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成骨细胞的骨吸收的复杂相互作用。 破骨细胞糖尿病相关的、环境的和内源性的因素，以及遗传异常， 改变体内平衡，导致骨质疏松症和其他与骨质流失有关的疾病。新的治疗 需要靶点，特别是成骨细胞合成代谢药物来治疗这些疾病。胰岛素样3（INSL 3） 最近已显示肽在骨代谢中具有重要作用。它通过G蛋白发出信号- 偶联受体RXFP 2控制成骨细胞分化和功能。RXFP 2的表达已经被证实是 在人类和小鼠成骨细胞和骨细胞中证实。RXFP 2突变患者发生 骨质减少和骨质疏松症。在小鼠中，RXFP 2的失活导致骨量减少，矿化 表面，骨形成，破骨细胞表面相比，野生型同窝。成骨细胞的治疗 与INSL 3诱导他们的完全分化与成骨细胞标志物的表达增加， 以及矿化细胞外基质的能力。这建立了INSL 3信号通路作为一个信号通路。 有前途的新的药理学靶标，特别是因为INSL 3受体是细胞膜GPCR。 然而，迄今为止，还没有小分子RXFP 2激动剂是已知的。目前的应用程序旨在填补这一 通过NIH NCGC的大的小分子化合物文库的高通量筛选（HTS）， 通过INSL 3激活RXFP 2导致cAMP产生的容易检测的增加。使用HEK 293 T细胞 用RXFP 2稳定转染后，我们优化了cAMP测定法，用于RXFP 2激动剂的定量HTS， 1536-好格式。该试验将用于RXFP 2激动剂筛选活动。在主屏幕之后， 活性化合物将在一系列旨在鉴定特异性INSL 3的次级试验中进行测试 受体激动剂二级测定包括针对亲本HEK 293 T细胞、细胞免疫原性的反筛选。 用相关GPCR转染，并使用正交cAMP检测方法进行确认筛选。 将进行与探针-受体相互作用建模相结合的结构-活性关系研究， 提高化合物的效力、功效和选择性。包括成骨细胞的三级细胞检测 增殖、分化和矿化作用，沿着已知的靶基因表达， 蛋白质组学研究将用于选择具有优选药理学特征的最有效的化合物。 RXFP 2激动剂的功能表征将进一步验证它们在调节骨形成中的作用。的 INSL 3受体激动剂的发现将为它们作为新的、安全的 抗骨质疏松症和其他与低骨量相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.