Elucidating the Molecular Basis of PPAR (Gamma) Agonist Driven Transcriptional Regulation by Microenvironment Mapping

该项目属于EPSRC物理科学研究领域。该项目是与诺和诺德公司合作的。2020年，在用于目标识别和相互作用组图绘制的光亲和标记方面，报告了一项革命性的物理科学进展。该方法使用与具有4 nm能量转移范围的光激活光催化剂连接的探针来激活基于卡宾的标记。与传统的光亲和标记技术相比，这种设计提供了两大优势：(1)催化信号放大导致每个探针发生多个标记事件，并大大提高了有助于检测低丰度蛋白质的信噪比(2)4 nm半径内的蛋白质的串联标记，允许识别相互作用伙伴。该项目将进一步发展这项物理科学技术，并将其应用于生物医学研究中尚未得到满足的需求。具体地说，为了了解PPA(Gamma)辅助因子招募和转录调节的机制，这是目前治疗2型糖尿病的目标。该项目的发现可能会对下一代糖尿病疗法的开发产生下游应用。目前许多核激素受体(NR)靶向治疗显示出不受欢迎的靶向副作用。例如，格列酮，PPAR(伽玛)激动剂，对2型糖尿病的使用受到体重增加、液体滞留和心血管影响的限制。配体结合引起PPAR(Gamma)的构象变化，导致共激活物和抑制物复合体的形成以及随后的转录活性的改变。虽然已知不同的配体通过PPAR(Gamma)刺激重叠但离散的转录程序和表型结果，但差异辅助因子结合的无偏见特征受到所涉及的技术挑战的限制。设计和合成光催化化学探针，以确定激动剂结合对PPAR(伽玛)S微环境的影响，包括辅因子募集。了解激动剂诱导的转录改变与PPAR(Gamma)蛋白复合体组成的依赖关系。基于一系列PPAR(Gamma)激动剂的结构-活性-关系数据，我们将合成一个光亲和型和光催化PPAR(Gamma)激动剂探针和结构匹配对照的文库。在确认PPAR(Gamma)结合和转录调控后，探针将应用于体外分化的人类脂肪细胞。接下来，样品将被生物素化的二氮杂环化合物剂量，并用蓝光脉冲来启动对光催化剂连接的PPAR(伽马)配体4 nm内的所有蛋白质的标记。标记的蛋白质将通过蛋白质组学进行浓缩和分析，同时进行转录分析。对体外分化的人类脂肪细胞进行的带有诱导性PPAR(Gamma)敲除的实验将被用来确保识别出的蛋白质的特异性，并区分非靶标标记和真正的相互作用组标记。配体将根据蛋白质组和转录组特征进行分类，以识别与转录变化相关的蛋白质。识别的蛋白质在介导配体诱导的转录改变中的必要性将通过评估激动剂诱导的细胞转录改变和识别的蛋白质被敲除来确定。该项目将为这一最先进的物理科学技术的新应用提供概念证明，该技术可以应用于其他受体或执行新的生物活性小分子或多肽的靶向去卷积。此外，这项工作将为PPAR(Gamma)对基因转录的调控提供基本的见解。这种洞察力可以为具有所需性质的PPAR(Gamma)激动剂疗法的开发提供信息，并使替代方法成为可能，例如抑制PPAR(Gamma)和特定辅助因子之间的相互作用。

This project falls within the EPSRC Physical Sciences research area. This project is in collaboration with Novo Nordisk. In 2020 a transformative physical science advance in photoaffinity labelling for target identification and interactome mapping was reported. The approach uses a probe conjugated to a light-activated photocatalyst with a 4 nm energy-transfer range to activate a carbene-based label. This design offers two major advantages over conventional photoaffinity labelling techniques: (1) catalytic signal amplification leads to multiple labelling events per probe and greatly enhanced signal-to-noise ratios aiding detection of low abundance proteins (2) tandem labelling of proteins within a 4 nm radius allowing interaction partners to be identified. This project will further develop this physical science technology and apply it to unmet needs in biomedical research. Specifically, to understand the mechanisms of PPA(Gamma) co-factor recruitment and transcriptional regulation, a current target for the treatment of type 2 diabetes. Findings from this project could have downstream applications for the development of next generation diabetes therapeutics. Many current nuclear hormone receptor (NR) targeting therapies display unwanted on-target side effects. For example, the use of glitazones, PPAR(Gamma) agonists, for type 2 diabetes has been limited by weight-gain, fluid retention and cardiovascular effects. Ligand binding causes a conformational shift in PPAR(Gamma) leading to alterations in co-activator and repressor complex formation and subsequent transcriptional activity. While different ligands are known to stimulate overlapping but discrete transcriptional programs and phenotypic outcomes through PPAR(Gamma), unbiased characterisation of differential co-factor binding has been limited by the technical challenges involved. To design and synthesize photocatalytic chemical probes to determine the impact of agonist binding on PPAR(Gamma)'s microenvironment, including co-factor recruitment. To understand the dependence of the agonist-induced transcriptomic changes on PPAR(Gamma) protein complex constitution. Based on the structure-activity-relationship data available for a range of PPAR(Gamma) agonists, we will synthesise a library of photoaffinity- and iridium photocatalytic PPAR(Gamma) agonist probes and structure-matched controls. Following confirmation of PPAR(Gamma) binding and transcriptional regulation, probes will be applied to in vitro differentiated human adipocytes. Next, the samples will be dosed with biotinylated diazirines and pulsed with blue light to initiate labelling of all proteins within 4nm of the photocatalyst-conjugated PPAR(Gamma) ligands. Labelled proteins will be enriched and analysed by proteomics with samples for transcriptomic analysis processed in parallel. Experiments on in vitro differentiated human adipocytes with inducible PPAR(Gamma) knock-out will be used to ensure the specificity of identified proteins and to distinguish off-target labelling from genuine interactome labelling. Ligands will be clustered based on proteomic and transcriptomic signatures to identify proteins correlating with transcriptional changes. The necessity of identified proteins in mediating the ligand induced transcriptomic changes will be determined by assessing agonist induced transcriptomic changes in cells with the identified proteins knocked out. This project will provide proof of concept for a novel application of this state-of-the-art physical science technology that could be applied to other receptors or to perform target deconvolution of novel bioactive small molecules or peptides. Additionally, this work will provide fundamental insight into the regulation of gene transcription by PPAR(Gamma). Such insight could inform the development of PPAR(Gamma) agonist therapeutics with desired properties and enable alternative approaches such as inhibiting interactions between PPAR(Gamma) and specific co-factors.