Fragment-based Screening of Cellular Proteomics for Multi-target Drug Discovery

基于片段的细胞蛋白质组筛选，用于多靶点药物发现

• 批准号: 1923540
• 金额: --
• 依托单位: University of Oxford
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2017
• 资助国家: 英国
• 起止时间: 2017 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-1923540
• 关键词: Fragment; based; Screening; Cellular; Proteomics

This project falls within the EPSRC Synthetic Organic Chemistry research area.Fragment-based ligand and drug discovery (FBLD) has been effective in generating lead compounds for various targets. Fragments are small molecules with molecular weight ranging between 120-250 Dalton, and have generally less functionality and lower binding affinity compared to normal hits. A major advantage of using fragment screening for hit identification instead of fully functionalized drug-like molecules is that much fewer compounds are needed to cover the same amount of chemical space, making the screening process much more efficient. The detection techniques, such as X-ray crystallography, used in fragment-based screening could also provide structural understanding on fragment binding.Fragment compounds were typically used for in-vitro assays with purified proteins for single-target screening. However, it has been recently demonstrated that differential fragment-protein interactions can be observed when cells are directly incubated with fragments in the medium. These exciting results have inspired us to apply similar principles to fragment screening using cellular proteomics as measures of efficacy. Depicting global cellular responses using gene expression signatures to better understand induced perturbations in biological pathways and key interactions caused by drug-like molecules has been gaining popularity in the field of biomedicine. In this project, we propose to apply the concept of "connectivity map" to cellular proteomics by quantitatively measuring the changes in whole-cell protein levels induced by fragment compounds.Overall, fragment-based screening using cellular proteomics as signatures can be extremely useful in providing insights in the absence of a distinct biological target. This project aims to demonstrate the effectiveness of such an approach in two separate cases.Our first target system was a mouse microglia cell line, BV2, which has been reported to play an active role in multiple neurodegenerative conditions including Alzheimer's disease (AD). Microglia cells are the most abundant macrophage-like cells in the central nervous system (CNS), but they exhibit diverse phenotypes based on cellular context, making target-based screening techniques relatively challenging. Proteomic analysis of microglia cells treated with different stimuli may be able to provide a global view of the cellular changes to better elucidate key proteins involved in the processes of neurodegeneration. Our next target would be the Wnt/b-catenin signaling pathway. Many of the current efforts have been focused on down-regulating b-catenin in order to reduce cancer proliferation. Nonetheless, b-catenin plays an active role in maintaining neurons and therefore also a crucial target for neurodegenerative diseases.Once a hit has been identified, analogues of the fragment structure will be synthesized and tested and reverse-matched against proteomics signatures generated by the disease model. Structure-activity relationship (SAR) may also be inferred if enough analogues are tested. The main part of the project will be dedicated to synthesizing functionally diverse molecules from fragment hits using various synthetic organic strategies. The ultimate goal would be to successfully develop lead compounds that reverses a majority of key changes in cellular proteomics of the disease model. These compounds could then be further tested in various animal models for in vivo efficacy.In summary, this project aims to develop cellular screening of fragment compounds using quantitative proteomics to identify multiple biological pathways and interactions that are relevant to diseases of interest, as well as finding "hit" compounds that can be further functioalized, tested and potentially developed into drug molecules or probes.

本项目福尔斯属于EPSRC合成有机化学研究领域。基于片段的配体和药物发现（FBLD）在产生各种靶点的先导化合物方面非常有效。片段是分子量范围在120-250道尔顿之间的小分子，并且与正常命中物相比通常具有较少的官能度和较低的结合亲和力。使用片段筛选而不是完全功能化的药物样分子进行命中识别的主要优点是，需要更少的化合物来覆盖相同量的化学空间，使筛选过程更加有效。在基于片段的筛选中使用的检测技术，如X射线晶体学，也可以提供对片段结合的结构理解。片段化合物通常用于与纯化蛋白进行单靶筛选的体外测定。然而，最近已经证明，当细胞与培养基中的片段直接孵育时，可以观察到差异片段-蛋白质相互作用。这些令人兴奋的结果启发了我们将类似的原理应用于使用细胞蛋白质组学作为功效测量的片段筛选。使用基因表达特征来描绘全局细胞反应以更好地理解由药物样分子引起的生物学途径和关键相互作用中的诱导扰动在生物医学领域中越来越受欢迎。在这个项目中，我们建议将“连接图”的概念应用到细胞蛋白质组学中，通过定量测量片段化合物诱导的全细胞蛋白质水平的变化。总体而言，使用细胞蛋白质组学作为签名的基于片段的筛选可以在缺乏明显的生物靶标的情况下提供非常有用的见解。我们的第一个靶系统是小鼠小胶质细胞系BV 2，据报道它在包括阿尔茨海默病（AD）在内的多种神经退行性疾病中起着积极的作用。小胶质细胞是中枢神经系统（CNS）中最丰富的巨噬细胞样细胞，但它们基于细胞环境表现出不同的表型，使得基于靶点的筛选技术相对具有挑战性。用不同刺激处理的小胶质细胞的蛋白质组学分析可能能够提供细胞变化的全局视图，以更好地阐明参与神经变性过程的关键蛋白质。我们的下一个目标是Wnt/b-连环蛋白信号通路。目前的许多努力都集中在下调b-连环蛋白以减少癌症增殖。尽管如此，b-连环蛋白在维持神经元中起着积极的作用，因此也是神经退行性疾病的关键靶点。一旦确定了命中，将合成和测试片段结构的类似物，并与疾病模型产生的蛋白质组学特征进行反向匹配。如果测试了足够的类似物，也可以推断结构-活性关系（SAR）。该项目的主要部分将致力于使用各种合成有机策略从片段命中合成功能多样的分子。最终目标是成功开发出先导化合物，逆转疾病模型细胞蛋白质组学中的大多数关键变化。总之，本项目的目标是利用定量蛋白质组学对片段化合物进行细胞筛选，以确定与感兴趣的疾病相关的多种生物学途径和相互作用，并找到可以进一步功能化、测试和潜在开发为药物分子或探针的“命中”化合物。