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Functional assays for osteoporosis therapeutics

骨质疏松症治疗的功能测定

基本信息

批准号：
7538083
负责人：
David E Sterner
金额：
$ 29.37万
依托单位：
PROGENRA, INC.
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-09-01 至 2010-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7538083
关键词：
Anabolic Agents Animal Model Animals Award BMP2 gene Biological Assay Biological Factors Biological Preservation Bone Diseases Bone Growth Cell Line Cells Cellular Assay Chemistry Clinical Collection Cultured Cells Development End Point Enzyme Inhibitor Drugs Enzyme Inhibitors Enzymes Evaluation Event Gene Expression Generic Drugs Genes Genetic Transcription Goals Human In Vitro Lead Libraries Ligase MG132 Marketing Measures Mediating Modeling Multiple Myeloma Mus Nobel Prize Osteoblasts Osteogenesis Osteoporosis Pathway interactions Patients Pharmaceutical Preparations Pharmacologic Substance Phase Phenotype Process Proteasome Inhibitor Proteins Public Health Refractory Relapse Reporter Genes Rosa Screening procedure Small Interfering RNA Source Technology Testing Therapeutic Therapeutic Agents Today Transcription Coactivator Transcriptional Activation Ubiquitin Ubiquitin-Proteasomal Pathway United States Food and Drug Administration Velcade Work antitumor drug assay development base bone bone loss bone sialoprotein drug discovery high throughput screening in vitro Assay in vitro Model inhibitor/antagonist multicatalytic endopeptidase complex pre-clinical prevent programs small molecule small molecule libraries transcription factor ubiquitin-protein ligase

项目摘要

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.

描述（由申请人提供）：目前可用的一种或两种骨质疏松症药物可以构建新骨而不是简单地防止进一步的骨质流失，但并非没有问题，并且合成代谢疗法的市场仍然开放。 Progenra 是一家基于泛素的药物发现公司，目前正在寻求这种治疗骨质疏松症的疗法。泛素途径为选择性治疗药物的发现提供了几个靶点，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 泛素化并导致转录共激活因子 MAGE-D1/Dlxin-1（转录因子 Dlx5 和 Dlx3）降解，干扰成骨基因程序（包括 Runx2、OC 和 BSP）的转录。 Praja1 抑制的最终结果是骨形成。 Progenra 正在筛选包含小分子和天然产物的文库，寻找这种连接酶的抑制剂，以开发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 E3 连接酶活性的特异性抑制剂。然而，接下来必须在基于细胞的功能测定中评估这种潜在的治疗方法，并在先导化合物优化和临床前开发/临床候选药物选择过程中在动物模型中进一步评估其中最好的方法。在本申请中，提出了基于细胞的体外测定，其中将评估来自Praja1抑制剂筛选的先导化合物的功能活性。这些测定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。这项工作将实现三个目标。首先，将使用培养的细胞系建立一种测定法来评估 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系列蛋白酶体抑制剂进行测试，以模拟被活性 Praja1 标记为降解的蛋白质的保存。在第二阶段，在这些基于细胞的功能测定中发现阳性的适当化合物将在骨形成动物模型中进行测试。公共健康相关性：用引起新骨生长的药物治疗骨质疏松症，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。如今，只有一两种常用的治疗骨质疏松症的药物通过这种机制发挥作用。大多数抑制进一步的骨质流失。 Progenra 开发了一种筛选技术，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形成。此类抑制剂有可能允许骨质疏松症患者形成新骨。在考虑将筛选中发现的抑制剂用于临床开发之前，必须对其进行测试，以确定其在细胞中以及在整个动物中的作用是否符合预期。该提案与筛选中发现的 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，测量化合物（筛选中的一种有效的 Praja1 抑制剂）允许通常被活性 Praja1 阻断的基因表达的能力，并促进小鼠细胞中与新骨形成一致的分化。所有考虑用于临床前的化合物都将在这些基于细胞的功能测定中进行测试。