Identification of a Small GTPase Inhibitor Using a High-Throughput Flow Cytometry Bead-Based Multiplex Assay

被引:52
|
作者
Surviladze, Zurab [1 ,2 ]
Waller, Anna [1 ]
Wu, Yang [1 ]
Romero, Elsa [3 ]
Edwards, Bruce S. [1 ,2 ,3 ]
Wandinger-Ness, Angela [2 ,3 ]
Sklar, Larry A. [1 ,2 ,3 ]
机构
[1] Univ New Mexico, Ctr Mol Discovery, Albuquerque, NM 87131 USA
[2] Univ New Mexico, Canc Res & Treatment Ctr, Sch Med, Albuquerque, NM 87131 USA
[3] Univ New Mexico, Dept Pathol, Sch Med, Albuquerque, NM 87131 USA
基金
美国国家科学基金会; 美国国家卫生研究院;
关键词
Ras; Rab; and Rho GTPases; actin cytoskeleton; bead-based multiplex assay; flow cytometry; fluorescent GTP binding; SMALL-MOLECULE INHIBITOR; REAL-TIME MEASUREMENT; ACTIN STRESS FIBERS; RHO-GTPASES; NUCLEOTIDE-BINDING; RAS ONCOGENES; MAST-CELLS; PROTEIN; RECEPTOR; CDC42;
D O I
10.1177/1087057109352240
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
Small GTPases are key regulators of cellular activity and represent novel targets for the treatment of human diseases using small-molecule inhibitors. The authors describe a multiplex, flow cytometry bead-based assay for the identification and characterization of inhibitors or activators of small GTPases. Six different glutathione-S-transferase (GST)-tagged small GTPases were bound to glutathione beads, each labeled with a different red fluorescence intensity. Subsequently, beads bearing different GTPase were mixed and dispensed into 384-well plates with test compounds, and fluorescent-guanosine triphosphate (GTP) binding was used as the readout. This novel multiplex assay allowed the authors to screen a library of almost 200,000 compounds and identify more than 1200 positive compounds, which were further verified by dose-response analyses, using 6- to 8-plex assays. After the elimination of false-positive and false-negative compounds, several small-molecule families with opposing effects on GTP binding activity were identified. The authors detail the characterization of MLS000532223, a general inhibitor that prevents GTP binding to several GTPases in a dose-dependent manner and is active in biochemical and cell-based secondary assays. Live-cell imaging and confocal microscopy studies revealed the inhibitor-induced actin reorganization and cell morphology changes, characteristic of Rho GTPases inhibition. Thus, high-throughput screening via flow cytometry provides a strategy for identifying novel compounds that are active against small GTPases. (Journal of Biomolecular Screening 2010: 10-20)
引用
收藏
页码:10 / 20
页数:11
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