TWEAK-Fn14 HTS compound screening

描述（由申请人提供）：尽管脑肿瘤治疗取得了进展，但在改善胶质母细胞瘤患者的预后方面进展甚微。 手术切除后残留的侵入性细胞显著促进了患者的死亡，而没有明显的肿块效应或复发性肿块疾病的证据。 因此，成功的治疗需要靶向肿瘤的侵袭性部分以及核心病变，这是当前治疗方案所不能满足的。该项目利用TWEAK-Fn 14靶点和信号通路作为治疗胶质瘤侵袭的创新方法。我们已经开发了基于细胞的测定和试剂，用于化学文库的高通量筛选（HTS），以鉴定TWEAK-Fn 14相互作用的药物样抑制剂。 我们的基于细胞的测定将询问在整个TWEAK-Fn 14信号通路中具有功能性结果的原位和变构调节剂。 利用Sanford-Burnham Prebys中心提供的资源，我们的目标是对NIH结构多样的化学库进行基于细胞的HTS格式，以鉴定抑制TWEAK-Fn 14信号通路的药物样化合物。 将使用基于二级细胞的TWEAK-Fn 14-NF-？B驱动荧光素酶试验和其他TNF？- TNFR驱动的NF-？B荧光素酶测定。作用机制（MOA）研究将评估TWEAK-Fn 14信号传导通路上经验证的命中沿着的影响。 最后，将完成经验证的命中系列的类似物的“通过购买”的结构-活性关系（SAR），沿着支持优化探针的药理学表征。一个重点将放在确定具有适当的物理化学性质，以促进通过血脑屏障（BBB）和交付到脑实质的高亲和力的小分子。 该项目的目标是确定靶向TWEAK-Fn 14的研究工具化合物，用于针对药物先导物开发的未来命中先导物优化活动，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TWEAK-Fn 14信号传导途径的调节剂进行NIH MLSMR > 365，000集合的HTS;目的2：确认初始活性，并使用现有的TWEAK-Fn 14途径依赖性和一般细胞毒性的二级细胞试验验证效价和选择性/非细胞毒性的命中率;目的3：通过“SAR-by-purchase”进行有限的构效关系（SAR）解析通过二级测定的命中验证级联来选择最有效和选择性的化学探针;目的4：在经验证的支架上进行真实的生物测定和三级测定， 阐明潜在的作用机制，阻断细胞迁移、侵袭和存活，并评估在其他细胞类型和临床相关原代细胞中的功效。 5.对最有前途的易处理探针进行ADME/T和体外BBB分析，并扩大（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): Despite advances in brain tumor therapy, little progress has been made to improve the prognosis of patients with glioblastoma. Invasive cells remaining after surgical resection significantly contribute to the demise of the patient without evidence of a discernable mass effect or recurrent bulk disease. Thus, successful treatment requires targeting the invasive portion of the tumor in addition to the core lesion, which is unmet by current treatment regimens. This project exploits the TWEAK-Fn14 targets and signaling pathways as an innovative approach to treat glioma invasion. We have developed cell-based assays and reagents for high throughput screening (HTS) of chemical libraries to identify drug-like inhibitors of TWEAK-Fn14 interactions. Our cell-based assay will interrogate both orthotopic and allosteric modulators that have a functional consequence throughout the TWEAK-Fn14 signaling pathway. Using the resources available through the Prebys Center at Sanford-Burnham, our objective is to perform a cell-based HTS format of the NIH's structurally diverse chemical library to identify drug-like compounds that inhibit the TWEAK-Fn14 signaling pathway. Selectivity profiles will be made using in-hand secondary cell-based TWEAK-Fn14-NF-?B driven luciferase assays and other TNF?-TNFR - driven NF-?B luciferase assays. Mechanism of action (MOA) studies will assess the impact of validated hits along the TWEAK-Fn14 signaling pathway. Finally, structure- activity relationship (SAR) "by purchase" of analog of validated hit series will be accomplished, along with supporting pharmacological characterization on optimized probe(s). One emphasis will be placed on identifying high affinity small molecules that possess appropriate physicochemical properties to promote passage across the blood-brain-barrier (BBB) and delivery into the brain parenchyma. The goal o the project is to identify research tool compounds targeting TWEAK-Fn14 for future hit-to-lead optimization campaigns directed towards development of drug leads, 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 TWEAK-Fn14 signaling pathway; Aim 2: Confirm initial activity and validate hits for potency and selectivity/non-cytotoicity with existing secondary cellular assays for TWEAK-Fn14 pathway dependence and general cytotoxicity; Aim 3: Perform a limited structure-activity relationship (SAR) elucidation through "SAR-by-purchase" of available commercial analogs through the hit validation cascade of secondary assays to select the most potent and selective chemical probe; Aim 4: Perform authentic biological assays and tertiary assays on validated scaffolds to elucidate the potential mechanisms of action, blockade of cellular migration, invasion and survival and evaluation of efficacy in other cell types and clinically relevant primary cells~ Aim 5. Perform 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.