Screening for RhoC Activation and its modulation by GEFs

描述（由申请方提供）：最近的研究结果表明Rho家族GTP酶在调节致癌进展中至关重要。特别重要的是，RhoC同种型的表达水平与癌症恶性程度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供了一个以前未知的靶点。在这里，我们建议设计一种新的检测系统，能够检测鸟嘌呤核苷酸状态的Rho GTdR和监测其效应器结合（激活）。我们将使用新型溶剂化显色染料，其可以产生作为局部蛋白质微环境变化的函数的荧光的大的变化，以检测效应域衍生的生物传感器对Rho C的结合。然后，我们将开发一个高通量筛选（HTS）分析系统，以筛选新的化合物，可以特异性地抑制RhoC活性，也是上游鸟嘌呤核苷酸交换因子调节Rho活性。我们将以提供高信噪比和低变异系数的方式多重化HTS，以及可靠且可重复的筛选策略，以发现能够影响RhoC功能的新型小分子和化合物。这将有可能识别出特异性干预RhoC并预防恶性癌症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.