QFRET-based primary biochemical high throughput screening assay to identify inhibitors of the SARS coronavirus 3C-like Protease (3CLPro)
Name: QFRET-based primary biochemical high throughput screening assay to identify inhibitors of the SARS coronavirus 3C-like Protease (3CLPro) ..more
BioActive Compounds: 405
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_%INH
Name: QFRET-based primary 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, primary screen, peptide cleavage, HTS, high throughput screen, 1536, inhibitor, QFRET, quenching fluorescence resonance energy transfer, 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 identify compounds that 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 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 singlicate at a final nominal concentration of 6 uM.
Prior to the start of the assay, 4 ul 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 uM 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 ul of 3Clpro peptide substrate (2 uM final concentration) in 50 mM HEPES at pH 7.5 to each well. After 30 minutes of incubation at room temperature, 1 ul 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.
A mathematical algorithm was used to determine nominally inhibiting compounds in the Primary screen. Two values were calculated: (1) the average percent inhibition of all compounds tested, and (2) three times their standard deviation. The sum of these two values was used as a cutoff parameter, i.e. any compound that exhibited greater % inhibition than the cutoff parameter was declared active.
The reported PubChem Activity Score has been normalized to 100% observed primary inhibition. Negative % inhibition values are reported as activity score zero.
The inactive compounds of this assay have activity score range of 0 to 15 and active compounds range of activity score is 15 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 case the results of each separate campaign were assigned "Active/Inactive" status based upon that campaign's specific compound activity cutoff value. 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.
Categorized Comment - additional comments and annotations
** Test Concentration.
Data Table (Concise)