Blangy Anne, Bouquier Nathalie, Gauthier-Rouvière Cécile, Schmidt Susanne, Debant Anne, Leonetti Jean-Paul, Fort Philippe
Centre de Recherches en Biochimie Macromoléculaire, CNRS (Centre National de la Recherche Scientifique) FRE2593, 1919 route de Mende, 34293 Montpellier Cedex 5, France.
Biol Cell. 2006 Sep;98(9):511-22. doi: 10.1042/BC20060023.
Rho GTPases are involved in many biological processes and participate in cancer development. Their activation is catalysed by exchange factors [RhoGEFs (Rho GTPase guanine nucleotide-exchange factor)] of the Dbl family. RhoGEFs display proto-oncogenic features, thus appearing as candidate targets for anticancer drugs. Dominant-negative Rho GTPase mutants have been widely used to block RhoGEF signalling. However, these tools suffer from limitations, due to the high number of RhoGEFs and the complex mechanisms that control Rho GTPase activation.
RhoG-T17N is a poor inhibitor of its exchange factor TRIO-GEFD1 (first exchange domain of the exchange factor TRIO) in vivo: although it binds to TRIO-GEFD1, RhoG-T17N does not block the downstream signalling. Using the yeast exchange assay, we show that in the presence of TRIO-GEFD1, RhoG-T17N can bind to its effectors, which illustrates how negative mutants may produce misleading interpretations and emphasizes the need for new types of RhoGEF inhibitors. In that prospect, we adapted the yeast exchange assay method to identify RhoGEF inhibitors. Using this novel approach, we screened a 3500-chemical-compound library and identified a potential inhibitor of TRIO-GEFD1. This molecule inhibited TRIO-GEFD1 in vitro. Among the chemical analogues of this compound, we identified two molecules with better inhibitory activity. The three TRIO-GEFD1 inhibitors had no effect on ARHGEF17 and ARNO [ARF (ADP-ribosylation factor) nucleotide-binding-site opener], two exchange factors for RhoA and Arf1 respectively.
The development of RhoGEF inhibitors appears as a valuable tool for the study of Rho GTPase signalling pathways. The yeast exchange assay adaptation we present here is suitable to screen for chemical or peptide libraries and identify candidate inhibitors.
Rho GTP酶参与许多生物学过程并参与癌症发展。它们的激活由Dbl家族的交换因子[RhoGEFs（Rho GTP酶鸟嘌呤核苷酸交换因子）]催化。RhoGEFs具有原癌基因特征，因此成为抗癌药物的候选靶点。显性负性Rho GTP酶突变体已被广泛用于阻断RhoGEF信号传导。然而，由于RhoGEFs数量众多以及控制Rho GTP酶激活的机制复杂，这些工具存在局限性。
RhoG-T17N在体内对其交换因子TRIO-GEFD1（交换因子TRIO的第一个交换结构域）的抑制作用较差：尽管它与TRIO-GEFD1结合，但RhoG-T17N并不能阻断下游信号传导。使用酵母交换试验，我们表明在存在TRIO-GEFD1的情况下，RhoG-T17N可以与其效应器结合，这说明了负性突变体可能如何产生误导性解释，并强调了新型RhoGEF抑制剂的必要性。在这一前景下，我们改进了酵母交换试验方法以鉴定RhoGEF抑制剂。使用这种新方法，我们筛选了一个包含3500种化合物的文库，并鉴定出一种潜在的TRIO-GEFD1抑制剂。该分子在体外抑制TRIO-GEFD1。在该化合物的化学类似物中，我们鉴定出两种具有更好抑制活性的分子。这三种TRIO-GEFD1抑制剂对ARHGEF17和ARNO[ARF（ADP-核糖基化因子）核苷酸结合位点开放剂]没有影响，它们分别是RhoA和Arf1的两种交换因子。
RhoGEF抑制剂的开发似乎是研究Rho GTP酶信号通路的一个有价值的工具。我们在此展示的酵母交换试验改进方法适用于筛选化学或肽文库并鉴定候选抑制剂。
