title: Development of a robust cell-based high-throughput screening assay to identify targets of HIV-1 viral protein R dimerization.
abstract: Targeting protein-protein interactions (PPI) is an emerging field in drug discovery. Dimerization and PPI are essential properties of human immunodeficiency virus (HIV)-1 proteins, their mediated functions, and virus biology. Additionally, dimerization is required for the functional interaction of HIV-1 proteins with many host cellular components. In this study, a bimolecular fluorescence complementation (BiFC)-based screening assay was developed that can quantify changes in dimerization, using HIV-1 viral protein R (Vpr) dimerization as a "proof of concept." Results demonstrated that Venus Vpr (generated by BiFC Vpr constructs) 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 small molecules for an effect on Vpr dimerization. Among the tested molecules, a few of the small molecules demonstrate an effect on Vpr dimerization in a dose-dependent manner.
authors.affiliation: Department of Infectious Diseases and Microbiology, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, PA 15261, USA.
authors.forename: Courtney
authors.initials: C
authors.lastname: Zych
authors.forename: Alexander
authors.initials: A
authors.lastname: Domling
authors.forename: Velpandi
authors.initials: V
authors.lastname: Ayyavoo
mesh_terms: D016336:Gene Products, vpr / Q000737:chemistry*; D006367:HeLa Cells; D057166:High-Throughput Screening Assays; D006801:Humans; D007700:Kinetics; D019151:Peptide Library; D055503:Protein Multimerization
chemical_list: D016336:Gene Products, vpr; D019151:Peptide Library
keywords: BiFC; HIV-1 Vpr; dimerization; drug targets; protein-protein interaction