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BioAssay: AID 488967

Late 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

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 assay for inhibitors of 3CLPro. ..more
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 Tested Compounds
 Tested Compounds
All(32)
 
 
Active(15)
 
 
Inactive(17)
 
 
 Tested Substances
 Tested Substances
All(32)
 
 
Active(15)
 
 
Inactive(17)
 
 
AID: 488967
Data Source: The Scripps Research Institute Molecular Screening Center (3CLPRO_INH_QFRET_96_3X%INH)
Depositor Category: NIH Molecular Libraries Probe Production Network, Assay Provider
Deposit Date: 2010-10-28
Hold-until Date: 2011-10-27
Modify Date: 2011-10-27

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 15
Depositor Specified Assays
Show more
AIDNameTypeComment
1706QFRET-based primary biochemical high throughput screening assay to identify inhibitors of the SARS coronavirus 3C-like Protease (3CLPro)screeningPrimary screen (3CLPro inhibitors in singlicate)
1859Summary of probe development efforts to identify inhibitors of the SARS coronavirus 3C-like Protease (3CLPro)summarySummary (3CLPro inhibitors)
1879QFRET-based confirmation biochemical high throughput screening assay for inhibitors of the SARS coronavirus 3C-like Protease (3CLPro)screeningConfirmation 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)confirmatoryDose 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)confirmatoryDose 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 cellsscreeningLate stage counterscreen (cytotoxic compounds in Vero E6 cells in triplicate)
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
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
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
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
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
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
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_3X%INH

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 assay for inhibitors of 3CLPro.

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, 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 unquenched on the peptide, resulting in no increase in well fluorescence. Compounds were tested in triplicate at a final nominal concentration of 100 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 - AverageRFU_Low_Control ) / ( AverageRFU_High_Control - AverageRFU_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.

PubChem Activity Outcome and Score:

Compounds with greater than 50% inhibition were considered active.

The reported PubChem Activity Score has been normalized to 100% observed inhibition. Negative % inhibition values are reported as activity score zero.

The PubChem Activity Score range for active compounds is 100-52, and for inactive compounds 43-0.

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 (Fisher, part NC9871802)
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.
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Average Inhibition at 100 uM (100μM**)Average value of % inhibition at 100 uM compound concentration.Float%
2Inhibition at 100 uM [1] (100μM**)Value of % Inhibiton at 100 uM compound concentration; replicate one.Float%
3Inhibition at 100 uM [2] (100μM**)Value of % Inhibiton at 100 uM compound concentration; replicate two.Float%
4Inhibition at 100 uM [3] (100μM**)Value of % Inhibiton at 100 uM compound concentration; replicate three.Float%

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

Data Table (Concise)
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