Small molecule antagonists of relaxin receptor

描述（由申请人提供）：肽激素松弛素（RLN）及其G蛋白偶联受体RXFP1在多种类型的癌细胞中表达，包括前列腺、子宫内膜、甲状腺等。研究表明，松弛素的过度表达常与晚期转移性疾病有关。刺激RLN/RXFP1信号可以增加细胞的增殖、侵袭、迁移、粘附，并减少细胞凋亡。RLN激活了一组信号通路和基因，这些信号通路和基因先前被证明与肿瘤发生有关。更重要的是，在前列腺癌细胞中通过siRNA敲低或肽拮抗剂表达抑制RLN或RXFP1对肿瘤生长和转移具有抑制作用。这表明RLN信号通路是一种很有前景的新型抗癌靶点。迄今为止，还没有发现松弛素受体的小分子或非肽拮抗剂。目前的应用程序旨在通过NIH NCGC的高通量筛选（HTS）来填补这一空白。RXFP1激活的一个容易检测和可靠的指标是cAMP产量的增加。使用稳定转染RXFP1的HEK293T细胞，我们优化了一种时间分辨荧光共振能量转移cAMP测定方法，用于1536孔格式的RXFP1拮抗剂的定量HTS。该试验将应用于RXFP1拮抗剂筛选活动。在初级筛选后，活性化合物将在一系列旨在确定特定松弛素受体拮抗剂的二级分析中选择。这些方法包括对用相关GPCR转染的细胞进行反筛，用非特异性cAMP激活剂刺激细胞，以及使用正交检测方法进行构象筛选。基于三级细胞的测定将用于选择影响前列腺癌细胞中rnn诱导反应的拮抗剂。最后，我们将分析拮抗剂的特异性、疗效、效力、作用方式及其对前列腺癌细胞的影响。在与NCGC的合作下，我们已经生成、开发和获得了所有必要的试剂，用于拟议的分析，并测试了所有的实验程序。松弛素受体拮抗剂的发现将为其作为抗癌药物的试验提供基础。

DESCRIPTION (provided by applicant): The peptide hormone relaxin (RLN) and its G protein-coupled receptor RXFP1 are expressed in several types of cancer cells, including prostate, endometrial, thyroid and others. It was shown that the overexpression of relaxin is often associated with advanced metastatic disease. Stimulation of RLN/RXFP1 signaling increases cell proliferation, invasion, migration, adhesion, and decreases cell apoptosis in vitro and in viv. RLN activates a set of signaling pathways and genes previously shown to be involved in tumorigenesis. More importantly, a suppression of RLN or RXFP1 by siRNA knockdown or through peptide antagonist expression in prostate cancer cells has an inhibitory effect on tumor growth and metastasis. This establishes the RLN signaling pathway as a promising novel anticancer target. To date no small molecule or non-peptide antagonists of the relaxin receptor are known. The current application is designed to fill this gap through high throughput screening (HTS) of a >400,000 small molecule compound library at NIH NCGC. An easily detectable and reliable indication of RXFP1 activation is an increase of cAMP production. Using HEK293T cells stably transfected with RXFP1 we have optimized a time-resolved fluorescence resonance energy transfer cAMP assay for quantitative HTS of RXFP1 antagonists in a 1536-well format. This assay will be applied for the RXFP1 antagonist screening campaign. After the primary screen the active compounds will be selected in a series of secondary assays designed to identify specific relaxin receptor antagonists. These include a counterscreen against cells transfected with related GPCR, cells stimulated with non-specific activator of cAMP, and a conformation screen using an orthogonal detection method. Tertiary cell-based assays will be used to select antagonists affecting RLN-induced responses in prostate cancer cells. Finally, we will analyze the antagonist specificity, efficacy, potency, mode of action, and their effects on prostate cancer cells. In collaboration with NCGC we have generated, developed, and obtained all reagents necessary for the proposed assays and tested all experimental procedures. The discovery of relaxin receptor antagonists will provide a basis for their testing as anticancer agents.