Surviladze Zurab, Waller Anna, Wu Yang, Romero Elsa, Edwards Bruce S, Wandinger-Ness Angela, Sklar Larry A
University of New Mexico Center for Molecular Discovery, Albuquerque, NM 87131, USA.
J Biomol Screen. 2010 Jan;15(1):10-20. doi: 10.1177/1087057109352240. Epub 2009 Dec 11.
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.
小GTP酶是细胞活性的关键调节因子，是使用小分子抑制剂治疗人类疾病的新靶点。作者描述了一种基于流式细胞术磁珠的多重检测方法，用于鉴定和表征小GTP酶的抑制剂或激活剂。六种不同的谷胱甘肽-S-转移酶（GST）标记的小GTP酶与谷胱甘肽磁珠结合，每种磁珠标记有不同的红色荧光强度。随后，将携带不同GTP酶的磁珠混合，并与测试化合物一起分配到384孔板中，以荧光鸟苷三磷酸（GTP）结合作为读数。这种新颖的多重检测方法使作者能够筛选近200,000种化合物的文库，并鉴定出1200多种阳性化合物，这些化合物通过剂量反应分析进一步验证，使用6至8重检测。在消除假阳性和假阴性化合物后，鉴定出了几个对GTP结合活性有相反作用的小分子家族。作者详细描述了MLS000532223的特性，它是一种通用抑制剂，以剂量依赖的方式阻止GTP与几种GTP酶结合，并且在生化和基于细胞的二次检测中具有活性。活细胞成像和共聚焦显微镜研究揭示了抑制剂诱导的肌动蛋白重组和细胞形态变化，这是Rho GTP酶抑制的特征。因此，通过流式细胞术进行高通量筛选提供了一种鉴定对小GTP酶有活性的新型化合物的策略。
