Functional assays for osteoporosis therapeutics

描述(由申请人提供)：目前可用的一两种骨质疏松症药物不是简单地防止进一步的骨丢失而是建立新的骨，并不是没有问题，合成代谢疗法的市场仍然是开放的。Progenra是一家基于泛素的药物发现公司，目前正在寻找治疗骨质疏松症的这种疗法。泛素途径为发现选择性治疗药物提供了几个靶点，包括E3连接酶，它将泛素连接到靶蛋白上，标记它们在蛋白酶体中降解。泛素E3连接酶Praja1泛素化并导致转录辅助激活因子MAGE-D1/Dlsin-1(针对转录因子Dlx5和DLX3)的降解，干扰成骨基因程序(包括Runx2、OC和BSP)的转录。因此，Praja1抑制的最终结果是骨形成。Progenra正在筛选小分子和含有天然产物的文库，寻找这种连接酶的抑制剂，作为治疗骨质疏松症的合成代谢药物。利用现有的体外筛选试验，Progenra正在鉴定Praja1的E3连接酶活性的特定抑制剂。然而，这种潜在的治疗方法接下来必须在基于细胞的功能分析中进行评估，其中最好的必须在先导优化和临床前开发/临床候选选择的过程中在动物模型中进一步评估。在本申请中，提出了基于细胞的体外试验，其中将评估来自Praja1抑制剂筛选的先导化合物的功能活性。这些检测将评估候选化合物在转录和骨形成水平上对细胞的影响，并将通过使用Praja1 siRNA和通用蛋白酶体抑制剂进行验证。这项工作将分三个目标完成。首先，将建立一种方法来评估Dlx2、3和5介导的Runx2和Osterix的转录和激活。接下来，将建立成骨细胞分化和功能的模型。最后，开发的体外模型将使用一组蛋白酶体抑制剂进行测试，以模拟保存被活性Praja1标记为降解的蛋白质。在第二阶段，在这些基于细胞的功能分析中发现阳性的适当化合物将在骨形成的动物模型中进行测试。与公共卫生相关：用引起新骨生长的药物来治疗使人衰弱的骨质疏松症，而不仅仅是防止进一步的骨丢失，仍然是许多药物努力的目标。如今，只有一两种通常用于治疗骨质疏松症的药物通过这种机制发挥作用；大多数药物都能抑制进一步的骨质流失。Progenra创造了一种筛选技术，可以搜索大量的化合物集合，以寻找一种名为Praja-1的酶的抑制剂，这种酶负责阻止新骨形成。这些抑制物有可能允许骨质疏松症患者新骨形成。在筛查中发现的一种抑制剂可以被考虑用于临床开发之前，必须进行测试，看看它是否在细胞中发挥预期的作用，然后在整个动物中发挥作用。这一建议与筛查中发现的Praja1抑制剂的细胞评估有关。这里建议使用培养细胞构建检测方法，以测量一种化合物(筛选出的一种有效的Praja1抑制剂)的能力，以允许通常被活性Praja1阻止的基因的表达，并促进小鼠细胞中与新骨形成一致的分化。所有正在考虑进行临床前研究的化合物都将在这些基于细胞的功能分析中进行测试。

DESCRIPTION (provided by applicant): The one or two osteoporosis drugs now available that build new bone rather than simply prevent further bone loss are not without issues, and the market for anabolic therapy remains open. Progenra, a ubiquitin based drug discovery company, is currently seeking such a therapy for treatment of osteoporosis. The ubiquitin pathway provides several targets for the discovery of selective therapeutic agents, including E3 ligases, which attach ubiquitin to target proteins, marking them for degradation in the proteasome. The ubiquitin E3 ligase Praja1 ubiquitylates and causes degradation of the transcriptional coactivator MAGE-D1/Dlxin-1 (for the transcription factors Dlx5 and Dlx3), interfering with the transcription of an osteogenic gene program (including Runx2, OC, and BSP). The end result of Praja1 inhibition is thus bone formation. Progenra is screening small molecule and natural product-containing libraries for inhibitors of this ligase for development as anabolic agents to treat osteoporosis. Using existing in vitro screening assays, Progenra is in the process of identifying specific inhibitors of the E3 ligase activity of Praja1. However, such potential therapeutics must next be evaluated in cell-based functional assays, and the best of these further evaluated in animal models in the course of lead optimization and preclinical development/clinical candidate selection. In the present application, cell-based in vitro assays are proposed wherein the functional activity of lead compounds from Praja1 inhibitor screens will be evaluated. The assays will assess the effects of candidate compounds on cells at both the transcriptional and bone-formation levels and will be validated by the use of Praja1 siRNA and generic proteasome inhibitors. The work will be accomplished in three aims. First, an assay will be established to evaluate Dlx2-, 3-, and 5-mediated transcription and activation of Runx2 and osterix, using cultured cell lines. Next, a model for osteoblast differentiation and function will be established. Finally, the in vitro models developed will be tested using a battery of proteasome inhibitors to mimick preservation of the proteins marked for degradation by active Praja1. In Phase II, appropriate compounds that have been found positive in these cell-based functional assays will be tested in animal models of bone formation. PUBLIC HEALTH RELEVANCE: Treating the debilitating bone disease osteoporosis with drugs that cause new bone growth, rather than merely prevent further bone loss, remains a goal of many pharmaceutical efforts. Today only one or two drugs commonly prescribed for osteoporosis work by this mechanism; most inhibit further bone loss. Progenra has created a screening technology that will allow it to search large compound collections to find inhibitors of an enzyme called Praja-1, which is responsible for blocking new bone formation. Such inhibitors have the potential to permit new bone formation in patients with osteoporosis. Before an inhibitor found in the screen can be considered for clinical development, it must be tested to see whether it acts as predicted in cells and then in whole animals. This proposal has to do with the cellular evaluation of Praja1 inhibitors found in screening. It is proposed here to construct assays using cultured cells that will measure the ability of a compound (a potent Praja1 inhibitor from the screen) to allow the expression of genes normally blocked by active Praja1, and to promote differentiation in mouse cells that is consistent with new bone formation. All compounds under consideration for preclinical will be tested in these cell-based functional assays.