Development of innovative label-free methods to investigate the binding properties and degradation efficiencies of PROTACs

Short Summary: Proteolysis-targeting chimeras (PROTACs) are small molecule degraders that connect a ubiquitin E3 ligase to a target protein, thus inducing its ubiquitination and degradation. A ternary complex must be formed between the target protein, E3 ligase and PROTAC molecule to achieve degradation, which is difficult to measure with most biophysical techniques. We will develop novel, label-free native mass spectrometry methods to study PROTACs in cell-like environments. This will reveal the interplay between ternary complex formation, target ubiquitination and target degradation in a single experiment. These methods will be faster and less labour intensive than current methods used to deduce the biophysical characteristics of PROTACs, and furthermore will provide information on their degradation efficiencies. The project will therefore catalyse the progression of PROTACs into clinical therapies that will have an impact on cancer treatment.
Key research questions: The overall aim of this PhD project is to develop, test and streamline new mass spectrometry tools to (i) quantify the formation of ternary complexes in cell lysates (ii) determine how efficiently the target proteins is ubiquitinated upon PROTAC engagement and (iii) measure the degradation rate of the target protein, all in a single experiment. The following key questions will be addressed with native mass spectrometry:
- How well do PROTACs form ternary complexes, how specific are the PROTACs for a given target, and is the binding mechanism cooperative in the cell lysate?
- What is the interplay between ternary complex formation, target ubiquitination and target degradation for a given PROTAC?
- How can experiments be streamlined into high-throughput, label-free methods to reveal the maximum information with minimum time and sample requirements?