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）：现在可用的一种或两种骨质疏松药物可以建立新的骨头而不是简单地防止进一步的骨质流失没有问题，而合成代谢疗法的市场仍然开放。 Gepenra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。泛素途径为发现选择性治疗剂（包括E3连接酶）提供了几个靶标，它们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泛素化合物并导致转录共激活因子MAGE-D1/DLXIN-1（对于转录因子DLX5和DLX3），干扰了成骨基因程序的转录（包括runx2，oc，oc，oc和bsp）。 praja1抑制的最终结果是骨形成。后代正在筛选小分子和含天然产物的文库，以作为该连接酶的抑制剂作为处理骨质疏松症的合成代谢剂。使用现有的体外筛选测定法，后代正在识别Praja1 E3连接酶活性的特定抑制剂。但是，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剂，并且在铅优化和临床前开发/临床候选选择的过程中，这些方法中的最好的方法在动物模型中进一步评估。在本应用中，提出了基于细胞的体外测定法，其中将评估来自PRAJA1抑制剂筛查的铅化合物的功能活性。该测定法将评估候选化合物对转录和骨形成水平的细胞的影响，并通过使用PRAJA1 siRNA和通用蛋白酶体抑制剂来验证。这项工作将以三个目标完成。首先，使用培养的细胞系将建立一个测定法以评估Runx2和Osterix的DLX2-，3-和5介导的转录和激活。接下来，将建立成骨细胞分化和功能的模型。最后，将使用一系列蛋白酶体抑制剂对开发的体外模型进行测试，以模仿活性praja1降解的蛋白质。在II期中，在这些基于细胞的功能测定中发现阳性的合适化合物将在骨形成动物模型中进行测试。公共卫生相关性：用导致新骨骼生长的药物（而不是仅仅防止进一步的骨质流失）治疗使人衰弱的骨病骨质疏松症，这仍然是许多药物努力的目标。如今，只有一种或两种药物通常用于骨质疏松症的工作。大多数抑制进一步的骨质流失。 Gepenra创建了一种筛选技术，它将允许其搜索大型化合物收集，以查找称为Praja-1的酶的抑制剂，该酶负责阻断新的骨形成。这种抑制剂有可能在骨质疏松症患者中允许新的骨形成。在筛选中发现的抑制剂进行临床发育之前，必须对其进行测试，以查看其是否如细胞中的预测，然后在整个动物中的预测。该建议与筛查中PRAJA1抑制剂的细胞评估有关。这里建议使用培养的细胞来构建测定法，以测量化合物的能力（从屏幕上使用有效的PRAJA1抑制剂），以允许通常被活性praja1阻断的基因表达，并促进与新骨形成一致的小鼠细胞中的分化。所有正在考虑的临床前考虑的化合物将在这些基于细胞的功能测定中进行测试。