenge studies as future work.
Animal Health Component
30%
Research Effort Categories
Basic
20%
Applied
30%
Developmental
50%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3113299110170%
3113299110030%
Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
3299 - Poultry, general/other;

Field Of Science
1101 - Virology; 1100 - Bacteriology;
Goals / Objectives
Major goal of the proposed work is to screen and identify natural antiviral and antibacterial cyanobacterial crude extracts/purified compounds for prophylactic and therapeutic control of highly infectious poultry viruses and bacteria while gaining a fundamental understanding of their mechanisms of action and their broad-spectrum activity to enhance the ongoing research and education programs at Tennessee State University (TSU). This overarching goal will be accomplished through the following supporting objectives:In silico screening of cyanobacterial metabolite database for potential target antiviral and antibacterial compounds and predict their pharmacokinetics.Identification of top hit compounds and corresponding cyanobacterial strains, preparation of crude extracts, purified cyanobioactives and target viral receptors for in vitro studies; In vitro approaches to identify inhibitory effect against target receptors, antiviral activity of purified cyanobioactives and crude extracts; Assessment of antibacterial and immunomodulatory properties of purified cyanobioactives and crude extracts; TSU, NCATDevelop formal education and training programs at the graduate and undergraduate levels in food and animal science, natural antimicrobial design, virology, bacteriology, homology modeling, molecular docking/molecular dynamics simulations,recombinant DNA technology, analytical chemistry for under-represented minorities students and establishing a multidisciplinary, collaborative, and competitive STEM field knowledge-based society;
Project Methods
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).

Progress 04/01/23 to 03/31/24

Outputs
Target Audience:USDA National Institute of Food and Agriculture (NIFA) and US Poultry & Egg Association (USPOULTRY): These agencies are interested in innovations that enhance food safety, animal health, and sustainable agricultural practices. This work aligns with their goals by predicting the natural antiviral and antibacterial compounds to control infectious diseases in poultry. Undergraduate and graduate students (Animal and Food Science, Pre-vet, Pre-med): These students are trained in insilico approaches to screening natural antimicrobial compounds that can be used in disease prevention and treatment in poultry. Students involved in biomedical research, microbiology, biochemistry, and related fields will find this project valuable for its innovative approach to natural product drug discovery. The detailed methodologies and findings can be a significant resource for their research projects. Collaborators from Tennessee State University (TSU), University of Texas (UT), and North Carolina A&T State University (NCAT): These collaborators will benefit from our findings and methodologies, potentially leading to joint research projects, shared resources, and collaborative publications. Researchers in antimicrobial feed additives development, natural products drug discovery, and cyanobacterial biorefinery: Researchers focusing on developing antimicrobial feed additives will find our project particularly relevant. Our discoveries can lead to new, natural feed additives that enhance poultry health and reduce reliance on synthetic antibiotics, addressing growing concerns about antibiotic resistance. Scientists working on drug discovery from natural products will be interested in our methods and findings, especially those involving cyanobacterial bioactives. Our results contribute valuable data on potential new compounds for therapeutic use and highlight the potential of cyanobacteria as sources of bioactive compounds, supporting their efforts in biorefinery and sustainable biotechnology. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? The project has provided the following opportunities for the training and professional development of graduate students: Training on preparing input receptor and ligand files, including downloading receptor PDB files from databases. Processing receptor files by removing water molecules, adding charges, repairing missing atoms, and performing energy minimization. Prepare pdbqt files, enhancing the understanding and application of these critical preprocessing steps. In-depth training on preparing grid configuration files using native ligand coordinates. Experience setting up and configuring simple and multiple docking simulations to ensure a comprehensive understanding of the setup process. Hands-on experience running simple and multiple docking simulations, providing practical skills and confidence in executing molecular docking studies. Complete training on the molecular docking virtual screening method, equipping the student with the knowledge and skills to conduct virtual screenings effectively and efficiently. The students have gained a robust understanding and practical expertise in molecular docking through these training opportunities, positioning them well for future research and professional roles in the field. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?Planned tasks for the next reporting period Validation of molecular docking results with molecular dynamics (MD) simulations In silico prediction of pharmacokinetics properties of compounds Identification of top-hit compounds and corresponding cyanobacterial strains Preparation of crude extracts and purified top-hit cyanobioactives. Heterologous expression and purification of selected viral and bacterial targets for invitro assays.

Impacts
What was accomplished under these goals? The proposed integrated project addresses the critical issue of controlling highly infectious poultry viruses and bacteria, which pose significant threats to poultry health, agricultural productivity, and food security. Poultry diseases caused by viral and bacterial pathogens can lead to severe economic losses due to high mortality rates, reduced productivity, and increased costs of disease management and control measures. Current treatment and prevention strategies, including vaccines and antibiotics, face challenges such as limited efficacy, the emergence of resistant strains, and potential adverse effects on poultry and human health. Therefore, there is an urgent need for innovative and effective alternatives. This project tackles the problem explicitly by focusing on discovering and characterizing antiviral and antibacterial compounds derived from cyanobacteria. These cyanobioactives have the potential to offer new, natural, and potent solutions for prophylactic and therapeutic applications. By understanding the mechanisms of action and assessing the broad-spectrum activity of these bioactive compounds, the project aims to develop novel interventions that could enhance disease control in poultry, thereby improving animal health, safeguarding the poultry industry, and contributing to global food security. The target audience for this work includes;poultry farmers and producers, veterinarians and animal health professionals, agricultural researchers, biotechnologists and pharmaceutical companies, government and regulatory agencies such as USDA NIFA, USPOULTRY, public health officials, and educational institutions. Major activities completed: Objective 1. In silico screening of cyanobacterial metabolite database for potential target antiviral and antibacterial compounds and predict their pharmacokinetics: 90 % of the activities of objective 1 are completed. Methodology:A set of cyanobacterial compounds (n=2605) from CyanoMetDB was filtered based on molecular weight (< 700 g/mol) and hydrogen bond acceptor and donor capacity, resulting in the selection of 1078 compounds. The SMILES strings of these selected compounds were then used to identify and download 3D conformer Structural Data Files (SDF) from the PubChem database. Some 3D Structural Data Files were also generated using Open Babel or Marvin. The best-resolution crystal structures of drug targets were obtained from the RCSB PDB database. For H5N1 and H3N2 influenza viruses, targets such as H5 and H3 haemagglutinin, N1 and N2 neuraminidases, PACPBN1, PB2, and PH1N1 RNA polymerase subunits were selected. For Infectious bronchitis virus (IBV), Mpro, PLpro proteases, and RdRP RNA polymerase were chosen. For Salmonella and Campylobacter, DNA gyrases were selected. Since the crystal structure of the Campylobacter DNA gyrase is not available, a homology model obtained from AlphaFold 2 was used for molecular docking. Open Babel and MGLTools were used to process and convert ligand and receptor PDB files to PDBQT format. Autodock Vina served as the docking engine, with the docking box defined at the center of the native ligand to include the residues of the entire cavity. The exhaustiveness level was set to 14, with 10 docking modes. Docked protein-ligand complex structures were visualized using PyMOL. Results:Based on the lower negative docking scores and key polar contacts with active site amino acids, several potential antimicrobial compounds were predicted and identified from CyanoMetDB. The study focused on their antiviral and antibacterial properties. Antiviral Compounds The following compounds were predicted as potential inhibitors against Influenza A Virus (IAV) and Influenza B Virus (IBV) targets: Calothrixin sp.: Calothrixin A, Calothrixin B, Spironostoic acid, 11,12-didehydrospironostoic acid, 12-hydroxy-2-oxo-11-epi-hinesol. Fischerella sp.: Tjipanazoles, 13-Hydroxy dechlorofontonamide, Hapalindolinone B. Hapalosiphon sp.: Dechlorofontonamide, Hapalindole J, Hapalindole J-formamide, Hapalocyclamide. Lyngbya sp.: Pukeleimides, Tetrahydroindol 5, 3-Acetyl-2'-deoxyuridine, 7-Formyl-3-methoxy-5-methylindanone, Ulongamides, Aplysiaenal, Biselyngbyaside, Asterina-330. Nostoc sp.: Nostodione A, Cryptophycins, Aulosirazole B, N-Acetyltryptamine, Nostoclide N1, 4,5-Dihydroxy-1-methyl-anthraquinone, Nb-p-Coumaroyltryptamine. Scytonema sp.: Palythine-serine, 12-dihydroxystigolone, Stigolone, Scytonemin, Scytoscalarol. Tolyprothrix sp.: Tjipanazoles, Tolyporphins. Antibacterial Compounds The following compounds were predicted as potential inhibitors of Salmonella and Campylobacter DNA gyrase B subunits: Calothrixin sp.: Spironostoic acid, 11,12-didehydrospironostoic acid, 12-hydroxy-2-oxo-11-epi-hinesol. Fischerella sp.: Tjipanazol B. Lyngbya sp.: Pukeleimides, 3-Acetyl-2'-deoxyuridine. Nostoc sp.: Nostodione A, Aulosirazole B, Nostoclide N1, 4,5-Dihydroxy-1-methyl-anthraquinone, Nb-p-Coumaroyltryptamine. Tolyprothrix sp.: Tjipanazole B. Eucapsis sp.: Eucapsitrione. Outcome:These compounds were selected based on their interaction profiles and predicted efficacy against specific viral and bacterial targets, and their source cyanobacterial strains were selected for further in-vitro validation studies.

Publications