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RXFP 1在几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，松弛素的过度表达通常与晚期转移性疾病相关。RLN/RXFP 1信号的刺激在体外和体内增加细胞增殖、侵袭、迁移、粘附，并减少细胞凋亡。RLN激活了一系列信号通路和基因，这些通路和基因先前被证明与肿瘤发生有关。更重要的是，通过siRNA敲低或通过肽拮抗剂在前列腺癌细胞中表达来抑制RLN或RXFP 1对肿瘤生长和转移具有抑制作用。这建立了RLN信号通路作为一个有前途的新的抗癌靶点。迄今为止，还不知道松弛素受体的小分子或非肽拮抗剂。目前的申请旨在通过在NIH NCGC对> 400，000个小分子化合物文库进行高通量筛选（HTS）来填补这一空白。RXFP 1激活的一个容易检测和可靠的指示是cAMP产生的增加。使用用RXFP 1稳定转染的HEK 293 T细胞，我们优化了时间分辨荧光共振能量转移cAMP测定，用于在1536孔格式中定量HTS RXFP 1拮抗剂。该试验将用于RXFP 1拮抗剂筛选活动。在初步筛选后，将在一系列设计用于鉴定特异性松弛素受体拮抗剂的次级测定中选择活性化合物。这些包括对用相关GPCR转染的细胞的反筛选，用cAMP的非特异性激活剂刺激的细胞，以及使用正交检测方法的构象筛选。基于细胞的三级测定将用于选择影响前列腺癌细胞中RLN诱导的应答的拮抗剂。最后，我们将分析拮抗剂的特异性，功效，效力，作用模式及其对前列腺癌细胞的影响。在与NCGC的合作中，我们已经生成、开发并获得了拟议测定所需的所有试剂，并测试了所有实验程序。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.