Functional assays for osteoporosis therapeutics

描述（由申请人提供）：现在可用的一种或两种骨质疏松症药物，建立新的骨，而不是简单地防止进一步的骨质流失不是没有问题，合成代谢治疗的市场仍然开放。Progenra是一家基于泛素的药物发现公司，目前正在寻找这种治疗骨质疏松症的疗法。泛素途径为发现选择性治疗剂提供了几个靶点，包括E3连接酶，其将泛素连接到靶蛋白，标记它们在蛋白酶体中降解。泛素E3连接酶Praja 1泛素化并导致转录辅激活因子MAGE-D1/Dlxin-1（转录因子Dlx 5和Dlx 3）降解，干扰成骨基因程序（包括Runx 2、OC和BSP）的转录。因此，Praja 1抑制的最终结果是骨形成。Progenra正在筛选含有这种连接酶抑制剂的小分子和天然产物库，以开发为治疗骨质疏松症的合成代谢药物。使用现有的体外筛选试验，Progenra正在鉴定Praja 1的E3连接酶活性的特异性抑制剂。然而，这些潜在的治疗方法必须在基于细胞的功能测定中进行评估，并且在先导物优化和临床前开发/临床候选物选择过程中，在动物模型中进一步评估这些方法中最好的。在本申请中，提出了基于细胞的体外测定，其中将评价来自Praja 1抑制剂筛选的先导化合物的功能活性。该试验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Praja 1 siRNA和通用蛋白酶体抑制剂进行验证。这项工作将在三个目标中完成。首先，使用培养的细胞系建立测定以评价Dlx 2、3和5介导的Runx 2和osterix的转录和活化。接下来，将建立成骨细胞分化和功能的模型。最后，将使用一组蛋白酶体抑制剂来模拟标记为活性Praja 1降解的蛋白质的保存，对开发的体外模型进行测试。在II期，将在骨形成动物模型中测试在这些基于细胞的功能测定中发现阳性的适当化合物。 公共卫生相关性：用能引起新骨生长的药物治疗使人衰弱的骨质疏松症，而不仅仅是防止进一步的骨质流失，仍然是许多制药工作的目标。今天，只有一种或两种常用的治疗骨质疏松症的药物是通过这种机制起作用的;大多数药物都抑制了进一步的骨质流失。Progenra开发了一种筛选技术，使其能够搜索大量化合物，以找到一种名为Praja-1的酶的抑制剂，这种酶负责阻止新骨的形成。这种抑制剂有可能使骨质疏松症患者的新骨形成。在筛选中发现的抑制剂可以考虑用于临床开发之前，必须对其进行测试，以确定它是否在细胞中以及在整个动物中发挥预测的作用。这项提议与筛选中发现的Praja 1抑制剂的细胞评价有关。本文提出使用培养的细胞构建测定法，该测定法将测量化合物（来自筛选的有效Praja 1抑制剂）允许通常被活性Praja 1阻断的基因表达的能力，以及促进与新骨形成一致的小鼠细胞分化的能力。将在这些基于细胞的功能试验中检测临床前考虑的所有化合物。

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.