Fluorescence-based biochemical high throughput screening primary assay to identify inhibitors of Crimean-Congo Hemorrhagic Fever (CCHF) viral ovarian tumor domain protease (vOTU): Pep-AMC substrate
Name: Fluorescence-based biochemical high throughput screening primary assay to identify inhibitors of Crimean-Congo Hemorrhagic Fever (CCHF) viral ovarian tumor domain protease (vOTU): Pep-AMC substrate. ..more
BioActive Compounds: 2045
Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center (SRIMSC)
Center Affiliation: The Scripps Research Institute (TSRI)
Assay Provider: Scott Pegan, University of Illinois at Chicago (UIC)
Network: Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Proposal Number: 1 R03 MH097507-01A1
Grant Proposal PI: Scott Pegan
External Assay ID: CCHFVOTU-PEP-AMC_INH_FLINT_1536_1X%INH PRUN
Name: Fluorescence-based biochemical high throughput screening primary assay to identify inhibitors of Crimean-Congo Hemorrhagic Fever (CCHF) viral ovarian tumor domain protease (vOTU): Pep-AMC substrate.
The Crimean-Congo Hemorrhagic Fever (CCHF) virus is a tick-borne negative strand RNA nairovirus of the family Bunyaviridae. CCHF virus (CCHFV) infection in humans is characterized by high fever, prostration, and severe hemorrhages (1, 2). There is no FDA approved vaccine or therapeutic treatment for CCHFV, which is considered one of the most dangerous emerging human pathogens as well as representing a significant bioterrorist threat agent (1). Investigators have identified an ovarian tumor domain protease (OTU) located in the L-protein of CCHFV and revealed its potential role in suppressing the innate immune response (3). This cysteine protease represents a new viral subclass of OTU domain proteases (vOTUs), which have also been located in economically devastating viruses such as porcine reproductive respiratory syndrome virus (PRRSV) and rice stripe virus (RSV) (4, 5). Predominantly, OTUs have been linked to ubiquitin (Ub) removal and/or remodeling of Ub-conjugated proteins, also known as deubiquitination, placing them among five protease superfamilies that facilitate signal transduction cascades and play key roles in protein stability (6). However, unlike all other members of the OTU superfamily, CCHF-vOTU has illustrated not only a preference for Ub-conjugated proteins but also for those proteins conjugated to Ub-like interferon-simulated gene product 15 (ISG15) (7, 8). As conjugation of ISG15 and Ub play crucial roles in the regulation of the human interferon type 1 (IFN1) response plus directly inactivating viral proteins through modification, the presence of a vOTU in CCHFV is currently viewed as a candidate virulence factor for its potential ability to assist CCHFV in evading the innate immune system (3, 9-11). vOTU molecular probes would allow an assessment of whether the molecular probe scaffold can serve as a basis to selectively inhibit other viral and eukaryotic ovarian tumor domain proteases, as well as potentially leading to the development of prophylactics targeting vOTUs and their eukaryotic superfamily relatives that negatively regulate the human innate response.
1. Weber, F. and A. Mirazimi, Interferon and cytokine responses to Crimean Congo hemorrhagic fever virus; an emerging and neglected viral zonoosis. Cytokine Growth Factor Rev, 2008. 19(5-6): p. 395-404.
2. Crimean-Congo haemorrhagic fever. WHO Media Centre, 2001.
3. Frias-Staheli, N., et al., Ovarian tumor domain-containing viral proteases evade ubiquitin- and ISG15-dependent innate immune responses. Cell Host Microbe, 2007. 2(6): p. 404-16.
4. Feng, Y., et al., Porcine respiratory and reproductive syndrome virus variants, Vietnam and China, 2007. Emerg Infect Dis, 2008. 14(11): p. 1774-6.
5. Zhang, H.M., et al., Genomic analysis of rice stripe virus Zhejiang isolate shows the presence of an OTU-like domain in the RNA1 protein and a novel sequence motif conserved within the intergenic regions of ambisense segments of tenuiviruses. Arch Virol, 2007. 152(10): p. 1917-23.
6. Komander, D., M.J. Clague, and S. Urbe, Breaking the chains: structure and function of the deubiquitinases. Nat Rev Mol Cell Biol, 2009. 10(8): p. 550-63.
7. Ha, B.H. and E.E. Kim, Structures of proteases for ubiqutin and ubiquitin-like modifiers. BMB Rep, 2008. 41(6): p. 435-43.
8. Zhao, C., et al., Human ISG15 conjugation targets both IFN-induced and constitutively expressed proteins functioning in diverse cellular pathways. Proc Natl Acad Sci U S A, 2005. 102(29): p. 10200-5.
9. Akutsu, M., et al., Molecular basis for ubiquitin and ISG15 cross-reactivity in viral ovarian tumor domains. Proc Natl Acad Sci U S A, 2011. 108(6): p. 2228-33.
10. Capodagli, G.C., et al., Structural analysis of a viral ovarian tumor domain protease from the Crimean-Congo hemorrhagic fever virus in complex with covalently bonded ubiquitin. J Virol, 2011. 85(7): p. 3621-30.
11. James, T.W., et al., Structural basis for the removal of ubiquitin and interferon-stimulated gene 15 by a viral ovarian tumor domain-containing protease. Proc Natl Acad Sci U S A, 2011. 108(6): p. 2222-7.
PRUN, primary screen, Crimean-Congo Hemorrhagic Fever, CCHF, CCHFV, viral ovarian tumor domain protease, vOTU, primary, singlicate, confirmation, triplicate, dose response, biochemical, inhibit, inhibitor, inhibition, inh, cysteine protease, cysteine, protease, peptide substrate, substrate, pep-AMC, AMC, fluorescence, HTS, high throughput screen, 1536, Scripps, Scripps Florida, MLSMR, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN.
The purpose of this assay is to identify compounds that act as inhibitors of the cysteine protease activity of CCHF-vOTU. In this biochemical assay, 40 uL of buffered CCHF-vOTU (1 uM final concentration) and test compound are added first. Then a 10 uL solution containing the peptide substrate Z-ARG-LEU-ARG-GLY-GLY-7-amino-4-methylcoumarin (pep-AMC, final concentration 50 uM) is added. Pep-AMC shares the last five amino acids with the C-terminus of both ISG15 and Ubiquitin (Ub). CCHF-vOTU like Plpro-CoV and other deubiqutinatases cleave this substrate between the last glycine and AMC, thus allowing AMC to be freed and fluoresce at 380 nm excitation and 460 nm emission. The assay is allowed to shake at 70 rpm for 30 seconds, and allowed to reach 5 minutes of total reaction time before reading fluorescence at 380 and 460 nm. As designed, test compounds that inhibit CCHF-vOTU protease activity will decrease the cleavage of the pep-AMC substrate, leading to decreasing well fluorescence. Compounds are tested in singlicate at a final nominal concentration of 11.5 uM.
Prior to the start of the assay, 4 ul of CCHF-vOTU enzyme (0.5 uM final concentration) in assay Buffer A (5 mM HEPES, 100 mM NaCl, 2 mM DTT) at pH 7.5 was dispensed into wells 4-48 of a 1536 microtiter plate and 4 ul of Buffer A alone into wells 1-3. Next, 30 nL of test compound in DMSO or DMSO alone (0.8% final concentration) was added to the appropriate wells. The plates were then incubated for 15 minutes at room temperature. The assay was started by dispensing 1 ul of Pep-AMC (Z-RLRGG-AMC) substrate (50 uM final concentration) in Buffer B (50 mM HEPES, 100 mM NaCl, 0.1%BSA, 5mM DTT) at pH 7.5 to each well. Plates were centrifuged and fluorescence was read (T0) on aPerkinElmer EnVision using umbelliferone filters: excitation wavelength of 355 nm (with 40 nm bandwidth) and emission wavelength of 460 nm (with 25 nm bandwidth). Plates were incubated for 30 min at room temperature and fluorescence was measured (T30).
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 only Pep AMC and DMSO.
Low_Control is defined as wells containing DMSO, CCHF-vOTU and PepAMC.
PubChem Activity Outcome and Score:
A mathematical algorithm was used to determine nominally inhibiting compounds in the Primary screen. Two values were calculated: (1) the average percent inhibition of 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. Note: only compounds that were within the assay window were taken into account for the calculations.
The reported PubChem Activity Score has been normalized to 100% observed primary inhibition. Negative % inhibition values are reported as activity score zero.
The PubChem Activity Score range for active compounds is 100-21, and for inactive compounds 21-0.
List of Reagents:
CCHF-vOTU (supplied by Assay Provider)
Pep AMC: ZRLRGG-AMC (Bachem, I-1690.001)
1536-well plates (Corning, 7261 )
HEPES (Acros, 17257-0250)
NaCl(Fisher, part 5640-500)
DTT (Acros part 16568-0250))
BSA (Sigma, part A7906)
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)