TROY HTS Compound Screening

 描述（由申请人提供）：目前胶质母细胞瘤（GB）患者的治疗选择有限，且大多无效。GB的高度侵袭性使其对标准治疗产生高度抵抗，几乎可以肯定会复发。新的治疗策略的发展加上治疗的选择性增加是必不可少的GB患者的管理和提高生存。该项目汇集了一个在生物靶标验证，高通量检测开发和分子化学库方面具有专业知识的现有团队，以靶向TROY信号通路作为治疗GB入侵的创新方法。我们已经开发了基于细胞的高通量筛选（HTS），以确定TROY依赖性信号转导的小分子抑制剂的测定。我们的基于细胞的测定将询问对TROY信号传导具有功能后果的原位和变构调节剂。我们将利用Sanford-Burnham Prebys中心的可用资源，在NIH结构多样的化学文库的1536孔格式中进行基于细胞的HTS，以鉴定抑制TROY信号传导途径的药物样化合物。选择性特征将使用基于细胞的反筛选试验进行。将进一步评价命中在TROY依赖性胶质瘤细胞系中的生物学效应，同时不影响TROY阴性细胞。作用机制（MOA）研究将评估经验证的命中对TROY寡聚化、TROY-TRAF 2相互作用和TROY依赖性细胞内信号传导途径破坏的影响。最后，沿着对最佳可用探针进行支持性药理学表征，对经验证的命中系列类似物进行“购买”构效关系（SAR）。研究将强调鉴定具有高亲和力的小分子， 合适的物理化学性质，以促进穿过血脑屏障（BBB）并递送到脑实质中。为了利用研究人员组成的团队之间强大的生物学/化学协同作用，以确定靶向TROY的研究工具化合物用于未来的命中-先导优化活动，提出了以下具体目标：目标1：实施基于细胞的报告基因测定，并对TROY信号传导途径的调节剂进行NIH MLSMR > 365，000收集的HTS;目标2：目的3：通过二级测定的命中验证级联，通过“目录类似物”进行有限的结构-活性关系（SAR）阐明，以选择最有效和最具选择性的化学探针;目标4：对经验证的支架进行功能生物学测定和三级测定，以阐明在临床相关原代GB细胞中抑制细胞迁移、侵袭和耐药性的潜在作用机制和功效;目的5：进行有限的 在最有前途的易处理探针上进行ADME/T和体外BBB分析，并扩大规模（25 - 50 mg），用于有限的啮齿动物PK研究以及未来的概念验证和研究。

 DESCRIPTION (provided by applicant): Current treatment options for glioblastoma (GB) patients are limited and largely ineffective. The highly invasive nature of GB instills a high resistance to standard therapies and recurrence is virtually assured. The development of new therapeutic strategies coupled with increased selectivity of the treatments is essential for the management and enhanced survival of GB patients. This project brings together an existing team with expertise in biological target validation, high-throughput assay development, and molecular chemical libraries to target the TROY signaling pathway(s) as an innovative approach to treat GB invasion. We have developed cell-based assays for high throughput screening (HTS) to identify small molecule inhibitors of TROY dependent signaling. Our cell-based assay will interrogate both orthotopic and allosteric modulators that have functional consequences for TROY signaling. We will utilize resources available at the Prebys Center at Sanford- Burnham to perform a cell-based HTS in 1536-well format of the NIH's structurally diverse chemical library to identify drug-like compounds that inhibit the TROY signaling pathway. Selectivity profiles will be made using in- hand cell-based counterscreen assays. Hits will be further evaluated for biological effects in TROY-dependent glioma cell lines while not affecting TROY negative cells. Mechanism of action (MOA) studies will assess the impact of validated hits on the disruption of TROY oligomerization, TROY-TRAF2 interaction, and TROY- dependent intracellular signaling pathways. Finally, structure-activity relationship (SAR) "by purchase" of analogs of validated hit series will be performed along with supporting pharmacological characterization on the best available probe(s). Studies will emphasize the identification of high affinity small molecules with appropriate physicochemical properties to promote passage across the blood-brain-barrier (BBB) and delivery into the brain parenchyma. To capitalize on the strong biology/chemistry synergy among the assembled team of investigators with a goal to identify research tool compounds targeting TROY for future hit-to-lead optimization campaigns, the following Specific Aims are proposed: Aim 1: Implement a cell-based reporter assay and perform a HTS of the NIH MLSMR >365,000 collection for modulators of the TROY signaling pathway; Aim 2: Confirm initial activity and validate hits for potency and selectivity/non-cytotoxicity with existing secondary cellular assays for TROY pathway dependence and general cytotoxicity; Aim 3: Perform a limited structure-activity relationship (SAR) elucidation through "analogs by catalogs" through the hit validation cascade of secondary assays to select the most potent and selective chemical probe; Aim 4: Perform functional biological assays and tertiary assays on validated scaffolds to elucidate the potential mechanisms of action and efficacy in inhibition of cellular migration, invasion, and resistance in clinically relevant primary GB cells; Aim 5: Perform limited ADME/T and in vitro BBB profiling on the most promising tractable probe(s) and scale up (25 - 50 mg) for a limited rodent PK study and future proof of concept and research studies.