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BioAssay: AID 651828

A screen for compounds that inhibit nucleocapsid/RNA interactions in Rift Valley Fever Virus

Rift Valley fever virus (RVFV) is an emerging infectious pathogen that causes severe disease in humans and livestock and has the potential for global spread. Currently, there is no proven effective treatment for RVFV infection, and there is no licensed vaccine. Inhibition of RNA binding to the essential viral nucleocapsid (N) protein represents a potential antiviral therapeutic strategy because more ..
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 Tested Compounds
 Tested Compounds
All(19421)
 
 
Active(25)
 
 
Inactive(19396)
 
 
 Tested Substances
 Tested Substances
All(19899)
 
 
Active(25)
 
 
Inactive(19874)
 
 
 Related BioAssays
 Related BioAssays
AID: 651828
Data Source: ICCB-Longwood/NSRB Screening Facility, Harvard Medical School (HMS1061)
Deposit Date: 2012-11-30

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 25
Description:
Rift Valley fever virus (RVFV) is an emerging infectious pathogen that causes severe disease in humans and livestock and has the potential for global spread. Currently, there is no proven effective treatment for RVFV infection, and there is no licensed vaccine. Inhibition of RNA binding to the essential viral nucleocapsid (N) protein represents a potential antiviral therapeutic strategy because all of the functions performed by N during infection involve RNA binding. To target this interaction, we developed a fluorescence polarization-based high-throughput drug-screening assay and tested 26,424 chemical compounds for their ability to disrupt an N-RNA complex. From libraries of Food and Drug Administration-approved drugs, druglike molecules, and natural product extracts, we identified several lead compounds that are promising candidates for medicinal chemistry.
Protocol
Aliquots of N protein were pooled together from frozen tubes on the first day of screening and kept at 4 degrees C as an 11-fold concentrated stock (130 uM). On the day of screening, columns 1-23 of assay plates (Corning 3820) were filled with 10 uL of N protein at a concentration of 14.2 uM in binding buffer 1x (10 mM Hepes pH8, 150 mM NaCl, 20 mM KCl, 5 mM MgCl2). Column 24 and any empty column (some plates have no compound in columns 1 and 2) were filled with 10 ul of binding buffer 1x.

100 nL of each compound were pin-transferred to each assay plate. For every compound library plate, there were either one or two assay plates. All wells then received 2 ul of fluorescein-labeled RNA oligonucleotide (final concentration = 10 nM). The final assay well volume was 12 uL. Plates were centrifuged for one minute at 1000 rpm, then stacked 10 high with the top plate covered with an empty assay plate, and incubated at 24 degrees C for 30 minutes in a closed bench drawer.

Viral N protein was produced by overexpression in Escherichia coli and purified by affinity chromatography. The sequence of the N protein corresponds to the M12 RVFV isolate with an extra hexameric His-Asn tag at its N-terminus. The RNA oligonucleotide was chemically synthetized with a fluorescein FAM label at its 3' end (TriLink Biotechnologies).

Final perturbator concentration: 42 ug/mL (for 5 mg/ml stocks); 125 ug/mL (for natural product extracts); various for known bioactives.

Negative control: N protein and fluorescent RNA oligo in all wells in column 23, with no experimental compound and no DMSO
Positive control: Fluorescent RNA oligo only in all wells of column 24

The assay plates were read using a PerkinElmer EnVision, with excitation at 480 nm and emission (P and S channels) at 535 nm. The first 25 library plates were screened in duplicate, with both assay plates (A and B) in a given set prepared together. Since the Z' value and assay reproducibility were high, the last 52 plates were screened in single copy.
Comment
The percent RNA oligo bound to N protein (% Bound) was calculated for each well as follows: well FP was subtracted from negative control plate average FP, divided by the difference between plate average negative and positive control FP, multiplied by 100 and subtracted from 100. Wells were considered positive if the % Bound for at least one replicate < 58 and > 0. Wells were excluded from further consideration if the sum of the P and S channel intensities for at least one replicate was greater than 20,000,000 or less than 10,000,000, indicating either that the compound was fluorescent or that insufficient fluorescent oligo RNA was added to the well. Activity scores were calculated by subtracting % Bound from 100. Average % Bound was used for wells with two replicates. Activity score result values < 0 or > 100 were set to 0.
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Fluorescence Polarization (mP)_AFloat
2P-Channel_AP-channel fluorescence intensity at 535 nm, replicate AInteger
3S-Channel_AS-channel fluorescence intensity at 535 nm, replicate AInteger
4Fluorescence Polarization (mP)_BFloat
5P-Channel_BP-channel fluorescence intensity at 535 nm, replicate BInteger
6S-Channel_BS-channel fluorescence intensity at 535 nm, replicate BInteger
7Sum channel[P+S]_ASum of P and S-channel fluorescence intensity, replicate AInteger
8Sum channel[P+S]_BSum of P and S-channel fluorescence intensity, replicate BInteger
9% Bound_APercent RNA oligo bound to N protein, replicate AFloat%
10% Bound_BPercent RNA oligo bound to N protein, replicate BFloat%

Data Table (Concise)
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