Small molecule agonists of insulin-like3 receptor for treatment of osteoporosis

正常的骨发育是成骨细胞的骨形成和骨吸收的复杂相互作用。 破骨细胞。与年龄相关的、环境和内源性因素，以及遗传异常可能 改变这种动态平衡，导致骨质疏松症和其他与骨质流失相关的疾病。新疗法 靶点，尤其是成骨细胞合成代谢药物是治疗此类疾病所必需的。胰岛素样3(INSL3) 最近发现，多肽在骨代谢中起着重要作用。它通过G蛋白发出信号- 偶联受体RXFP2控制成骨细胞的分化和功能。RXFP2的表达已被 在人和小鼠成骨细胞和骨细胞中得到证实。携带RXFP2突变的患者会发生 骨量减少和骨质疏松症。在小鼠中，RXFP2的失活会导致骨量减少，矿化 表面、骨形成和破骨细胞表面与野生型窝种的比较。成骨细胞的治疗 INSL3诱导其完全分化，成骨细胞标志物表达增加，AS 以及矿化细胞外基质的能力。这建立了INSL3信号通路作为一个 很有希望成为新的药理靶点，特别是因为INSL3受体是一种细胞膜gpr。 然而，到目前为止，还没有已知的小分子RXFP2激动剂。当前的应用程序就是为满足这一需求而设计的 NIH NCGC通过高通量筛选(HTS)大小分子化合物文库获得GAP。 INSL3激活RXFP2导致cAMP产量明显增加。使用HEK293T细胞 稳定地转导RXFP2，我们优化了cAMP法测定RXFP2激动剂在 1536-井格式。该分析将用于RXFP2激动剂的筛选活动。在主屏幕之后， 活性化合物将在一系列二次检测中进行测试，以确定特定的INSL3 受体激动剂。二次检测包括针对亲本HEK293T细胞、细胞 并使用正交cAMP检测方法进行确认筛选。 结合探针-受体相互作用模型的结构-活性关系研究将被用于 提高化合物的效力、效力和选择性。包括成骨细胞在内的第三代细胞化验 增殖、分化和矿化效应，以及已知的靶基因表达和 蛋白质组学研究将被用来选择具有首选药理特征的最活跃的化合物。 RXFP2激动剂的功能特征将进一步验证它们在调节骨形成中的作用。这个 INSL3受体激动剂的发现将为其作为新的、安全的测试提供基础 治疗骨质疏松症和其他与骨量减少有关的疾病的合成代谢治疗药物。

Normal bone development is a complex interplay of bone formation by osteoblasts and bone resorption by osteoclasts. Age-associated, environmental, and endogenous factors, as well as genetic abnormalities can alter that homeostasis, leading to osteoporosis and other diseases associated with bone loss. New therapeutic targets and especially osteoblast anabolic drugs are needed to treat such disorders. Insulin-like3 (INSL3) peptide has recently been shown to have an important role in bone metabolism. It signals through its G protein- coupled receptor RXFP2 to control osteoblast differentiation and function. Expression of RXFP2 has been demonstrated in human and mouse osteoblasts and osteocytes. Patients with RXFP2 mutations develop osteopenia and osteoporosis. In mice, inactivation of RXFP2 causes a decrease in bone mass, mineralizing surface, bone formation, and osteoclast surface compared with wild-type littermates. Treatment of osteoblasts with INSL3 induced their complete differentiation coupled with increased expression of osteoblast markers, as well as the ability to mineralize the extracellular matrix. This establishes the INSL3 signaling pathway as a promising novel pharmacological target, especially because the INSL3 receptor is a cellular membrane GPCR. However, to date no small molecule RXFP2 agonists are known. The current application is designed to fill this gap through high throughput screening (HTS) of a large small molecule compound library at NIH NCGC. RXFP2 activation by INSL3 causes an easily detectable increase in cAMP production. Using HEK293T cells stably transfected with RXFP2, we have optimized a cAMP assay for quantitative HTS of RXFP2 agonists in a 1536-well format. The assay will be used for the RXFP2 agonist screening campaign. After the primary screen, the active compounds will be tested in a series of secondary assays designed to identify specific INSL3 receptor agonists. The secondary assays include a counterscreen against parental HEK293T cells, cells transfected with related GPCRs, and a confirmation screen using an orthogonal cAMP detection method. Structure-activity relationship studies coupled with probe-receptor interaction modeling will be performed to improve potency, efficacy and selectivity of the compounds. Tertiary cell-based assays that include osteoblast proliferation, differentiation, and mineralization effects, along with known target gene expression and proteomics studies will be used to select the most active compounds with preferred pharmacological profiles. Functional characterization of RXFP2 agonists will further verify their role in regulating bone formation. The discovery of INSL3 receptor agonists will provide a basis for their testing as novel, safe anabolic therapeutic drugs against osteoporosis and other diseases associated with low bone mass.