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袭性细胞对患者的死亡有很大影响，没有明显的肿块效应或复发的大块性疾病的证据。 因此，成功的治疗需要靶向肿瘤的侵袭性部分，而不是核心病变，这是目前的治疗方案所不能满足的。该项目利用TWEEP-Fn14靶点和信号通路作为治疗胶质瘤侵袭性的创新方法。我们已经开发了基于细胞的分析和试剂，用于高通量筛选化学文库(HTS)，以识别TWEEP-Fn14相互作用的类药物抑制剂。我们的基于细胞的分析将询问原位和变构调节剂，这些调节剂在整个TWEEP-Fn14信号通路中具有功能结果。利用位于Sanford-Burnham的Prebys中心提供的资源，我们的目标是对NIH结构多样化的化学库进行基于细胞的HTS格式，以识别抑制TWEEP-Fn14信号通路的类药物化合物。选择性图谱将使用手二次细胞为基础的TWEAY-Fn14-NF-？B驱动的荧光素酶分析和其他肿瘤坏死因子受体-驱动的NF-？B荧光素酶分析。作用机制(MOA)研究将评估TWEEP-Fn14信号通路上有效的HITS的影响。最后，将完成验证的HIT系列类似物的结构-活性关系(SAR)，并在优化的探针上支持药理表征(S)。一个重点将放在识别具有适当物理化学性质的高亲和力小分子，以促进通过血脑屏障(BBB)并进入脑实质。该项目的目标是确定针对TWEWE-Fn14的研究工具化合物，用于未来针对药物先导开发的Hit-to-Lead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分析，并执行NIH MLSMR&GT；365,000集合的HTS，用于TWAKE-Fn14信号通路的调节器；目标2：确认初始活性，并利用现有的二级细胞分析验证HITS的效力和选择性/非细胞毒性，用于TWAKE-Fn14途径依赖和一般细胞毒性；目标3：通过二次分析的HIT验证级联选择最有效和选择性最高的化学探针，通过可获得的商业类似物进行有限的结构-活性关系(SAR)阐明；目标4：在已验证的支架上进行真实的生物分析和三次分析，以 阐明潜在的作用机制，阻断细胞的迁移、侵袭和存活，以及对其他类型细胞和临床相关原代细胞的疗效评估~AIM 5.对最有希望的易处理探针(S)进行ADME/T和体外血脑屏障分析，并放大(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.