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
BioActive Compounds: 19
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)
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. 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.
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.
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.
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
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)
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.
* Activity Concentration. ** Test Concentration.
Data Table (Concise)