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BioAssay: AID 588786

Late 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 4

Name: Late 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 4. ..more
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 Tested Compounds
 Tested Compounds
All(10)
 
 
Active(3)
 
 
Inactive(7)
 
 
 Tested Substances
 Tested Substances
All(10)
 
 
Active(3)
 
 
Inactive(7)
 
 
AID: 588786
Data Source: The Scripps Research Institute Molecular Screening Center (3CLPRO_INH_QFRET_96_3XIC50_SET 4)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network, Assay Provider
BioAssay Version:
Deposit Date: 2011-11-12
Hold-until Date: 2012-10-28
Modify Date: 2012-10-29

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 3
Related Experiments
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AIDNameTypeComment
1706QFRET-based primary biochemical high throughput screening assay to identify inhibitors of the SARS coronavirus 3C-like Protease (3CLPro)Screeningdepositor-specified cross reference: Primary screen (3CLPro inhibitors in singlicate)
1859Summary of probe development efforts to identify inhibitors of the SARS coronavirus 3C-like Protease (3CLPro)Summarydepositor-specified cross reference: Summary (3CLPro inhibitors)
1879QFRET-based confirmation biochemical high throughput screening assay for inhibitors of the SARS coronavirus 3C-like Protease (3CLPro)Screeningdepositor-specified cross reference: Confirmation screen (3CLPro inhibitors in triplicate)
1890QFRET-based dose response biochemical high throughput screening assay to identify inhibitors of the SARS coronavirus 3C-like Protease (3CLPro)Confirmatorydepositor-specified cross reference: Dose response (3CLPro inhibitors in triplicate)
1944Luminescence-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)Confirmatorydepositor-specified cross reference: Dose response counterscreen (papain-like protease inhibitors in triplicate)
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 cellsScreeningdepositor-specified cross reference: Late stage counterscreen (cytotoxic compounds in Vero E6 cells in triplicate)
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 cellsConfirmatorydepositor-specified cross reference: Late stage counterscreen dose response (SARS-CoV-infected Vero cells restored viability in triplicat
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 3CLProConfirmatorydepositor-specified cross reference: Late stage dose response (3CLPro inhibitors in triplicate)
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 3CLProOtherdepositor-specified cross reference: Late stage screen (3CLPro inhibitors in triplicate)
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 2Otherdepositor-specified cross reference: Late stage assay (3CLPro inhibitors in triplicate)
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 2Confirmatorydepositor-specified cross reference: Late stage dose response (3CLPro inhibitors in triplicate)
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 3Otherdepositor-specified cross reference: Late stage assay (3CLPro inhibitors in triplicate)
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 5Confirmatorydepositor-specified cross reference
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 5Otherdepositor-specified cross reference
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 3Confirmatorysame project related to Summary assay
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 4Othersame project related to Summary assay
623944ML300 Competition in Radioligand Binding assays (Ricerca)Othersame project related to Summary assay
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: 3CLPRO_INH_QFRET_96_3XIC50_SET 4

Name: Late 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 4.

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 3CLpro 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, 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.

Keywords:

late stage, late stage AID, SAR, powder, synthesized, synthesis, 96, fluorescence, quench, peptide substrate, 3CLpro, 3C-like protease, protease, cysteine protease, coronavirus, virus, SARS, SARS-CoV, peptide cleavage, inhibitor, inhibition, dose response, assay, assay provider, Vanderbilt, Vanderbilt Specialized Chemistry Center, VSCC, VU, 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 biochemical assay is to test the ability of a set of synthesized compounds to inhibit SARS-3CLpro-mediated peptide cleavage. In this assay, a fluorescent compound, HiLyte fluor TM 488, is attached at the N-terminus of a 3CLpro peptide substrate and is FRET quenched by a QXLTM520 moiety attached at the C-terminus. When the peptide is cleaved by SARS-3CLpro the fluorescent compound and quencher separate, leading to an increase in well fluorescence when measured at an excitation wavelength of 485 nm and an emission wavelength of 535 nm. As designed, compounds that inhibit 3CLpro activity will prevent cleavage of the labeled peptide substrate, thus leaving the fluorescent tag quenched on the peptide, resulting in no increase in well fluorescence. Compounds were tested in triplicate at 8 final concentrations (in serial dilution) from 120 to 0.313 uM.
Protocol Summary:
Prior to the start of the assay, 1 uL of test compound in DMSO or DMSO alone (1% final concentration) was added to the wells of a 96-well black flat-bottomed half-area plate. Next, 80 uL of 3CLpro enzyme (250 nM final concentration) in assay buffer (50 mM HEPES, 0.1 mg/ml BSA, 0.01% Triton-X 100, 2 mM DTT) at pH 7.5 was dispensed into each well. Negative control wells contained buffer without enzyme. The plates were then incubated for 10 minutes at room temperature. The assay was started by dispensing 20 uL of 3CLpro peptide substrate (2 uM final concentration) in 50 mM HEPES at pH 7.5 to each well. Well fluorescence was read continuously using fluorescein filters: excitation wavelength of 480 nm (with 20 nm bandwidth) and emission wavelength of 535 nm (with 20 nm bandwidth).
The % inhibition for each well was then calculated as follows:
%_Inhibition = ( 1 - ( RFU_Test_Compound - Average_RFU_Low_Control ) / ( Average_RFU_High_Control - Average_RFU_Low_Control ) ) * 100
Where:
Test_Compound is defined as wells containing test compound and enzyme.
High_Control is defined as wells containing DMSO and enzyme.
Low_Control is defined as wells containing DMSO and no enzyme.
The reported IC50 value was generated from a dose response curve.
For some compounds, an artifact in the curve fit possibly due to limited compound solubility and/or induced aggregation of the enzyme was observed, resulting in incomplete inhibition at the highest concentration tested.
PubChem Activity Outcome and Score:
Compounds with an IC50 less than 10 uM were considered active.
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 active compounds is 100-84, and for inactive compounds 1-1.
List of Reagents:
Recombinant SARS-3CLpro (supplied by Assay Provider)
3CLpro peptide substrate: HiLyte Fluor 488-ESATLQSGLRKAK(QXL520)-NH2 (AnaSpec, part UIVT-3)
96-well plates (Corning, part 3694)
HEPES (Invitrogen, part 15630)
Triton-X 100 (Fisher, part BP151-100)
DTT (Fisher, part BP172-5)
BSA (Sigma, part A35090)
Comment
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 compounds tested in this assay were synthesized by The Vanderbilt Specialized Chemistry Center.
Categorized Comment - additional comments and annotations
From BioAssay Depositor:
BAO: assay format: biochemical format: protein format: single protein format
BAO: bioassay specification: assay biosafety level: bsl1
BAO: bioassay specification: assay measurement type: endpoint assay
BAO: bioassay specification: assay readout content: assay readout method: regular screening
BAO: bioassay specification: assay readout content: content readout type: single readout
BAO: bioassay specification: assay stage: compound profiling
BAO: detection technology: fluorescence: fluorescence intensity
BAO: meta target: biological process target: viral genome replication
BAO: meta target: molecular target: protein target: enzyme: protease
BAO: version: 1.4b1090
From PubChem:
Assay Format: Biochemical
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Average IC50*The concentration at which 50 percent of the activity in the inhibition assay is observed; (IC50) shown in micromolar.FloatμM
2Standard ErrorStandard error for value of IC50.Float
3Inhibition at 120 uM [1] (120μM**)Value of % Inhibiton at 120 uM compound concentration; replicate one.Float%
4Inhibition at 100 uM [1] (100μM**)Value of % Inhibiton at 100 uM compound concentration; replicate one.Float%
5Inhibition at 80 uM [1] (80μM**)Value of % Inhibiton at 80 uM compound concentration; replicate one.Float%
6Inhibition at 60 uM [1] (60μM**)Value of % Inhibiton at 60 uM compound concentration; replicate one.Float%
7Inhibition at 40 uM [1] (40μM**)Value of % Inhibiton at 40 uM compound concentration; replicate one.Float%
8Inhibition at 20 uM [1] (20μM**)Value of % Inhibiton at 20 uM compound concentration; replicate one.Float%
9Inhibition at 10 uM [1] (10μM**)Value of % Inhibiton at 10 uM compound concentration; replicate one.Float%
10Inhibition at 5 uM [1] (5μM**)Value of % Inhibiton at 5 uM compound concentration; replicate one.Float%
11Inhibition at 2.5 uM [1] (2.5μM**)Value of % Inhibiton at 2.5 uM compound concentration; replicate one.Float%
12Inhibition at 1.25 uM [1] (1.25μM**)Value of % Inhibiton at 1.25 uM compound concentration; replicate one.Float%
13Inhibition at 0.625 uM [1] (0.625μM**)Value of % Inhibiton at 0.625 uM compound concentration; replicate one.Float%
14Inhibition at 0.313 uM [1] (0.313μM**)Value of % Inhibiton at 0.313 uM compound concentration; replicate one.Float%
15Inhibition at 120 uM [2] (120μM**)Value of % Inhibiton at 120 uM compound concentration; replicate two.Float%
16Inhibition at 100 uM [2] (100μM**)Value of % Inhibiton at 100 uM compound concentration; replicate two.Float%
17Inhibition at 80 uM [2] (80μM**)Value of % Inhibiton at 80 uM compound concentration; replicate two.Float%
18Inhibition at 60 uM [2] (60μM**)Value of % Inhibiton at 60 uM compound concentration; replicate two.Float%
19Inhibition at 40 uM [2] (40μM**)Value of % Inhibiton at 40 uM compound concentration; replicate two.Float%
20Inhibition at 20 uM [2] (20μM**)Value of % Inhibiton at 20 uM compound concentration; replicate two.Float%
21Inhibition at 10 uM [2] (10μM**)Value of % Inhibiton at 10 uM compound concentration; replicate two.Float%
22Inhibition at 5 uM [2] (5μM**)Value of % Inhibiton at 5 uM compound concentration; replicate two.Float%
23Inhibition at 2.5 uM [2] (2.5μM**)Value of % Inhibiton at 2.5 uM compound concentration; replicate two.Float%
24Inhibition at 1.25 uM [2] (1.25μM**)Value of % Inhibiton at 1.25 uM compound concentration; replicate two.Float%
25Inhibition at 0.625 uM [2] (0.625μM**)Value of % Inhibiton at 0.625 uM compound concentration; replicate two.Float%
26Inhibition at 0.313 uM [2] (0.313μM**)Value of % Inhibiton at 0.313 uM compound concentration; replicate two.Float%
27Inhibition at 120 uM [3] (120μM**)Value of % Inhibiton at 120 uM compound concentration; replicate three.Float%
28Inhibition at 100 uM [3] (100μM**)Value of % Inhibiton at 100 uM compound concentration; replicate three.Float%
29Inhibition at 80 uM [3] (80μM**)Value of % Inhibiton at 80 uM compound concentration; replicate three.Float%
30Inhibition at 60 uM [3] (60μM**)Value of % Inhibiton at 60 uM compound concentration; replicate three.Float%
31Inhibition at 40 uM [3] (40μM**)Value of % Inhibiton at 40 uM compound concentration; replicate three.Float%
32Inhibition at 20 uM [3] (20μM**)Value of % Inhibiton at 20 uM compound concentration; replicate three.Float%
33Inhibition at 10 uM [3] (10μM**)Value of % Inhibiton at 10 uM compound concentration; replicate three.Float%
34Inhibition at 5 uM [3] (5μM**)Value of % Inhibiton at 5 uM compound concentration; replicate three.Float%
35Inhibition at 2.5 uM [3] (2.5μM**)Value of % Inhibiton at 2.5 uM compound concentration; replicate three.Float%
36Inhibition at 1.25 uM [3] (1.25μM**)Value of % Inhibiton at 1.25 uM compound concentration; replicate three.Float%
37Inhibition at 0.625 uM [3] (0.625μM**)Value of % Inhibiton at 0.625 uM compound concentration; replicate three.Float%
38Inhibition at 0.313 uM [3] (0.313μM**)Value of % Inhibiton at 0.313 uM compound concentration; replicate three.Float%
39Incomplete Inhibition at Highest Concentration TestedWhether an artifact in the curve fit due to limited compound solubility and/or induced aggregation of the enzyme was observed, resulting in incomplete inhibition at the highest concentration tested, one of true or false.Boolean

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

Data Table (Concise)
Data Table ( Complete ):     View Active Data    View All Data
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