Drug Discovery for Multiple Hereditary Exostoses

多发性遗传性外生骨疣 (MHE) 是一种常染色体显性遗传疾病，其特征是形成 长骨和其他骨骼元件旁边的异位软骨帽生长板状外生骨疣。微量元素HE 基因 Ext1 或 Ext2 突变的结果，会降低生长板中细胞的能力 周围的软骨膜产生硫酸乙酰肝素。硫酸乙酰肝素变化的机制 含量导致异位骨软骨瘤的原因尚不清楚，但有证据表明乙酰肝素的减少 硫酸盐影响多种信号通路，生长因子通过这些信号通路调节组织和 生长板中软骨细胞的增殖。无论机制如何，主要缺陷在于 硫酸乙酰肝素的组装，表明恢复硫酸乙酰肝素的水平会减少 外生骨疣的频率。所有细胞都通过共同的机制产生硫酸乙酰肝素。因此，我们建议 使用具有 Ext1 功能的半合子的中国仓鼠卵巢 (CHO) 细胞，并采用原代细胞 基于筛选来寻找增强硫酸乙酰肝素表达的潜在候选药物。试点研究有 与康拉德普雷比斯化学中心的高内涵筛选核心合作完成 桑福德伯纳姆研究所的基因组学。分析优化、验证和最终实施 所提出的基于图像的高通量筛选测定将得以完成。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、工件过滤、重复命中确认和 剂量反应曲线的生成。二次检测将测试阳性结果对乙酰肝素的影响 硫酸盐含量和结构。第三次测定将测量命中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类软骨细胞。得到的排序结果为 已确认的命中集和化学型与额外的二级和三级测定结果合并的效力将 帮助识别先导化合物。化学信息学核心将寻找商业上可用的类似物 支持有限的结构活性分析。增强硫酸乙酰肝素合成的药物将通过以下方式进行评估 制剂、稳定性、药代动力学和毒性及其减少 Ext1 /-;Ext2 /- 中外生骨疣的能力 老鼠。中心假设是改变硫酸乙酰肝素代谢中涉及的关键酶可以导致 恢复小鼠硫酸乙酰肝素的功能正常水平并减少外生骨疣，这将 作为 MHE 患者外生骨疣形成的药理学操作的原理验证。

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.