Drug Discovery for Multiple Hereditary Exostoses

多种遗传性外生骨疣的药物发现

• 批准号: 9283197
• 负责人: Jeffrey D Esko
• 金额: $ 5.32万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-16 至 2018-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9283197
• 关键词: Affect; Biological Assay; Cartilage; Cartilaginous exostosis; Cataloging; Catalogs; Cell model; Cell surface; Cells; Cellular biology; Chemicals; Chinese Hamster Ovary Cell; Chondrocytes; Collaborations; Data Analyses; Defect; Development; Disaccharides; Disease; Dose; Drug Kinetics; Elements; Enzymes; Epiphysial cartilage; Exostoses; Family; Femur; Flow Cytometry; Formulation; Frequencies; Generations; Genes; Genomics; Growth; Health; Heparan Sulfate Biosynthesis; Heparitin Sulfate; Hereditary Multiple Exostoses; Heterozygote; Human; Image; Image Analysis; Informatics; Institutes; Lead; Libraries; Link; Liquid Chromatography; Mass Spectrum Analysis; Measures; Mesenchymal; Metabolism; Molecular Weight; Morphologic artifacts; Mus; Mutation; Natural History; Outcome; Patients; Pharmaceutical Chemistry; Pharmaceutical Preparations; Phenocopy; Physiology; Pilot Projects; Rare Diseases; Reducing Agents; Signal Pathway; Signal Transduction; Source; Structure; Testing; Therapeutic; Toxic effect; Toxicology; Validation; analog; base; cheminformatics; drug candidate; drug discovery; high throughput screening; human disease; in vivo; innovation; insight; long bone; monolayer; mouse model; neglect; pre-clinical; research study; response; restoration; rib bone structure; screening; skeletal; stem

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.

描述(申请人提供)：多发性遗传性外骨瘤(MHE)是一种常染色体显性遗传性疾病，其特征是在长骨和其他骨骼元素旁边形成异位软骨帽生长板样外骨瘤。MHE是由基因ext1或ext2的突变引起的，这会降低生长板和周围软骨膜中细胞制造硫酸乙酰肝素的能力。硫酸肝素含量变化导致异位骨软骨瘤的机制尚不清楚，但有证据表明，硫酸肝素含量的降低影响了多个信号通路，生长因子通过这些信号通路调节生长板中软骨细胞的组织和增殖。不管机制如何，主要的缺陷是在硫酸肝素的组装中，这表明恢复硫酸肝素的水平将减少外生骨病的频率。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，我们建议使用中国仓鼠卵巢(CHO)细胞，这些细胞在功能上是Ext1的半合子，并采用基于细胞的原代筛选来寻找潜在的候选药物来增强硫酸乙酰肝素的表达。已经与桑福德-伯纳姆研究所康拉德·普雷比化学基因组学中心的高含量筛选核心合作进行了试点研究。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。桑福德-伯纳姆的化学信息学和信息学核心将协助数据分析、人工产物过滤、复制命中确认和生成剂量响应简档。二次化验将检测对硫酸乙酰肝素含量和结构的影响。第三级测试将测量HITS是否调节了小鼠软骨细胞、软骨膜细胞和人类软骨细胞中硫酸肝素的表达。由此得到的确认命中集和化学类型的效力排序与额外的二级和三级检测结果相结合将有助于Hit-to-Lead鉴定。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-活性图谱。促进硫酸乙酰肝素合成的药物将通过配方、稳定性、药代动力学和毒性以及它们减少Ext1+/-；Ext2+/-小鼠外生骨痂的能力进行评估。中心假说是，改变参与硫酸肝素代谢的关键酶可以恢复功能正常的硫酸肝素水平，减少小鼠的外生骨痂，这将为MHE患者外骨瘤形成的药物操作提供原则证明。