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

该项目属于EPSRC物理科学研究领域的福尔斯。该项目与诺和诺德公司合作。2020年，报告了用于靶标识别和相互作用组作图的光亲和标记的变革性物理科学进展。该方法使用与具有4 nm能量转移范围的光活化光催化剂缀合的探针来激活基于卡宾的标记。这种设计提供了两个主要的优势，超过传统的光亲和标记技术：（1）催化信号放大导致多个标记事件每个探针和大大增强的信噪比，帮助检测低丰度蛋白质（2）串联标记的蛋白质在4 nm半径内，允许相互作用的配偶体被识别。该项目将进一步发展这种物理科学技术，并将其应用于生物医学研究中未满足的需求。具体而言，了解PPA（γ）辅因子募集和转录调控的机制，这是目前治疗2型糖尿病的目标。该项目的发现可能会在下一代糖尿病治疗药物的开发中产生下游应用。许多目前的核激素受体（NR）靶向疗法显示出不希望的靶向副作用。例如，格列酮、过氧化物酶体增殖体激活受体（γ）激动剂用于2型糖尿病受到体重增加、体液潴留和心血管效应的限制。配体结合引起PPAR（γ）的构象转变，导致共激活因子和阻遏物复合物形成和随后的转录活性的改变。虽然已知不同的配体通过PPAR（γ）刺激重叠但离散的转录程序和表型结果，但是差异辅因子结合的无偏表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（Gamma）微环境的影响，包括辅因子募集。了解激动剂诱导的转录组学变化对过氧化物酶体增殖物激活受体（γ）蛋白复合物组成的依赖性。基于一系列PPAR（γ）激动剂的结构-活性-关系数据，我们将合成一个光亲和性和铱光催化PPAR（γ）激动剂探针和结构匹配对照的库。在确认PPAR（γ）结合和转录调控后，将探针应用于体外分化的人脂肪细胞。接下来，将用生物素化的二氮杂环丙烷给药样品，并用蓝光脉冲以开始标记光催化剂缀合的PPAR（γ）配体的4 nm内的所有蛋白质。将通过蛋白质组学富集和分析标记的蛋白质，同时处理用于转录组学分析的样品。对具有诱导型PPAR（γ）敲除的体外分化的人脂肪细胞的实验将用于确保鉴定的蛋白质的特异性，并区分脱靶标记与真正的相互作用物组标记。将基于蛋白质组学和转录组学特征对配体进行聚类，以鉴定与转录变化相关的蛋白质。鉴定的蛋白质在介导配体诱导的转录组学变化中的必要性将通过评估具有鉴定的蛋白质敲除的细胞中激动剂诱导的转录组学变化来确定。该项目将为这种最先进的物理科学技术的新应用提供概念证明，该技术可应用于其他受体或对新型生物活性小分子或肽进行靶向去卷积。此外，这项工作将提供基本的见解基因转录的调控PPAR（γ）。这种见解可以为开发具有所需特性的PPAR（γ）激动剂治疗提供信息，并实现替代方法，例如抑制PPAR（γ）与特定辅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.