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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)
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页码:10 / 20
页数:11
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