TY  - UNPB
ID  - pittir6753
UR  - http://d-scholarship-dev.library.pitt.edu/6753/
A1  - Zych, Courtney
TI  - Development of a high throughput cell-based assay to screen for inhibitors of HIV-1 Vpr oligomerization
Y1  - 2011/06/29/
N2  - Highly active anti-retroviral treatment (HAART) targets less than a third of the proteinsproduced during HIV-1 infection. Testing the effectiveness of an anti-retroviral drug requiresassays specific for the individual target that take into account its mechanism of action. MostHIV-1 proteins need to undergo dimerization in order to become functional in the viral life cycle. Historically, it has been difficult to visualize and quantify changes in a protein-protein interaction, which has left this characteristic of proteins unexplored as potential antiviral targets. In this study, a bimolecular fluorescence complementation based screening assay is developed that can quantify a change in dimerization, using the HIV-1 accessory protein Vpr as a "proof of concept".Results demonstrated that bimolecular fluorescence complementation of Vpr could be competed off in a dose-dependent manner using untagged, full length Vpr as a competitor molecule. The change in signal intensity was measured quantitatively through flow cytometry and fluorescence microscopy in a high content screening assay. High content imaging was used to screen a library of peptides and a library of small molecules for an effect on Vpr dimerization. None of the Vpr peptides were shown to have an effect; however, one of the small molecules was shown to interfere with Vpr dimerization in a dose-dependent manner.Statement of Public Health relevance: Dimerization is a unique property of many HIV-1 viral proteins and is necessary to complete the viral life cycle, thus it has been identified as a potential drug target. By developing an assay that screens for inhibition of HIV-1 protein dimerization, high throughput screening can be performed to detect inhibitors of a new target in HIV-1 replication. Small molecules identified using this screening method could be developed into a novel anti-retroviral drug.
AV  - public
KW  -  drug screening; high content screening; HIV; Vpr; assay development; dimerization
ER  -