Use of Viral Entry Assays and Molecular Docking Analysis for the Identification of Antiviral Candidates against Coxsackievirus A16

This protocol can aid in the discovery of potential antiviral small molecules through a mechanism-driven approach. The time of addition assay determines at which step of the infection the small molecule exhibits its antiviral activity. Molecular docking predicts the interaction between the small molecule and the viral proteins.

To evaluate the influence of drugs on host cells prior to viral infection, seed RD cells in 12-well plates at a two times 10 to the fifth cells per well plating density. Incubate the cells at 37 degrees Celsius in a 5%carbon dioxide incubator overnight. The next morning, treat each well of the RD monolayer with a test compound of interest, in triplicate, at non-toxic concentrations in one milliliter of basal medium for one or four hours.

At the end of the treatment incubation, wash the cells with one milliliter of PBS before adding 50 plaque-forming units of virus in 300 microliters of basal medium per well for one hour, with rocking every 15 minutes. At the end of the infection incubation, wash the cells with PBS and overlay the cells with one milliliter of fresh basal medium containing 0.8%methylcellulose. After 72 hours in the cell culture incubator, wash each well with two milliliters of PBS and fix the cells with 0.5 milliliters of 37%formaldehyde per well for 15 minutes.

At the end of the fixation, wash the wells with PBS and stain the cells with 0.5 milliliters of 0.5%crystal violet solution per well. After two minutes, wash the wells with a gentle stream of water and allow the plate to air dry. Then place the plate on a white light box for counting and calculate the percent of coxsackievirus infected cells according to the formula.

For molecular docking analysis, download 3D molecules of the test compounds from PubChem. If a molecule does not have a 3D structure uploaded, download the 2D structure or use the SMILE string sequence to transform the structure into a 3D molecule via an appropriate molecular program. Next, download a viral biological assembly unit from RCSB Protein Data Bank.

Using an appropriate bio-computing program, delete the solvents from the Protein Data Bank file, replace the incomplete side chains using data from the Dunbrack 2010 rotamer library and add hydrogens and charges to the structure as previously reported. To dock the test compounds onto the prepared virus unit, upload the test compound file into University of California San Francisco Chimera as the ligand and select the whole prepared viral protein as the receptor to perform blind docking. For additional docking, confine the docking site onto the viral protein to regions of interest derived from the blind docking results by further reducing the search volume.

Then upload the docking file into an appropriate molecular graphics system to analyze the binding mode positions. Select the ligand to find polar contacts from the compound to the viral protein, identifying the polar contacts with the to any atoms option. Both of the small molecules tested in this representative experiment, only produced a marginal impact against the coxsackievirus A16 infectivity, whether in the pretreatment of the host cells prior to viral infection or in the post-infection treatment.

In contrast, the molecules efficiently abrogated the infection by greater than 80%in the co-addition treatment, suggesting that the two compounds are the most effective when they are concurrently present with the virus particles on the host cell surface during the infection. Flow cytometry based binding analysis confirms that the two tannins prevented coxsackievirus A16 infectivity entry by preventing viral particle binding to the host cells. Molecular docking of the tannins indicates that they are both predicted to bind in the canyon region of the coxsackievirus pentamer, just above the pocket entrance that holds the pocket factor and plays an important role for mediating coxsackievirus binding and entry into the host cell.

In these surface projections, the unique residues predicted from the polar contacts of the small molecules around the pocket entrance can be observed, with asparagine-85, lysine-257, and asparagine-417 in common between the two tannins. For molecular docking, the topology of the viral protein needs to be taken into account when ranking the binding frames. Additional experiments could include testing the antiviral activity of the compound on recombinant viruses, with mutations on the amino acids discovered to validate their importance to the drug's efficacy.