Development of native ion mobility mass spectrometry methods to study PROTAC systems

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Small molecule inhibitors have traditionally been developed to bind to a specific protein, thereby preventing it from carrying out its function in the cell. In recent biotechnological approaches, molecules have been created that degrade a target protein, rather than inhibit it. Major advantages of protein degraders over inhibitors are the longer-lasting effects of degraders and the lower concentrations required to achieve efficacy. Moreover, degraders are applicable to a wider spectrum of proteins since binding is not limited to a specific active site. The most popular type of degraders to date are called proteolysis targeting chimeras (PROTACs), which are bifunctional ligands that bind simultaneously to the targeted protein and an E3 ligase, bringing the proteins into a complex, so that the E3 ligase labels the targeted protein for degradation by the cell. PROTACs have been developed against a variety of medically relevant proteins, such as the tumorigenic Androgen Receptor and Estrogen Receptor, as explored in clinical trials. A current limitation in the development of novel PROTACs is the ability to directly measure the formation of three-component complexes in a fast and efficient manner. Native mass spectrometry (nMS) is an efficient method for analysing the species present in complex mixtures involving E3 ligases, PROTACs, and substrate proteins. This is due to its ability to report on multiple binding stoichiometries present in dynamic protein mixtures, including species populated to a low extent. When coupled with ion mobility, which is a complementary gas-phase technique, different conformations of proteins or protein complexes can also be separated. The aim of this PhD project is to further develop native ion mobility mass spectrometry methods for the analysis of PROTACs and other protein degraders, which have strong potential as therapies for cancer and other diseases.