Drug Discovery for Multiple Hereditary Exostoses

描述（由申请人提供）：多发性遗传性外生骨疣（MHE）是一种常染色体显性遗传疾病，其特征是在长骨和其他骨骼成分附近形成异位软骨覆盖的生长板样外生骨疣。MHE是由Ext 1或Ext 2基因突变引起的，这会降低生长板和周围软骨膜中细胞产生硫酸乙酰肝素的能力。硫酸乙酰肝素含量的变化导致异位骨软骨瘤的机制尚不清楚，但有证据表明，硫酸乙酰肝素的减少影响多种信号通路，生长因子通过这些通路调节生长板中软骨细胞的组织和增殖。无论机制如何，主要缺陷在于硫酸乙酰肝素的组装，这表明恢复硫酸乙酰肝素水平将减少外生骨疣的发生率。所有细胞通过共同的机制产生硫酸乙酰肝素。因此，我们建议使用中国仓鼠卵巢（CHO）细胞，功能上是半合子的Ext 1和采用原代细胞为基础的筛选，以找到潜在的候选药物，增加硫酸乙酰肝素的表达。试点研究已经完成了与高内容筛选核心在康拉德Prebys中心化学基因组学在桑福德伯纳姆研究所。将完成拟议的基于图像的高通量筛选试验的试验优化、验证和最终实施。Sanford-Burnham的化学信息学和信息学核心将协助数据分析、伪影过滤、重复命中确认和剂量反应曲线的生成。二次试验将检测阳性命中对硫酸乙酰肝素含量和结构的影响。三级测定将测量命中物是否调节小鼠软骨细胞和软骨膜细胞以及人软骨细胞中的硫酸乙酰肝素表达。确认的命中集和化学型的效力的所得等级排序与额外的二级和三级测定结果合并，将有助于命中-导联鉴别。化学信息学核心将搜索商业上可用的类似物，以支持有限的结构-活性分析。将通过制剂、稳定性、药代动力学和毒性及其减少Ext 1 +/-; Ext 2 +/-小鼠中外生骨疣的能力来评价增强硫酸乙酰肝素合成的药剂。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硫酸乙酰肝素功能正常水平的恢复和小鼠外生骨疣的减少，这将作为MHE患者外生骨疣形成的药理学操作的原理证明。

DESCRIPTION (provided by applicant): 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.