Proteasomal Degradation of XPB as a Novel Mechanism for Treating Inflammation

与NCATS功能基因组实验室合作，我们创建并验证了一种高通量发光报告试验，适用于识别XPB的小分子降解物和全基因组RNAi筛选。 使用这一最先进的高通量分析方法，我们将： 1)寻找具有强大抗炎作用的新型小分子XPB降解剂，可加速进入临床试验；2)表征药物诱导XPB降解所需的分子伙伴，以构建更具普适性的药物范例。这一创新的分析平台将使临床上相关的XPB降解物能够调节PAH患者的肺血管炎症，而NIH临床中心非常适合用于测试这些一流抗炎药物的先锋试验。 项目里程碑： -在NCATS小分子资料库(多达150,000种化合物)中筛选导致XPB-HiBiT降解的药物。候选药物将通过生物/化学信息学分析进行选择，在11个浓度下进行验证，并进行反筛选，以排除细胞毒性作用和/或分析干扰。排名靠前的候选人将被重新合成，他们的活动将得到确认。 -对候选XPB降解物进行第二次高通量筛选，以获得跨不同信号通路的强大抗炎效果。在两个筛选中发现的新药将进一步检验它们对PAH相关内皮细胞功能障碍细胞模型中炎症基因转录和细胞因子产生的影响。 -进行全基因组RNAi筛选，以确定药物诱导的XPB降解的分子机制。诱导蛋白酶体依赖的XPB降解的候选药物的目标去卷积和NCATS药理学注释的化学工具箱将被用于证实基因沉默效应。候选XPB降解基因将在原代人肺血管内皮细胞中得到证实。 -应用最先进的药物化学技术，优化主要抗炎药的药代动力学、效力和疗效，以便转化为临床。

In collaboration with the NCATS Functional Genomics Lab, we have created and validated a high-throughput luminescent reporter assay suitable for identifying small molecule degraders of XPB and genome-wide RNAi screening. Using this state-of-the-art high-throughput assay we will: 1) Identify novel, small-molecule XPB degraders with potent anti-inflammatory effects that can be accelerated into clinical trials; and 2) Characterize molecular partners necessary for drug-induced XPB degradation in order to construct a more generalizable pharmaceutical paradigm. This innovative assay platform will enable discovery of clinically relevant XPB degraders that modulate lung vascular inflammation in PAH patients, and the NIH Clinical Center is ideally suited for spearheading trials testing these first-in-class anti-inflammatory drugs. Project Milestones: - Screen the NCATS Small Molecule Library (up to 150,000 compounds) for drugs that induce XPB-HiBiT degradation. Candidate drugs will be selected through bio/chemi-informatic analyses, validated at 11 concentrations, and undergo counter screens to exclude cytotoxic effects and/or assay interference. Top candidates will be resynthesized and their activity confirmed. - Perform a second, high-throughput screen of candidate XPB degraders for potent anti-inflammatory effects across heterogeneous signaling pathways. Novel drugs identified in both screens will be further examined for their effects on inflammatory gene transcription and cytokine production in cellular models of PAH-associated endothelial cell dysfunction. - Perform a genome-wide RNAi screen to define the molecular mechanism underlying drug-induced XPB degradation. Target deconvolution of candidate drugs that induce proteasome-dependent XPB degradation and the NCATS pharmacologically annotated chemical toolbox will be used to substantiate gene silencing effects. Candidate XPB degradation genes will be confirmed in primary human pulmonary vascular endothelium. - Apply state-of-the-art medicinal chemistry techniques to optimize pharmacokinetics, potency, and efficacy of lead anti-inflammatory drugs for translation into the clinic.