Primary biochemical high-throughput screening assay for inhibitors of the HIV Rev RRE RNA interaction (disruption of protein-RNA interaction)
Primary biochemical high-throughput screening assay for inhibitors of the HIV Rev - RRE RNA interaction (disruption of protein-RNA interaction) ..more
BioActive Compounds: 769
Source (MLSCN Center Name): The Scripps Research Institute Molecular Screening Center
Center Affiliation: The Scripps Research Institute, TSRI
Assay Provider: The Scripps Research Institute, TSRI
Network: Molecular Library Screening Center Network (MLSCN)
Proposal Number: 1 X01 MH078935-01
External Assay ID: HIVREVRRE_INH_FRET_1536_%INH
Primary biochemical high-throughput screening assay for inhibitors of the HIV Rev - RRE RNA interaction (disruption of protein-RNA interaction)
Rev is a small basic protein that is critical for HIV replication (1).
Early in infection, before synthesis of significant amounts of Rev, mRNA transcripts are processed by a default pathway that fully splices both introns. Export of these mRNAs to the cytoplasm for translation produces a set of small regulatory proteins, including Tat and Rev. Rev binds to the Rev-Responsive Element (RRE) on the viral mRNA (2), which results in efficient export of singly spliced and unspliced mRNAs to the cytoplasm by the machinery used for cellular protein and RNA export. Translation of these larger mRNAs gives rise to the structural proteins gag, pol, and env required for assembly of new viral particles. Thus, binding of Rev to the RRE serves as a switch from the early to the late pattern of gene expression (3).
Inhibitors of Rev-RRE interaction have the potential to provide a novel therapeutic avenue for combating HIV infection at a target that has not yet been exploited (4). In addition, such inhibitors would provide great insights into how RNA-protein complex formation might be inhibited in the general sense.
 Cook, K.S., Fisk, G.J., Hauber, J., Usman, N., Daly, T.J. and Rusche, J.R. (1991) Characterization of HIV-1 REV protein: binding stoichiometry and minimal RNA substrate. Nucleic Acids Research, 19, 1577-1582.
 Kjems, J., Brown, M., Chang, D.D. and Sharp, P.A. (1991) Structural analysis of the interaction between the human immunodeficiency virus Rev protein and the Rev response element. Proc Natl. Acad. Sci. USA, 88, 683-687.
 Battiste, J.L., Mao, H., Rao, N.S., Tan, R., Muhandiram, D.R., Kay, L.E., Frankel, A.D. and Williamson, J.R. (1996) a-helix-RNA major groove recognition in an HIV-1 Rev peptide-RRE RNA complex. Science, 273, 1547-1551.
 Zapp, M.L., Stern, S. and Green, M.R. (1993) Small Molecules That Selectively Block RNA-Binding of Hiv-1 Rev Protein Inhibit Rev Function and Viral Production. Cell, 74, 969-978.
HIV, Rev-RRE, Scripps, HTS, assay, Rev, Rev Responsive Element, ribosome, mRNA, inhibition, viral production, fluorescence resonance energy transfer, primary screen
This assay is based on the ability of the Rev peptide to form a complex with the Rev-Responsive Element (RRE) of the double-stranded viral mRNA. Both binding partners have been labeled with appropriate fluorophores, allowing monitoring of their interaction by fluorescence resonance energy transfer (FRET). A Cy5- labeled RNA target harboring the Rev Response Element (named Cy5-RRE) is first incubated with test compounds. A Fluorescein-labeled Rev peptide (named Fl-Rev) is then added to the mixture. After a 15 minute incubation time, fluorescence is measured. A high FRET is indicative of the proper binding of Rev to the RRE (no inhibition), whereas a low FRET might indicate an inhibition of the Rev-RRE interaction caused by the tested compound.
The primary HTS campaign was conducted in 1536 well plate format. 96,891 compounds were tested once at a final nominal concentration of 8 micromolar.
Prior to the assay, a 1:2 mixture of 5 micromolar Cy5-labeled single-stranded RNA (RRE1) and 10 micromolar unlabeled complementary RNA strand (RRE2) were allowed to anneal by heating up the mixture for 5 minutes at 90 degrees Celsius and cooling it down to room temperature.
2.5 microliters of solution containing 180 nanomolar RNA (Dharmacon, Inc., Lafayette, CO) in assay buffer 30 mM HEPES pH 7.5 (Invitrogen, Carlsbad, CA), 100 mM KCl, 2 mM MgCl2, 20 mM NaCl (Sigma-Aldrich, St.Louis, MO), 0.5 mM EDTA (Invitrogen, Carlsbad, CA) were dispensed in a 1536 microtiter plate. 40 nanoliters of test compound or positive and negative control (10 millimolar Neomycin B (Sigma-Aldrich, St.Louis, MO) and DMSO, respectively) were then added to the appropriate wells. Plates were incubated for 5 minutes at 25 degrees Celsius. The assay was started by dispensing 2.5 microliters of 20 nanomolar Fl-Rev (BioPeptide, San Diego, CA) solution in assay buffer supplemented with 10 mM NaH2PO4, 10 mM NH4OAc, 10 mM guanidinium HCl, and 0.01% Igepal CA-630. After 15 minutes of incubation at 25 degrees Celsius, plates were centrifuged and fluorescence was read on the Viewlux microplate reader (Perkin-Elmer, Turku, Finland) with excitation = 480nm, donor emission = 540nm (Fluorescein), acceptor emission = 671nm (Cy5).
Values measured from both wavelengths were used to calculate a ratio for each well, according to the following mathematical expression:
Ratio = RFU 671nm / RFU 540nm
where RFU 671nm represents the measured fluorescence "acceptor" emission at 671 nm and RFU 540nm represents the measured fluorescence "donor" emission at 540nm.
The percent inhibition for each compound was calculated as follows:
Per cent inhibition = (test_compound_Ratio -median_negative_control_Ratio)/(median_positive_control_Ratio - median_negative_control_Ratio)*100
with positive_control: 80 micromolar of Neomycin B
and negative control: DMSO
A mathematical algorithm was used to determine nominally inhibitory 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 per cent inhibition than the cutoff parameter was declared active.
The reported Pubchem_Activity_Score has been normalized to 100% of the highest observed primary inhibition. In other words, we divided the inhibition value for each compound with the highest observed inhibition and report it as a percent.
As per Primary Investigator's request, all compounds were also analyzed by two additional hit criteria:
1. The Relative FRET (RF) criterion. This was calculated according to the following mathematical expression:
RF = (DF - [DF]) x ([AF] - AF)/ [DF] x [DF]
where RF is the Relative FRET, DF is the donor fluorescence for the well, AF is the acceptor fluorescence for the well, [DF] is the median donor fluorescence for the plate, and [AF] is the median acceptor fluorescence for the plate. The RF quantity is positive only for anti-correlated changes (i.e. simultaneous increase in Acceptor fluorescence and decrease in Donor fluorescence). Furthermore, the RF quantity is normalized for the fluorescence values of each individual plate, which facilitates plate to plate comparison. We used a cutoff of RF > 0.01 to score primary hits by this approach.
2. Acceptor channel criterion. A drop in Acceptor fluorescence of more than 30 per cent as compared to Median RFU value of Acceptor channel of Sample Field was scored as a primary hit by this approach.
To determine possible fluorescent artifacts the combined list of compounds that were independently scored as hits by three approaches was compared to the list of the actives from "qHTS Assay for Spectroscopic Profiling in Fluorescein Spectral Region" (Pubchem AID 593). Compounds suspected to be affected by intrinsic or extraneous fluorescence (lint/dust) were annotated as "Possible fluorescent artifact". Selected hits from Primary screen will be validated in follow-up assays.
All data reported were normalized on a per-plate basis.
Possible artifacts of this assay can include, but are not limited to: fluorescent dust/lint, compounds that quench or enhance fluorescence.
Categorized Comment - additional comments and annotations
Data Table (Concise)