Drug Discovery for Multiple Hereditary Exostoses

描述（由申请人提供）：多发性遗传性外生骨病（Multiple Hereditary Exostoses， MHE）是一种常染色体显性遗传病，其特征是在长骨和其他骨骼部件旁边形成异位软骨帽生长板样外生骨病。MHE是由Ext1或Ext2基因突变引起的，这些突变会降低生长板和周围软骨膜中细胞制造硫酸肝素的能力。硫酸肝素含量的变化导致异位性骨软骨瘤的机制尚不清楚，但有证据表明，硫酸肝素含量的降低影响生长板中生长因子调节软骨细胞组织和增殖的多种信号通路。无论机制如何，主要缺陷是在硫酸肝素的组装中，这表明恢复硫酸肝素水平将减少外植骨的频率。所有细胞通过一种共同的机制制造硫酸肝素。因此，我们建议使用具有Ext1半合子功能的中国仓鼠卵巢（CHO）细胞，并采用基于原代细胞的筛选来寻找增加硫酸肝素表达的潜在候选药物。试点研究是与斯坦福-伯纳姆研究所康拉德·普雷比斯化学基因组学中心的高含量筛选核心合作进行的。将完成所提出的基于图像的高通量筛选分析的分析优化，验证和最终实施。斯坦福-伯纳姆大学的化学信息学和信息学核心将协助进行数据分析、伪影过滤、重复命中确认和剂量反应谱的生成。二级分析将检测其对硫酸肝素含量和结构的影响。三级分析将测量hit是否调节硫酸肝素在小鼠软骨细胞和软骨外细胞以及人软骨细胞中的表达。将确认的命中集和化学型的效力排序与额外的二级和三级分析结果合并，将有助于命中-先导鉴定。化学信息学核心将寻找商业上可用的类似物来支持有限的结构-活性分析。促进硫酸肝素合成的药物将通过配方、稳定性、药代动力学、毒性及其减少Ext1+/-外生菌的能力进行评估；Ext2 + / -老鼠。核心假设是，改变参与硫酸肝素代谢的关键酶可以导致小鼠硫酸肝素功能正常水平的恢复和外生骨量的减少，这将作为MHE患者外生骨量形成的药理操作的原理证明。

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.