Zych Courtney, Domling Alexander, Ayyavoo Velpandi
Department of Infectious Diseases and Microbiology, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, PA 15261, USA.
Drug Des Devel Ther. 2013 May 24;7:403-12. doi: 10.2147/DDDT.S44139. Print 2013.
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.
靶向蛋白质-蛋白质相互作用（PPI）是药物研发中一个新兴的领域。二聚化和PPI是人类免疫缺陷病毒（HIV）-1蛋白、其介导的功能以及病毒生物学的基本特性。此外，HIV-1蛋白与许多宿主细胞成分的功能相互作用需要二聚化。在本研究中，开发了一种基于双分子荧光互补（BiFC）的筛选测定法，该方法可以利用HIV-1病毒蛋白R（Vpr）的二聚化作为“概念验证”来量化二聚化的变化。结果表明，使用未标记的全长Vpr作为竞争分子，Venus Vpr（由BiFC Vpr构建体产生）可以以剂量依赖的方式被竞争掉。通过流式细胞术和荧光显微镜在高内涵筛选测定法中对信号强度的变化进行了定量测量。利用高内涵成像筛选小分子文库对Vpr二聚化的影响。在测试的分子中，有一些小分子以剂量依赖的方式对Vpr二聚化产生影响。
