qHTS for inhibitors of Vif-A3G interactions: Cherry picks counterscreen
The recent discovery and characterization of host restriction factors, which provide a potent defense to invading retroviruses, has provided new targets for antiviral drug development. APOBEC3G (A3G) and APOBEC3F (A3F) are potent cytidine deaminases that block HIV-1 replication; HIV-1 expresses the Vif protein, which counteracts A3G and A3F by binding to them and inducing their proteasomal degradation. ..more
BioActive Compounds: 1107
The recent discovery and characterization of host restriction factors, which provide a potent defense to invading retroviruses, has provided new targets for antiviral drug development. APOBEC3G (A3G) and APOBEC3F (A3F) are potent cytidine deaminases that block HIV-1 replication; HIV-1 expresses the Vif protein, which counteracts A3G and A3F by binding to them and inducing their proteasomal degradation.
The Vif-A3G and Vif-A3F interactions provide two potential targets for development of small molecules which can interfere with the ability of Vif to degrade A3G or A3F. Structural determinants of these interactions have been mapped to 1 to 5 amino acids in A3G, A3F, and Vif, suggesting that small molecules could potentially inhibit these interactions. To identify inhibitors that can interfere with Vif mediated degradation of A3G and A3F, we have developed Hela cell lines that stably express A3G or A3F tagged with eYFP at the C-terminal end (A3G-eYFP or A3F-eYFP, respectively) and Vif. In the absence of an inhibitor, Vif induces degradation of A3G-eYFP and A3F-eYFP, and the cells are not fluorescent. In the presence of an inhibitor, it is expected that Vif will be unable to degrade A3G-eYFP and/or A3F-eYFP, resulting in an increase in yellow fluorescence. In this counterscreen assay, Vif A3G was screened against the cherry picks ordered from the MLSMR.
NIH Chemical Genomics Center [NCGC]
NIH Molecular Libraries Probe Centers Network [MLPCN]
MLPCN Grant: MH093245
Assay Submitter (PI): Vinay Pathak, National Cancer Institute
The qHTS was performed with Hela_A3G_Vif cells (A3G-Vif), that showed enhanced yellow fluorescent protein (eYFP) after compound treatment during automated liquid handling in 1,536-well format. A3G-Vif cells were harvested from T225 flask and resuspended in phenol free OPTI-MEM medium with 5% FBS. Then 5 uL of 140,000 cells/mL resuspended cells was dispensed into each well of low base Aurora COC 1,536-well plates (black walled, clear bottom) using a Multidrop Combi dispenser. After overnight culture at 37 deg C with 5% CO2, a total of 23 nL of compounds at 5 selected concentrations from the NPC or positive control (2 mM stock of MG132) in DMSO was transferred to each well of the assay plate using a pintool (Kalypsys, San Diego, CA), and the plates were further incubated at 37 deg C with 5% CO2 for 24 hours. After that, the plates were measured on Acumen Explorer (TTP Lab Tech). The fluorescence was detected by 488 nm/Ex and 500-530nM/Em.
1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. See data field "Curve Description". For this assay, apparent inhibitors are ranked higher than compounds that showed apparent activation.
2. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given for each curve class type given above. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39. Fit_LogAC50 was used for determining relative score and was scaled to each curve class' score range.
Categorized Comment - additional comments and annotations
* Activity Concentration. ** Test Concentration.
Data Table (Concise)