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

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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.