TROY HTS Compound Screening

 描述(由申请人提供)：目前对胶质母细胞瘤(GB)患者的治疗选择有限，而且大部分无效。GB的高度侵袭性给标准治疗灌输了高度的抵抗力，而且几乎可以保证复发。开发新的治疗策略，并增加治疗的选择性，对于GB患者的管理和提高存活率至关重要。该项目汇集了一支在生物靶标验证、高通量分析开发和分子化学库方面拥有专业知识的现有团队，将特洛伊信号通路(S)作为治疗GB侵袭的创新方法。我们已经开发了基于细胞的高通量筛选(HTS)方法，以确定Troy依赖信号的小分子抑制物。我们的基于细胞的分析将询问对Troy信号具有功能影响的原位和变构调节器。我们将利用桑福德-伯纳姆的Prebys中心可用的资源，在NIH结构多样化的化学库的1536孔格式中进行基于细胞的HTS，以识别抑制Troy信号通路的类药物化合物。将使用手把手的基于细胞的反筛选分析来制作选择性图谱。HITS将进一步评估特洛伊依赖胶质瘤细胞系的生物学效应，而不影响特洛伊阴性细胞。作用机制(MOA)研究将评估有效的HITS对Troy寡聚、Troy-TRAF2相互作用和Troy依赖的细胞内信号通路的破坏的影响。最后，验证的HIT系列类似物的结构-活性关系(SAR)将与最佳可用探针(S)上的支持药理学特征一起执行。研究将侧重于鉴定具有高亲和力的小分子 适当的物理化学性质，以促进通过血脑屏障(BBB)并进入脑实质。为了利用聚集的研究团队之间强大的生物/化学协同作用，目标是为未来的Hit-to-Lead优化活动确定针对Troy的研究工具化合物，提出了以下具体目标：目标1：实施基于细胞的报告分析，并执行NIH MLSMR&GT；365,000收集的Troy信号途径调节器的HTS；目标2：利用现有的针对Troy途径依赖和一般细胞毒性的二级细胞分析，确认初始活性和有效性和选择性/非细胞毒性；目的3：通过二次分析的HIT验证级联，通过“目录类似物”进行有限结构-活性关系(SAR)的阐明，以选择最有效和选择性最高的化学探针；目标4：在经过验证的支架上进行功能生物学分析和三级分析，以阐明在抑制临床相关的原代GB细胞中细胞迁移、侵袭和耐药方面的潜在作用机制和有效性；目标5：执行有限的 ADME/T和体外血脑屏障分析上最有前景的易处理探针(S)和放大(25-50毫克)有限的啮齿动物PK研究和未来的概念验证和研究研究。

 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.