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

该项目属于EPSRC合成有机化学研究领域。基于碎片的配体和药物发现(FBLD)在为各种目标生成先导化合物方面一直是有效的。片段是分子量在120-250道尔顿之间的小分子，与正常的HITS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类药物分子进行HIT鉴定的一个主要优势是，需要更少的化合物来覆盖相同数量的化学空间，从而使筛选过程更加高效。在基于片段的筛选中使用的检测技术，如X射线结晶学，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的体外分析，以进行单靶点筛选。然而，最近已经证明，当细胞直接与培养液中的片段孵育时，可以观察到不同的片段与蛋白质的相互作用。这些令人兴奋的结果激励我们将类似的原则应用于片段筛选，使用细胞蛋白质组学作为有效性的衡量标准。使用基因表达特征描述全球细胞反应，以更好地理解由类药物分子引起的生物通路和关键相互作用中的诱导扰动，在生物医学领域得到了越来越多的欢迎。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引起的整个细胞蛋白质水平的变化。总而言之，使用细胞蛋白质组学作为特征的基于片段的筛选在缺乏明显的生物靶点的情况下可以非常有用地提供见解。这个项目的目的是在两个不同的案例中展示这种方法的有效性。我们的第一个目标系统是小鼠小胶质细胞系BV2，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发挥积极作用。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，但根据细胞环境的不同表现出不同的表型，这使得基于靶点的筛选技术相对具有挑战性。对不同刺激处理的小胶质细胞进行蛋白质组学分析，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退行性变过程的关键蛋白质。我们的下一个目标将是Wnt/b-catenin信号通路。目前的许多努力都集中在下调b-连环蛋白，以减少癌症的扩散。尽管如此，b-catenin在维持神经元方面发挥着积极的作用，因此也是神经退行性疾病的关键靶点。一旦确定了命中结果，将合成和测试片段结构的类似物，并与疾病模型生成的蛋白质组特征进行反向匹配。如果测试足够多的类似物，也可以推断构效关系(SAR)。该项目的主要部分将致力于使用各种合成有机策略从碎片中合成不同功能的分子。最终目标将是成功地开发出能够逆转疾病模型细胞蛋白质组学中大多数关键变化的先导化合物。总之，这个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个生物学途径和相互作用，并找到可以进一步功能化、测试并有可能开发成药物分子或探针的“热门”化合物。

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.