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Antiviral Screening

We routinely perform antiviral testing against SARS CoV-2 via high throughput screening (HTS), titer reduction, and time of addition.

Antiviral assays can be developed and performed for most viruses. Please inquire.

 

We screen compounds prophylactically for antiviral activity against SARS CoV-2 using a cell-  based assay based on the publication from the Jonsson lab: Development and Validation of  a    High-Throughput Screen for Inhibitors of SARS CoV and Its Application in Screening of a 100,000-Compound Library. J Biomol Screen 2007 12: 33. DOI: 10.1177/1087057106296688. We invite you to learn more about Dr. Jonsson’s HTS program. 

Is a positive control run for HTS assay? Yes. HTS plates are examined for acceptability in each run using Z score and CV statistics. HTS Assay Validation is managed according to the NIH Assay Guidance Manual. From this table, we calculate the Z score, CV, S/B and S/N for each plate. The positive control is the cells only and the negative control is virus. This are set to 100 and 0 respectively to normalize the data. In line with the HTS approach, an inhibitor is added to each plate at a single dose. The identity of this compound will not be provided as it is not required for review of the data. Any plate that doesn’t meet the criteria below will be rerun for no cost.

 

Parameter Definition Range
Z value
1-(3* s.d. of cell control (σc) + 3* s.d. of the virus control (σv)/
[mean cell control signal (µc) - mean virus control signal (µv)].
> 0.5
A value of 1.0 is the maximum
signal/background (S/B) mean cell control signal (µc)/mean virus control signal (µv) >10
signal/noise (S/N) mean cell control signal (µc) - mean virus control signal (µv) / (s.d. of the cell control signal (σc)2 - s.d. of the virus control signal (σv) >3
Coefficient of Variance (CV) Percentage of s.d. per mean of cell or virus control <10%
Correlation coefficient (r2)
Does a straight line fit the data?
∑[(x0 - xi)2 + (y0 - yi)2]1/2
x0 and y0 = line of best fit
xi and yi = coordinates for each data point
 >0.7

To run this assay, it is essential to know the cytotoxicity of the compound in the cell line chosen. Without the information the assay’s data is not qualified.

For titer reduction assays, Vero E6 TMPRESS or A549 ACE2 or any other cells (primary etc) are grown in 12 to 24-well plates overnight and then infected with virus at a MOI of 0.1 diluted in the appropriate media (depends on the cell line). The cells are infected for one hour of adsorption at 37C, cells are washed with PBS twice and media containing 5 μM of compound (or as directed) is added to each well. After two days, the supernatant is harvested and the amount of virus in each well is measured using a TCID50 or plaque assay (as directed).

Drug can also be added 2 hours prior for prophylactic assays which we recommend people to test first. 

Drug concentration can be modified as needed and multiple doses can be tested. Other drugs can be provided as a control drug, but inclusion will incur an additional cost. 

Other types of cell line (e.g. A549) including primary cells (epithelial or endothelial) can be employed in titer reduction assays using a TCID50 with MTT or CTG or plaque titer as an endpoint.

Once you have identified an effective concentration of inhibitor using the HTS and titer reduction assays, one can measure the reduction of virus titer in a time of addition experiment . This will give you information on the antiviral effectiveness of your compound if it is added before infection, or early, middle or late in infection. While the assay is continuing to be refined, addition of compound at 2 hours prior to infection, 2 hours after are a good starting point in a pilot. Advanced studies may measure addition of compound at 2, 4, 6-, 8-, 12- and 24-hours post-infection. 

As in the titer reduction assay,  the chosen cells are grown in 12 to 24-well plates overnight and then infected with virus at a MOI of 0.1 diluted in the appropriate media 

As with the titer reduction, any cell line can be used that confers infection. We have two human cell lines available; 293T and A549 which harbor the hACE2 receptor.

Prophylactic addition at 2 hours prior to infection: In three wells (3 biological replicates) two hours prior to infection, a specific amount of compound. i.e. 5 μM, is added to each well.  The cells are washed, the cells are infected for one hour of adsorption at 37C, and then cells are washed with PBS twice and media containing X μM of compound is added to each well. 

Therapeutic addition at 2 hours post-infection: The cells are washed, the cells are infected for one hour of adsorption at 37C, and then cells are washed with PBS twice. At the designated times, in three wells (3 biological replicates) per time point at designated times post infection, a specific amount of compound. i.e. 5 μM, is added to each well.  

After two days, the supernatant is harvested and the amount of virus in each well is measured using a TCID50 or plaque assay.

 

Screening Frequency

Screening is conducted on a first come, first serve basis.

  • High throughput screening (HTS): Monday, Tuesday, and/or Wednesday, depending on volume and other studies. Compounds need to arrive at least 2 days week prior to the screening date. Assays will be scheduled according holidays and volume of incoming work.
  • Titer Reduction: Every Wednesday
  • Time of Addition: Once every two weeks

Shipping Information

Send by FEDEX or UPS to:

Jyothi Parvathareddy
UNIVERSITY OF TENNESSEE HEALTH SCIENCE CENTER
Regional Biocontainment Laboratory
858 Madison | Memphis, TN  38163
 

 

 

Frequently Asked Questions

When can I expect my results?

The HTS entire process takes approximately 7-10 days while the HTS assay can run at least 14 days. Once we review and analyze the data, we will prepare the results and send to you. To help us manage the client volume, we ask that you do not contact us until 14 days has passed since your submission and signing of the quote.

What do the tests tell us?

HTS: The percent protection of cytopathic effect of SARS CoV-2.

Titer reduction: the reduction in virus titer as compared to virus plus cells with no drug.

What concentrations of compound are needed?

HTS: We will test across 7, two-fold dilutions, starting at 33 micromolar in a total reaction volume of 30 microliters. The dose response will be run in duplicate for:

1.     Duplicate DR of Cells plus compound for – No Virus

2.     Duplicate DR of Cells plus compound for – Plus Virus

Titer reduction and time of addition: We will run concentrations as requested. Typically, one runs these at 1 and 5 micromolar.

How much material and in what form should I send?

We accept dry powders that can be made up to 10 mM solutions or prepared solutions. We accept 10 mM, 1 mM, 200 uM stock solutions. For the HTS, we will use 5 microliters of 200 micromolar solution to reach a starting concentration of 33 micromolar in 0.5% DMSO. The starting material must not exceed 0.5% DMSO in a volume of 30 microliters.

What concentration of DMSO is allowed?

We ask that final concentrations of DMSO not exceed 0.5%. In the HTS assay we are limited to add 5 microliters of compound from the working stock. If your compound is in 100% DMSO, we ask you send a 10mM stock. If it is at 1mM it needs to be in less than or equal to 10%DMSO.

What is the virus?

SARS-CoV-2 UTHSC passage 3, (SARS-Related Coronavirus 2 Isolate USA-WA1/2020)

What is the cell line?

Vero E6 or as directed

How do I request an MTA/NDA or send an MTA/NDA? 

Please send to Dr. Jonsson and Na Yeon at nyeon@uthsc.edu. Ms. Yeon will upload into the UTHSC system. This process takes approximately one week to complete.

Selected Resources in Antiviral Discovery from Dr. Jonsson’s Research Program

High throughput screening, Titer Reduction, and Other

  1. Severson, W.E., Shindo, N., Sosa, M., Fletcher, T., White, L. and C. Jonsson. (2007) Development, validation and optimization of a luminescence-based high throughput screen for inhibitors of Severe Acute Respiratory Syndrome. J. Biomol. Scr. 12:33-40.
  2. Noah, J., Severson, W., Noah, D. Rasmussen, L., White, E. L. and C. B. Jonsson. (2007) A cell based luminescence assay is effective for high throughput screening of potential influenza antivirals. Antiviral Res. 73:50-59.
  3. Severson, W., Rasmussen, L., White, E. L. and C. B. Jonsson (2008) High-Throughput Screening of a 100,000 Compound Library for Inhibitors of Influenza A virus (H3N2). J. Biomol. Scr. 13:879-387
  4. Jia, F., Maddox, C., Gao, A., Tran, L., Severson, W., White, E.L., Rasmussen, L., Dang, A., Jonsson, C.B. (2010) A novel cell-based 384-well, label-free assay for discovery of inhibitors of influenza virus. Inter. J. High Throughput Screening 1:57-67.
  5. Maddry, J.A., Chen, X., Jonsson, C.B., Ananthan, S., Hobrath, J., Smee, D.E., Noah, J.W., Noah, D., Xu, X., Jia, F., Maddox, C., Sosa , M.I., White, E.L. and Severson W. (2011) Discovery of Novel Benzoquinazolinones and Thiazoloimidazoles, Inhibitors of Influenza H5N1 and H1N1 viruses, from a Cell-Based High-throughput Screen. J. Biomol. Scr. 16:73-81. PMID: 21059874
  6. Moore, B.P., Chung, D.H., Matharu, D.S., Golden, J.E., Maddox, C., Rasmussen, L., Noah, J.W., Sosa, M.I., Ananthan, S., Tower, N.A., White, E.L., Jia, F. , Prisinzano, T.E., Aubé, J., Jonsson, C.B., Severson, W.E. (2012) (S)-N-(2,5-Dimethylphenyl)-1-(quinoline-8-ylsulfonyl)pyrrolidine-2-carboxamide as a Small Molecule Inhibitor Probe for the Study of Respiratory Syncytial Virus Infection. J. Med. Chem. 55(20): 8582–8587. PMID: 23043370
  7. Chung, D., Moore, B.P., Matharu, D.S., Golden, J.E., Maddox, C., Rasmussen, L, Sosa, M.I., White, E.L., Ananthan, S., Jia, F., Jonsson, C.B., and W.E. Severson (2013). A cell based high-throughput screening approach for the discovery of new inhibitors of respiratory syncytial virus. Virology Journal 10:19.
  8. Chung, D.H.C., Jonsson, C.B. et al (2014) Discovery of a Novel Quinazolinone with Anti-Venezuelan Equine Encephalitis Virus Activity that Targets the Non-structural Protein 2. PLoS Path. 10(6):e1004213. doi: 10.1371. PMID: 24967809
  9. Zheng M, Williams EP, Malireddi RKS, Karki R, Banoth B, Burton A, Webby R, Channappanavar R, Jonsson CB, Kanneganti TD. Impaired NLRP3 inflammasome activation/pyroptosis leads to robust inflammatory cell death via caspase-8/RIPK3 during coronavirus infection. J Biol Chem. 2020 Oct 9;295(41):14040-14052. doi: 10.1074/jbc.RA120.015036. Epub 2020 Aug 6. PMID: 32763970; PMCID: PMC7549031.
  10. Bocci, G., Bradfute, S. B., Ye, C., Garcia, M. J., Parvathareddy, J., Reichard, W., Surendranathan, S., Bansal, S., Bologa, C. G., Perkins, D. J., Jonsson, C. B., Sklar, L. A., & Oprea, T. I. (2020). Virtual and In Vitro Antiviral Screening Revive Therapeutic Drugs for COVID-19. ACS Pharmacology & Translational Science, acsptsci.0c00131. https://doi.org/10.1021/acsptsci.0c00131
  11. Karki, R., Sharma, B.R., Tuladhar, S., Williams, E.P., Zalduondo, L., Samir, P., Zheng, M., Sundaram, B., Banoth, B., Malireddi, R. K. S., Schreiner, P., Neale, G., Vogel, P., Webby, R., Jonsson, C.B., Kanneganti, T. (2021) COVID-19 cytokines and the hyperactive immune response: Synergism of TNF-α and IFN-γ in triggering inflammation, tissue damage, and death. Cell 184, 149–168
  12. Kneller, Daniel; Li, Hui; Galanie, Stephanie; Phillips, Gwyndalyn; Labbe, Audrey; Weiss, Kevin; Zhang, Qiu; Arnould, Mark; Clyde, Austin; Ma, Heng; Ramanathan, Arvind; Jonsson, Colleen; Head, Martha; Coates, Leighton; Louis, John M.; Bonnesen, Peter; Kovalevsky, Andrey (2021) Structural, electronic and electrostatic determinants for inhibitor binding to subsites S1 and S2 in SARS-CoV-2 main protease. J Med Chem. 64(23):17366-17383. doi: 10.1021/acs.jmedchem.1c01475
  13. Bansal S, Jonsson CB, Taylor SL, Figueroa JM, Dugour AV, Palacios C, Vega JC. Iota-carrageenan and xylitol inhibit SARS-CoV-2 in Vero cell culture. PLoS One. 2021 16:e0259943. doi: 10.1371/journal.pone.0259943. PMID: 34797868; PMCID: PMC8604354.
  14. Williams EP, Taylor MK, Demchyshyna I, Nebogatkin I, Nesterova O, Khuda I, Chernenko L, Hluzd OA, Kutseva VV, Glass GE, Yanko N, Jonsson CB. Prevalence of Hantaviruses Harbored by Murid Rodents in Northwestern Ukraine and Discovery of a Novel Puumala Virus Strain. Viruses. 2021 Aug 18;13(8):1640. doi: 10.3390/v13081640. PMID: 34452504; PMCID: PMC8402871.
  15. Banerjee S, Yadav S, Banerjee S, Fakayode SO, Parvathareddy J, Reichard W, Surendranathan S, Mahmud F, Whatcott R, Thammathong J, Meibohm B, Miller DD, Jonsson CB, Dubey KD. Drug Repurposing to Identify Nilotinib as a Potential SARS-CoV-2 Main Protease Inhibitor: Insights from a Computational and Vitro Study. J Chem Inf Model. 2021 Nov 22;61(11):5469-5483. doi:10.1021/acs.jcim.1c00524. Epub 2021 Oct 20. PMID: 34666487; PMCID: PMC8547516.
  16. Karki R, Lee S, Mall R, Pandian N, Wang Y, Sharma BR, Malireddi RS, Yang D, Trifkovic S, Steele JA, Connelly JP, Vishwanath G, Sasikala M, Reddy DN, Vogel P, Pruett-Miller SM, Webby R, Jonsson CB, Kanneganti TD. ZBP1-dependent inflammatory cell death, PANoptosis, and cytokine storm disrupt IFN therapeutic efficacy during coronavirus infection. Sci Immunol. 2022 Aug 26;7(74):eabo6294. doi: 10.1126/sciimmunol.abo6294. Epub 2022 Aug 26. PMID: 35587515; PMCID:PMC9161373.
  17. Kneller DW, Li H, Phillips G, Weiss KL, Zhang Q, Arnould MA, Jonsson CB, Surendranathan S, Parvathareddy J, Blakeley MP, Coates L, Louis JM, Bonnesen PV, Kovalevsky A. Covalent narlaprevir- and boceprevir-derived hybrid inhibitors of SARS-CoV-2 main protease. Nat Commun. 2022 Apr 27;13(1):2268. doi: 10.1038/s41467-022-29915-z. PMID: 35477935; PMCID: PMC9046211.
  18. Hu H, Mady Traore MD, Li R, Yuan H, He M, Wen B, Gao W, Jonsson CB, Fitzpatrick EA, Sun D. Optimization of the Prodrug Moiety of Remdesivir to Improve Lung Exposure/Selectivity and Enhance Anti-SARS-CoV-2 Activity. J Med Chem. 2022 Sep 22;65(18):12044-12054. doi: 10.1021/acs.jmedchem.2c00758. Epub 2022 Sep 7. PMID: 36070561; PMCID: PMC9469953.
  19. Ryan MC, Kim E, Cao X, Reichard W, Ogorek TJ, Das P, Jonsson CB, Baudry J, Chung D, Golden JE. Piperazinobenzodiazepinones: New Encephalitic Alphavirus Inhibitors via Ring Expansion of 2-Dichloromethylquinazolinones. ACS Med Chem Lett. 2022 Mar 14;13(4):546-553. doi: 10.1021/acsmedchemlett.1c00539. PMID: 35450382; PMCID: PMC9014857.

 

Feb 13, 2023