Late stage assay provider counterscreen results from the probe development effort to identify inhibitors of the SARS coronavirus 3C-like Protease (3CLPro): luminescence-based dose-response cell-based assay for restoration of viability of SARS-CoV-infected Vero cells
Name: Late stage assay provider counterscreen results from the probe development effort to identify inhibitors of the SARS coronavirus 3C-like Protease (3CLPro): luminescence-based dose-response cell-based assay for restoration of viability of SARS-CoV-infected Vero cells. ..more
Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center (SRIMSC)
Center Affiliation: The Scripps Research Institute (TSRI)
Assay Provider: Valerie Tokars and Andrew Mesecar, University of Illinois at Chicago (UIC)
Network: Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Proposal Number: 1-R03-MH084162-01A1
Grant Proposal PI: Valerie Tokars and Andrew Mesecar, UIC
External Assay ID: SARS-CoV-INFECTED VERO CELLS_INH_LUMI_96_3XIC50
Name: Late stage assay provider counterscreen results from the probe development effort to identify inhibitors of the SARS coronavirus 3C-like Protease (3CLPro): luminescence-based dose-response cell-based assay for restoration of viability of SARS-CoV-infected Vero cells.
Coronaviruses are enveloped, large plus-strand RNA viruses that cause the common cold and other disorders such as lower respiratory tract infections and diarrhea (1). In 2003, the novel SARS coronavirus (SARS-CoV) was identified (2, 3) as the etiological agent of the global epidemic of severe acute respiratory syndrome (SARS), an atypical pneumonia that led to progressive respiratory failure in 8000 individuals and 800 deaths by July of that year (4). The SARS-CoV genome encodes a polypeptide that is proteolytically processed by two main proteases, one of which is the 3C-like protease (3CLpro). This chemotrypsin-like cysteine protease is essential for proteolytic processing of the coronavirus polyprotein and thus viral gene expression (5). The protein exists as a dimer/monomer mixture in solution and the dimer was confirmed to be the active species for the enzyme reaction (6). The current absence of a vaccine to prevent SARS infection, the possibility of future SARS epidemics, the recent cloning and expression of recombinant SARS 3CLpro (7), along with studies showing that 3CLpro is essential for viral life cycle, support a role for 3CL-pro as an important pathogenic component of SARS-CoV. The identification of specific inhibitors of 3CLpro will add insights into the biology of SARS-CoV infection of avian and mammalian cells, and serve as valuable tools for inhibiting SARS-CoV replication.
1. Myint, S.H., Human coronavirus infections, in The Coronaviridae, S.G. Siddell, Editor. 1995, Plenum Press. p. 389-401.
2. Ksiazek, T.G., Erdman, D., Goldsmith, C.S., Zaki, S.R., Peret, T., Emery, S., Tong, S., Urbani, C., Comer, J.A., Lim, W., Rollin, P.E., Dowell, S.F., Ling, A.E., Humphrey, C.D., Shieh, W.J., Guarner, J., Paddock, C.D., Rota, P., Fields, B., DeRisi, J., Yang, J.Y., Cox, N., Hughes, J.M., LeDuc, J.W., Bellini, W.J., and Anderson, L.J., A novel coronavirus associated with severe acute respiratory syndrome. N Engl J Med, 2003. 348(20): p. 1953-66.
3. Drosten, C., Gunther, S., Preiser, W., van der Werf, S., Brodt, H.R., Becker, S., Rabenau, H., Panning, M., Kolesnikova, L., Fouchier, R.A., Berger, A., Burguiere, A.M., Cinatl, J., Eickmann, M., Escriou, N., Grywna, K., Kramme, S., Manuguerra, J.C., Muller, S., Rickerts, V., Sturmer, M., Vieth, S., Klenk, H.D., Osterhaus, A.D., Schmitz, H., and Doerr, H.W., Identification of a novel coronavirus in patients with severe acute respiratory syndrome. N Engl J Med, 2003. 348(20): p. 1967-76.
4. Ziebuhr, J., Molecular biology of severe acute respiratory syndrome coronavirus. Curr Opin Microbiol, 2004. 7(4): p. 412-9.
5. Yang, H., Bartlam, M., and Rao, Z., Drug design targeting the main protease, the Achilles' heel of coronaviruses. Curr Pharm Des, 2006. 12(35): p. 4573-90.
6. Lai, L., Han, X., Chen, H., Wei, P., Huang, C., Liu, S., Fan, K., Zhou, L., Liu, Z., Pei, J., and Liu, Y., Quaternary structure, substrate selectivity and inhibitor design for SARS 3C-like proteinase. Curr Pharm Des, 2006. 12(35): p. 4555-64.
7. Fan, K., Wei, P., Feng, Q., Chen, S., Huang, C., Ma, L., Lai, B., Pei, J., Liu, Y., Chen, J., and Lai, L., Biosynthesis, purification, and substrate specificity of severe acute respiratory syndrome coronavirus 3C-like proteinase. J Biol Chem, 2004. 279(3): p. 1637-42.
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The purpose of this luminescent cell-based assay is to determine whether powder samples of compounds identified as possible 3C-like Protease (3CLpro) inhibitor probe candidates have the ability to restore viability to Vero cells infected with the SARS-CoV virus. In this assay, test compounds are incubated with SARS-CoV infected cells, and cell viability compared to that of mock-infected cells. The assay utilizes the CellTiter-Glo luminescent reagent (Promega) to measure intracellular ATP in viable cells. The luminescent signal is proportional to the amount of ATP present, which is an indicator of metabolically active cells. As designed, compounds that inhibit 3CLpro will decrease SARS-CoV infection, increase the number of viable cells, and increase well luminescence. Compounds that fail to restore at least 50% of cell viability at 10 uM when luciferase values from SARS-CoV infected cells are compared to mock infected cells will be considered inactive. Compounds were tested in triplicate using a 4-point, 1:2 dilution series starting at a nominal concentration of 40 uM.
The Vero cell line was routinely cultured in T75 tissue culture flasks at 37 C and 95% relative humidity. To assess the antiviral activity of the test compound, a 96-well tissue culture plate is seeded with Vero E6 cells at 10,000 cells/well in 100mL of Minimal Essential Media (MEM) supplemented with 10% FCS and 1X antibiotics (100 U/mL penicillin and 100 ug/mL streptomycin). Half of the 96-well plate is infected at the BSL3 facility with SARS-CoV Urbani at 100 times the TCID50. Following a one-hour incubation to allow the virus to enter the cells, the media is replaced with fresh MEM supplemented with 2% FCS containing the compound of interest at decreasing concentrations in triplicate wells. Wells containing SARS-CoV infected cells containing no compound serve as a positive control for the ability of SARS-CoV to kill the cells. Mock infected cells lacking compound provide a reference for 100% cell viability. Following a 48-hour incubation at 37 C with 5% CO2, cell viability is determined using Promega's CellTiterGloR assay. Briefly, the media is removed and wells washed with 100uL of phosphate buffered saline (PBS). After removal of PBS, cells receive 50uL of fresh MEM supplemented with 2% FCS and an equal amount of the CellTiterGloR reagent. Following a two minute mixing on an orbital shaker, the lysates are allowed to incubate for 30 minutes at room temperature. 75 uL of cellular lysate is transferred to a 96 well assay plate and luminescence is measured on the Glomax 96 Microplate Luminometer.
% Inhibition = ( ( Control_1 - Test_Compound ) / ( Control_1 - Control_2 ) * 100
Test_Compound is defined as SARS-CoV infected cells in presence of compound.
Control_1 is defined mock infected cells in the presence of DMSO.
Control_2 is defined as SARS-CoV infected cells in the presence of DMSO.
The % recovery of cells representing cell viability = 100 - % inhibition
% Inhibition is plotted against concentration and the data visually examined. If a value of 50 % viability or less is observed at 10uM of compound, the compound is reported to have an IC50 greater than 10uM, and inactive. In this assay a compound demonstrating 100% inhibition represents cells that have minimal viability and have not recovered from the viral infection in the presence of compound. Conversely, 0% inhibition represents maximum viability.
The reported IC50 value was generated from a dose response curve with each concentration measured in triplicate.
PubChem Activity Outcome and Score:
Compounds with an IC50 less than 10 uM were considered active.
Any compound with a percent activity value < 50% at all test concentrations was assigned an activity score of zero. Any compound with a percent activity value >= 50% at any test concentration was assigned an activity score greater than zero.
Activity score was then ranked by the potency of the compounds with fitted curves, with the most potent compounds assigned the highest activity scores.
The PubChem Activity Score range for inactive compounds is 0-0. There are no active compounds.
List of Reagents:
SARS-CoV Urbani virus (supplied by Assay Provider)
Vero E6 Cell line (supplied by Assay Provider)
Minimal Essential Media (Gibco, part 61100-061)
L-Glutamine 200 mM (HyClone, part SH30034.01)
10,000 U/ml Penicillin-10,000 ug/ml Streptomycin (HyClone , part SV30010)
PBS (Gibco, part 70011-044)
Fetal Bovine Serum (Atlas Biologicals, part F-0500A)
CellTiter-Glo (Promega, part G75729)
T75 tissue culture flasks (Corning, part 430720)
DMSO (Sigma, part D2650)
96-well tissue culture plates (Zellkulter, Test Plate 92696)
96-well assay plate (Costar, part 3912)
Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, compounds that modulate well luminescence. This assay was performed by the assay provider, and submitted to PubChem by the Scripps Research Institute Molecular Screening Center (SRIMSC) on behalf of The Vanderbilt Specialized Chemistry Center. The compound tested in this assay was synthesized by The Vanderbilt Specialized Chemistry Center.
* Activity Concentration. ** Test Concentration.
Data Table (Concise)