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 在长骨和其他骨骼元素旁边的异位软骨覆盖的生长板样外生骨疣。MHE 由Ext 1或Ext 2基因突变引起，这会降低生长板中细胞的能力， 制造硫酸乙酰肝素硫酸乙酰肝素变化的机制 内容导致异位骨软骨瘤是未知的，但有证据表明，乙酰肝素的减少， 硫酸盐影响多种信号传导途径，通过这些途径生长因子调节组织， 生长板中软骨细胞的增殖。不管机制如何，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将减少 外生骨疣的频率。所有细胞通过共同的机制产生硫酸乙酰肝素。因此，我们建议 使用对Ext 1功能性半合子的中国仓鼠卵巢（CHO）细胞，并使用原代细胞- 基于筛选，以寻找增加硫酸乙酰肝素表达的潜在候选药物。试点研究有 与康拉德·普里比斯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。分析优化、验证和最终实施 将完成所提出的基于图像的高通量筛选测定。化学信息学和 Sanford-Burnham的信息学核心将协助数据分析、伪影过滤、复制命中确认， 生成剂量反应曲线。二次试验将检测阳性命中对乙酰肝素的影响 硫酸盐含量和结构。第三次测定将测量命中物是否调节细胞中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。的结果秩排序 确认的命中集和化学型的效力与额外的二级和三级检测结果合并， 帮助识别命中到引线。化学信息学核心将搜索商业上可获得的类似物， 支持有限的结构活性分析。将通过以下方法评价增强硫酸乙酰肝素合成的药物： 制剂、稳定性、药代动力学和毒性及其减少Ext 1 +/-; Ext 2 +/-中外生骨疣的能力 小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 恢复硫酸乙酰肝素的功能正常水平，减少小鼠的外生骨疣， 作为MHE患者外生骨疣形成的药理学操作的原理证明。

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.