Screening for RhoC Activation and its modulation by GEFs

描述（由申请人提供）：最近的研究结果表明，Rho家族GTPases在调节癌性进展中起关键作用。特别重要的是，RhoC异构体的表达水平与癌症恶性的预后指标直接相关。这为控制恶性肿瘤转移的治疗干预提出了一个以前未知的目标。在这里，我们建议设计一种新的检测系统，能够检测Rho GTPase的鸟嘌呤核苷酸状态，并监测其效应物的结合（激活）。我们将使用新型的溶剂变色染料，这种染料可以产生大量的荧光变化，作为局部蛋白质微环境变化的功能，来检测Rho c的效应域衍生生物传感器的结合。然后，我们将开发一种高通量筛选（HTS）分析系统，以筛选能够特异性抑制RhoC活性的新型化合物，以及调节Rho活性的上游鸟嘌呤核苷酸交换因子。我们将以这样一种方式复用HTS，以提供高信噪比和低变异系数，以及可靠和可重复的筛选策略，以发现能够影响RhoC功能的新型小分子和化合物。这将有可能鉴定出特异性干预RhoC并预防恶性肿瘤转化的未知治疗化合物。

DESCRIPTION (provided by applicant): Recent findings indicate Rho family GTPases are critical in regulating the oncogenic progression. Of particular importance, the expression level of RhoC isoform has been directly correlated to a prognostic indicator of cancer malignancy. This poses a previously unknown target for therapeutic intervention in controlling the malignant cancer metastasis. Here, we propose to devise a novel assay system capable of detecting the guanine nucleotide states of Rho GTPase and monitor its effector binding (activation). We will use novel, solvatochromic dyes that can produce large changes in fluorescence as a function of changes in local protein microenvironment to detect binding of effector-domain derived biosensor for Rho C. We will then develop a high-throughput screening (HTS) assay system to screen for novel compounds that can specifically inhibit RhoC activity, and also the upstream guanine nucleotide exchange factors which modulate Rho activity. We will multiplex the HTS in such a way as to provide high signal to noise ratio and low coefficient of variation, and a reliable and repeatable screening strategy to discover novel small molecules and compounds capable of affecting RhoC function. This will potentially identify yet-unknown therapeutic compounds that intervene specifically with RhoC and prevent malignant cancer transformation.