Late stage counterscreen results from the probe development effort to identify inhibitors of the SARS coronavirus 3C-like Protease (3CLPro); luminescence-based cell-based assay to identify cytotoxic compounds in Vero E6 cells
Name: Late stage counterscreen results from the probe development effort to identify inhibitors of the SARS coronavirus 3C-like Protease (3CLPro); luminescence-based cell-based assay to identify cytotoxic compounds in Vero E6 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: VERO-E6-CYTOX_INH_LUMI_96_3X%INH MDCSRUN Assay Provider
Name: Late stage counterscreen results from the probe development effort to identify inhibitors of the SARS coronavirus 3C-like Protease (3CLPro); luminescence-based cell-based assay to identify cytotoxic compounds in Vero E6 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 chymotrypsin-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-CoV infection, the possibility of future SARS-CoV epidemics, the recent cloning and expression of recombinant SARS-CoV 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 coronavirus 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 assay is to determine whether powder sample of a compound identified as a possible 3CLPro probe candidate are cytotoxic to Vero E6 cells. In this assay, test compounds are incubated with Vero E6 cells. Control cells are measured in the presence of DMSO alone. The assay utilizes the CellTiter-Glo luminescent reagent (Promega) to measure intracellular ATP in viable cells. The number of viable cells in culture is based on the quantitation of the ATP present. The luminescent signal is proportional to the amount of ATP present which is an indicator of metabolically active cells. If the luciferase values in the presence of inhibitor drop below that of DMSO alone, then the inhibitor is considered toxic. Compounds were tested in triplicate at a nominal concentration of 20 uM.
The Vero cell line was routinely cultured in T75 tissue culture flasks at 37 degrees C and 95% relative humidity. The growth media consisted of Minimal Essential Media supplemented with 10% fetal bovine serum, 4 mM L-Glutamine and 1x antibiotics (100 U/ml penicillin and 100 ug/ml streptomycin).
Vero E6 cells at 10000 cells/well are dispensed into a 96-well tissue culture plate in 100 uL of growth media. After one hour incubation at 37 C and 5% CO2, the media is replaced with fresh MEM supplemented with 2% FCS containing test compound (20 uM final concentration). The final DMSO concentration is 0.25%.
Following 48 hour incubation at 37 degrees C with 5% CO2, cell viability is determined using CellTiter-Glo assay (Promega). Briefly, the media is removed and wells are washed with 100 uL of phosphate buffered saline (PBS). After removal of PBS, cells receive 50 uL of fresh MEM and an equal amount of the CellTiter-Glo reagent. Following two minutes mixing on an orbital shaker, the lysates are incubated 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.
The percent inhibition for each compound was calculated as follows:
% Inhibition = ( ( Low_Control - Test_Compound ) / ( Low_Control ) ) * 100
Test_Compound is defined as wells containing test compound.
Low_Control is defined as wells containing 0.25% DMSO.
The reported % values were generated from a single concentration measured in triplicate. Compounds with a % inhibition greater than 25% were considered cytotoxic.
PubChem Activity Score and Outcome:
The PubChem Activity Score range for inactive compounds 0-0. There are no active compounds.
List of Reagents:
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 μg/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)
This assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. 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. All test compound concentrations reported above and below are nominal; the specific test concentration(s) for a particular compound may vary based upon the actual sample provided. This assay was performed by the assay provider, and submitted to PubChem by the Scripps Research Institute Molecular Screening Center (SRIMSC). Compound(s) tested in this assay were synthesized by the Vanderbilt Specialized Chemistry Center.
Categorized Comment - additional comments and annotations
** Test Concentration.
Data Table (Concise)