Drug Discovery for Multiple Hereditary Exostoses

多发性遗传性骨软骨病(MHE)是一种常染色体显性遗传性疾病，其特征是 异位软骨帽的生长板状外骨突，靠近长骨和其他骨骼元素。MHE 由基因ext1或ext2突变所致，这会降低生长板中细胞的能力 使周围的软骨膜产生硫酸乙酰肝素。硫酸乙酰肝素的变化机制 内容物导致异位骨软骨瘤尚不清楚，但有证据表明肝素的减少 硫酸盐影响多个信号通路，生长因子通过这些信号通路调节组织和 生长板内软骨细胞增殖。不管是什么机制，主要的缺陷是在 硫酸肝素的组装，这表明恢复硫酸肝素的水平将减少 外生骨软骨病的频率。所有的细胞都通过一个共同的机制产生硫酸乙酰肝素。因此，我们建议 使用Ext1功能半合子的中国仓鼠卵巢(CHO)细胞，并采用原代细胞- 基于筛选，以寻找增强硫酸乙酰肝素表达的潜在候选药物。试点研究已经 已与康拉德·普雷比化学中心的高含量筛选核心合作完成 桑福德-伯纳姆研究所的基因组学研究。检测优化、验证和最终实施 所提出的基于图像的高通量筛选分析将实现。化学信息学和 桑福德-伯纳姆的信息学核心将协助数据分析、人工产物过滤、复制命中确认以及 生成剂量响应配置文件。二次化验将对肝素的影响进行阳性检测 硫酸盐含量和结构。三级检测将测量HITS是否调节硫酸乙酰肝素的表达 小鼠软骨细胞和软骨膜细胞，以及人软骨细胞。的合成秩序 确认的命中集和化学类型的效力与额外的二次和三次化验结果合并将 帮助确认命中到线索。化学信息学核心将搜索商业上可用的类似物 支持有限的结构-活动分析。促进硫酸乙酰肝素合成的药物将通过 Ext1/-；Ext2/-的制剂、稳定性、药代动力学、毒性及其减少外生骨痂的能力 老鼠。中心假设是改变参与硫酸乙酰肝素代谢的关键酶可以导致 恢复小鼠功能正常的硫酸乙酰肝素水平并减少外生骨痂，这将 作为MHE患者外生性骨疣形成的药理操作的原则证明。

Multiple Hereditary Exostoses (MHE) is an autosomal dominant disorder characterized by the formation of ectopic cartilage-capped growth plate-like exostoses next to long bones and other skeletal elements. MHE results from mutations in the genes Ext1 or Ext2, which diminish the capacity of cells in the growth plate and the surrounding perichondrium to make heparan sulfate. The mechanism by which a change in heparan sulfate content causes ectopic osteochondromas is unknown, but evidence suggests that the decrease in heparan sulfate affects multiple signaling pathways through which growth factors regulate the organization and proliferation of chondrocytes in the growth plate. Regardless of the mechanism, the primary defect is in the assembly of heparan sulfate, suggesting that restoring the level of heparan sulfate would diminish the frequency of exostoses. All cells make heparan sulfate through a common mechanism. Thus, we propose to use Chinese hamster ovary (CHO) cells that are functionally hemizygous for Ext1 and to employ a primary cell- based screen to find potential drug candidates that augment heparan sulfate expression. Pilot studies have been done in collaboration with the High Content Screening Core in the Conrad Prebys Center for Chemical Genomics at the Sanford-Burnham Institute. Assay optimization, validation and final implementation of the proposed image-based high-throughput screening assay will be accomplished. The Cheminformatics and Informatics Core at Sanford-Burnham will assist in data analysis, artifact filtering, replicate hit confirmation, and generation of dose response profiles. Secondary assays will test positive hits for their impact on heparan sulfate content and structure. Tertiary assays will measure if the hits modulate heparan sulfate expression in mouse chondrocytes and perichondrial cells and in human chondrocytes. The resultant rank ordering of potency of confirmed hit sets and chemotypes merged with additional secondary and tertiary assay results will aid in hit-to-lead identification. The Cheminformatics Core will search for commercially available analogs to support limited structure-activity profiling. Agents that enhance heparan sulfate synthesis will be evaluated by formulation, stability, pharmacokinetics, and toxicity and their capacity to reduce exostoses in Ext1+/-;Ext2+/- mice. The central hypothesis is that altering key enzymes involved in heparan sulfate metabolism can result in restoration of functionally normal levels of heparan sulfate and reduction of exostoses in mice, which would serve as a proof-of-principle for pharmacological manipulation of exostosis formation in MHE patients.