Screening for anti-cancer agents against the smoothened receptor using novel mass spectrometry-based methodology

使用基于质谱的新型方法筛选针对平滑受体的抗癌药物

• 批准号: 2748015
• 金额: --
• 依托单位: University of Nottingham
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2022
• 资助国家: 英国
• 起止时间: 2022 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2748015
• 关键词: Screening; anti; cancer; agents; against

Background The smoothened receptor is an integral transmembrane protein that forms a key component of the so-called hedgehog cell-cell signalling pathway. Mutations in the smoothened protein can lead to overactivation of the pathway, which results in cell proliferation and cancer, including medulloblastoma, basal-cell carcinoma and pancreatic cancer. The drug vismodegib, which targets smoothened, is FDA-approved for use against basal-cell carcinoma, and is currently in trials against other cancers, but the emergence of vismodegib-resistant smoothened mutants makes the search for alternatives a high priority. This project centres on the development and of a novel screening platform and its application to the discovery of smoothened inhibitors.Methodology and Experimental Approaches We have shown that photochemical carbene footprinting is a powerful method for mapping protein binding sites (Nat. Commun. 2016, Angew. Chem. Int Ed. 2017, Nat. Chem. Biol. 2018, Nat. Chem. 2019, Proteomics 2021). The method uses differential labelling with a footprinting probe in the presence and absence of a binding partner (protein or small molecule) to show where masking of the protein surface occurs and hence identify the binding site. The DTP student develop a novel screening platform by using differential labelling of a reporter peptide in the smoothened binding site to inform the presence of a bound ligand (Fig 1). A reference peptide will provide a control for labelling efficiency.Research-based training The Oldham group provides an excellent environment for interdisciplinary research and training, with a strong collaborative ethos. Current joint projects with colleagues in the Schools of Chemistry (Soultanas, Hayes), Life Sciences (Layfield) and Biosciences (Holdsworth) illustrate the breadth of collaboration. As part of their research project, the student will gain key skills in the handling and characterisation of membrane proteins (see training from non-academic partner for expression and purification training). A key tool in the protein footprinting methodology is the application of mass spectrometry for mapping the sites of protein modification, and reporting binding in the screen. The student will receive high-level training in protein mass spectrometry, including MALDI-MS, LC-MS and MS/MS to enable them to conduct the footprinting project. These are transferable skills for protein identification and PTM characterisation, and are thus highly sought-after generally. Data handling and interpretation is crucial to footprinting methodology. The CASE student will be instructed in how to extract information from the raw MS data and judge its significance through the application of statistical analysis. The student will receive health and safety training through the School of Chemistry's induction sessions. These are enabled throughout the year by use of video lectures, supporting documents, and online resources. Specific health and safety training for the mass spectrometry lab is provided by the academic supervisor, as is a set of SOPs for each instrument and technique in the lab.

背景Smoothened受体是一种完整的跨膜蛋白，形成了所谓的刺猬细胞-细胞信号通路的关键组成部分。smoothened蛋白的突变会导致该通路的过度激活，从而导致细胞增殖和癌症，包括成神经管细胞瘤、基底细胞癌和胰腺癌。针对smoothened的药物vismodegib已被FDA批准用于治疗基底细胞癌，目前正在对其他癌症进行试验，但vismodegib耐药smoothened突变体的出现使得寻找替代品成为高度优先事项。该项目的发展和一个新的筛选平台及其应用的smoothened inhibitors.Methodology和实验方法的发现中心我们已经表明，光化学卡宾足迹是一个强大的方法映射蛋白质结合位点（Nat. Commun. 2016年，Angew。2017，Nat.Chem.Biol.2018，Nat.Chem.2019，Proteomics 2021）。该方法在存在和不存在结合伴侣（蛋白质或小分子）的情况下使用足迹探针进行差异标记，以显示蛋白质表面发生掩蔽的位置，从而鉴定结合位点。DTP学生开发了一种新的筛选平台，通过使用平滑结合位点中报告肽的差异标记来告知结合配体的存在（图1）。参考肽将为标记效率提供控制。基于研究的培训奥尔德姆集团提供了一个跨学科研究和培训的良好环境，具有强大的协作精神。目前与化学学院（Soultanas，Hayes），生命科学学院（Layfield）和生物科学学院（Holdsworth）的合作项目说明了合作的广度。作为研究项目的一部分，学生将获得膜蛋白处理和表征的关键技能（参见非学术合作伙伴的表达和纯化培训）。蛋白质足迹法中的一个关键工具是应用质谱法来绘制蛋白质修饰的位点，并报告筛选中的结合。学生将接受蛋白质质谱的高水平培训，包括MALDI-MS，LC-MS和MS/MS，使他们能够进行足迹项目。这些都是可转移的蛋白质鉴定和PTM表征的技能，因此普遍受到高度追捧。数据处理和解释对足迹法至关重要。CASE学生将学习如何从原始MS数据中提取信息，并通过统计分析的应用判断其意义。学生将通过化学学院的入门课程接受健康和安全培训。通过使用视频讲座，支持文件和在线资源，全年都可以实现这些目标。质谱实验室的具体健康和安全培训由学术主管提供，实验室中的每种仪器和技术都有一套SOP。