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BioAssay: AID 1944

Luminescence-based counterscreen for inhibitors of the SARS coronavirus 3C-like Protease (3CLPro): dose response biochemical high throughput screening assay to identify inhibitors of the papain-like protease (PLpro)

Name: Luminescence-based counterscreen for inhibitors of the SARS coronavirus 3C-like Protease (3CLPro): dose response biochemical high throughput screening assay to identify inhibitors of the papain-like protease (PLpro) ..more
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 Tested Compounds
 Tested Compounds
All(101)
 
 
Active(19)
 
 
Inactive(82)
 
 
 Tested Substances
 Tested Substances
All(101)
 
 
Active(19)
 
 
Inactive(82)
 
 
 Related BioAssays
 Related BioAssays
AID: 1944
Data Source: The Scripps Research Institute Molecular Screening Center (PLPRO_INH_LUMI_1536_3XIC50)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2009-09-09

Data Table ( Complete ):           Active    All
BioActive Compounds: 19
Depositor Specified Assays
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AIDNameTypeComment
1859Summary of probe development efforts to identify inhibitors of the SARS coronavirus 3C-like Protease (3CLPro)summarySummary AID.
1890QFRET-based dose response biochemical high throughput screening assay to identify inhibitors of the SARS coronavirus 3C-like Protease (3CLPro)confirmatoryDose response assay.
435015Late 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 cellsscreening
488877Late 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 cellsconfirmatory
488958Late stage assay provider results from the probe development effort to identify inhibitors of the SARS coronavirus 3C-like Protease (3CLPro): fluorescence-based biochemical dose-response assay for inhibitors of 3CLProconfirmatory
488967Late stage assay provider results from the probe development effort to identify inhibitors of the SARS coronavirus 3C-like Protease (3CLPro): fluorescence-based biochemical assay for inhibitors of 3CLProother
488984Late stage assay provider results from the probe development effort to identify inhibitors of the SARS coronavirus 3C-like Protease (3CLPro): fluorescence-based biochemical assay for inhibitors of 3CLPro: Set 2other
488999Late stage assay provider results from the probe development effort to identify inhibitors of the SARS coronavirus 3C-like Protease (3CLPro): fluorescence-based biochemical dose-response assay for inhibitors of 3CLPro; Set 2confirmatory
493245Late stage assay provider results from the probe development effort to identify inhibitors of the SARS coronavirus 3C-like Protease (3CLPro): fluorescence-based biochemical assay for inhibitors of 3CLPro: Set 3other
588771Late stage assay provider results from the probe development effort to identify inhibitors of the SARS coronavirus 3C-like Protease (3CLPro): fluorescence-based biochemical dose-response assay for inhibitors of 3CLPro; Set 3confirmatory
588772Late stage assay provider results from the probe development effort to identify inhibitors of the SARS coronavirus 3C-like Protease (3CLPro): fluorescence-based biochemical assay for inhibitors of 3CLPro: Set 4other
588786Late stage assay provider results from the probe development effort to identify inhibitors of the SARS coronavirus 3C-like Protease (3CLPro): fluorescence-based biochemical dose-response assay for inhibitors of 3CLPro; Set 4confirmatory
602486Late stage assay provider results from the probe development effort to identify inhibitors of the SARS coronavirus 3C-like Protease (3CLPro): fluorescence-based biochemical dose-response assay for inhibitors of 3CLPro; Set 5confirmatory
602487Late stage assay provider results from the probe development effort to identify inhibitors of the SARS coronavirus 3C-like Protease (3CLPro): fluorescence-based biochemical assay for inhibitors of 3CLPro: Set 5other
Description:
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: PLPRO_INH_LUMI_1536_3XIC50

Name: Luminescence-based counterscreen for inhibitors of the SARS coronavirus 3C-like Protease (3CLPro): dose response biochemical high throughput screening assay to identify inhibitors of the papain-like protease (PLpro)

Description:

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.

References:

1. Myint, S.H., Human coronavirus infections, in The Coronaviridae, S.G. Siddell, Editor. 1995. p. 389-401. Plenum Press, London. Book in the series, The Viruses. Fraenkel-Conrat, H., Wagner, R.R. (series Eds.), Plenum Press, New York.
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.

Keywords:

PLpro, papain-like protease, 3CLpro,protease, cysteine protease, coronavirus, virus, SARS, SARS-CoV, peptide cleavage, inhibitor, inhibition, QFRET, luminescence, quenching fluorescence resonance energy transfer, dose response, counterscreen, HTS, high throughput screen, 1536, assay, Scripps, Scripps Florida, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN.
Protocol
Assay Overview:

The purpose of this bioluminescent biochemical assay is to identify compounds that inhibit the activity of the deubiquitinating enzyme papain-like protease (PLpro) of SARS-CoV or Luciferase. This assay also serves as a counterscreen for a set of previous experiments entitled, "QFRET-based dose response biochemical high throughput screening assay to identify inhibitors of the SARS coronavirus 3C-like Protease (3CLPro)," (PubChem AID 1890). In this coupled-enzyme assay, test compounds are incubated with recombinant PLpro enzyme and an ubiquitin C-terminal RLRGG-derivatized aminoluciferin substrate, in the presence of luciferase and ATP. PLpro-mediated deubiquitination and cleavage of substrate releases aminoluciferin, which acts as a luciferase substrate, leading to an increase in well luminescence. The light signal is proportional to PLpro activity. As designed, compounds that act as PLpro inhibitors will prevent PLpro-mediated substrate deubiquitination and cleavage, thus preventing release of aminoluciferin, leading to no increase in well luminescence. Compounds were tested in triplicate using a 10-point, 1:3 dilution series starting at a nominal concentration of 60 micromolar.

Protocol Summary:

Prior to the start of the assay, a mixture of PLpro peptide substrate and luciferase detection reagent was made up in assay buffer (50mM HEPES, 0.1 mg/ml BSA, 5 mM DTT, 0.5 mM EDTA and 1 mM Magnesium sulfate) at pH 7.5 and incubated for 60 min. The assay was begun by dispensing 2.5 microliters of PLpro enzyme (7.5 nM final concentration) in assay buffer or assay buffer alone into each well of a 1536 microtiter plate. Next, 30 nL of test compound in DMSO, or DMSO alone (0.6% final concentration) was added to the appropriate wells. The plates were then incubated for 10 minutes at room temperature. Next, the enzyme reaction was initiated by dispensing 2.5 microliters of the preincubated mixture containing PLpro peptide substrate and luciferase detection reagent (1 micromolar final substrate concentration). Finally, well luminescence was read on a PerkinElmer Viewlux after 60 minutes of incubation at room temperature.

The % inhibition for each well was then calculated as follows:

% Inhibition = ( RLU_Test_Compound - MedianRLU_Low_Control ) / ( MedianRLU_High_Control - MedianRLU_Low_Control ) * 100

Where:

Test_Compound is defined as wells containing test compound.
High_Control is defined as wells containing no enzyme added.
Low_Control is defined as wells containing DMSO.

For each test compound, percent inhibition was plotted against compound concentration. A four parameter equation describing a sigmoidal dose-response curve was then fitted with adjustable baseline using Assay Explorer software (MDL Information Systems). The reported IC50 values were generated from fitted curves by solving for the X-intercept value at the 50% inhibition level of the Y-intercept value. In cases where the highest concentration tested (i.e. 60 micromolar) did not result in greater than 50% inhibition, the IC50 was determined manually as greater than 60 uM. Compounds with an IC50 greater than 10 uM were considered inactive. Compounds with an IC50 equal to or 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, with the most potent compounds assigned the highest activity scores.

The activity score range for active compounds is 100-84, for inactive 79-0.

List of Reagents:

Recombinant SARS-PLpro (supplied by Assay Provider)
Luciferase detection reagent (Promega, part V8920)
PLpro peptide substrate: (provided by Promega)
1536-well plates (Greiner, part 789173)
HEPES (Sigma, H4034)
Magnesium sulfate (Sigma, 203726)
EDTA (Sigma, E7889)
DTT (Fisher, part BP172-5)
BSA (Fisher, part NC9871802)
Comment
Due to the increasing size of the MLPCN compound library, this assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. All data reported were normalized on a per-plate basis. 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 by the MLSMR. The MLSMR was not able to provide all compounds selected for testing in this AID.
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1QualifierActivity Qualifier identifies if the resultant data IC50 came from a fitted curve or was determined manually to be less than or greater than its listed IC50 concentration.String
2IC50*The concentration at which 50 percent of the activity in the inhibitor assay is observed; (IC50) shown in micromolar.FloatμM
3LogIC50Log10 of the qualified IC50 (IC50) from the inhibitor assay in M concentration.Float
4Hill SlopeThe variable HillSlope describes the steepness of the curve. This variable is called the Hill slope, the slope factor, or the Hill coefficient. If it is positive, the curve increases as X increases. If it is negative, the curve decreases as X increases. A standard sigmoid dose-response curve (previous equation) has a Hill Slope of 1.0. When HillSlope is less than 1.0, the curve is more shallow. When HillSlope is greater than 1.0, the curve is steeper. The Hill slope has no units.Float
5Hill S0Y-min of the curve.Float
6Hill SinfY-max of the curve.Float
7Hill dSThe range of Y.Float
8Chi SquareA measure for the 'goodness' of a fit. The chi-square test (Snedecor and Cochran, 1989) is used to test if a sample of data came from a population with a specific distribution.Float
9RsquareThis statistic measures how successful the fit explains the variation of the data; R-square is the square of the correlation between the response values and the predicted response values.Float
10Number of DataPointsOverall number of data points of normalized percent inhibition that was used for calculations (includes all concentration points); in some cases a data point can be excluded as outlier.Integer
11Inhibition at 3.0 nM (0.003μM**)Value of %inhibition at 3.0 nanomolar inhibitor concentration; average of triplicate measurement.Float%
12Inhibition at 9.1 nM (0.009μM**)Value of %inhibition at 9.0 nanomolar inhibitor concentration; average of triplicate measurement.Float%
13Inhibition at 27.3 nM (0.0273μM**)Value of %inhibition at 27.3 nanomolar inhibitor concentration; average of triplicate measurement.Float%
14Inhibition at 81.8 nM (0.0818μM**)Value of %inhibition at 81.8 nanomolar inhibitor concentration; average of triplicate measurement.Float%
15Inhibition at 245.4 nM (0.245μM**)Value of %inhibition at 245 nanomolar inhibitor concentration; average of triplicate measurement.Float%
16Inhibition at 736.3 nM (0.736μM**)Value of %inhibition at 736 nanomolar inhibitor concentration; average of triplicate measurement.Float%
17Inhibition at 2.2 uM (2.2μM**)Value of %inhibition at 2.2 micromolar inhibitor concentration; average of triplicate measurement.Float%
18Inhibition at 6.6 uM (6.6μM**)Value of %inhibition at 6.6 micromolar inhibitor concentration; average of triplicate measurement.Float%
19Inhibition at 19.9 uM (19.9μM**)Value of %inhibition at 19.9 micromolar inhibitor concentration; average of triplicate measurement.Float%
20Inhibition at 59.6 uM (59.6μM**)Value of %inhibition at 59.6 micromolar inhibitor concentration; average of triplicate measurement.Float%

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: 1-R03-MH084162-01A1

Data Table (Concise)
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