QFRET-based dose response biochemical high throughput screening assay to identify inhibitors of the SARS coronavirus 3C-like Protease (3CLPro)
Name: QFRET-based dose response biochemical high throughput screening assay to identify inhibitors of the SARS coronavirus 3C-like Protease (3CLPro). ..more
BioActive Compounds: 44
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_1536_3XIC50
Name: QFRET-based dose response biochemical high throughput screening assay to identify inhibitors of the SARS coronavirus 3C-like Protease (3CLPro).
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, 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.
3CLpro, 3C-like protease, protease, cysteine protease, coronavirus, virus, SARS, SARS-CoV, peptide cleavage, inhibitor, inhibition, QFRET, quenching fluorescence resonance energy transfer, dose response, 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 biochemical assay is to confirm activity of compounds identified as active in a set of previous experiments entitled, "QFRET-based primary biochemical high throughput screening assay to identify inhibitors of the SARS coronavirus 3C-like Protease (3CLPro)", (PubChem AID 1706). 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 485nm and an emission wavelength of 535nm. 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 using a 10-point, 1:3 dilution series starting at a nominal concentration of 60 micromolar.
Prior to the start of the assay, 4 microliters of 3CLpro enzyme (150 nM final concentration) in assay buffer (50mM HEPES, 0.1 mg/ml BSA, 0.01% Triton-X 100, 2 mM DTT) at pH 7.5 was dispensed into each well of a 1536 microtiter plate. Next, 30 nL of test compound in DMSO, 3CLpro inhibitor (300 micromolar final concentration) 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. The assay was started by dispensing 1 microliter of 3Clpro peptide substrate (2 micromolar final concentration) in 50 mM HEPES at pH 7.5 to each well. After 30 minutes of incubation at room temperature, 1 microliter of 500 mM acetic acid was added to each well to terminate the assay and well fluorescence was read on a PerkinElmer Viewlux using fluorescein filters: excitation wavelength of 480 nm (with 20 nm bandwidth) and emission wavelength of 540 nm (with 20 nm bandwidth).
The % inhibition for each well was then calculated as follows:
% Inhibition = ( RFU_Test_Compound - MedianRFU_Low_Control ) / ( MedianRFU_High_Control - MedianRFU_Low_Control ) * 100
Test_Compound is defined as wells containing test compound.
High_Control is defined as wells containing 3CLpro inhibitor.
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 inactive compounds of this assay have an activity score range of 0 to 79 and the active compounds have an activity score range of 80 to 100.
List of Reagents:
Recombinant SARS-3CLpro (supplied by Assay Provider)
3CLpro peptide substrate: HiLyte Fluor 488-ESATLQSGLRKAK(QXL520)-NH2 (AnaSpec, part UIVT-3)
3CLpro Inhibitor: 5-chloro-3-pyridyl thiophene-2-carboxylate (Maybridge, part BTB07418)
1536-well plates (Greiner, part 789173)
HEPES (Invitrogen, part 15630)
Triton-X 100 (Fisher, part BP151-100)
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. In this assay 3CLPro Inhibitor had an IC50 of approximately 1.2 micromolar. 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 fluorescence. 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.
Categorized Comment - additional comments and annotations
* Activity Concentration. ** Test Concentration.
Data Table (Concise)