Molecular glues are a new class of drug which, as the name suggests, “stick” proteins together. They do this to bring proteins called E3 ligases into contact with cancer-causing proteins. When this happens, the E3 ligase adds a ‘label’ to the cancerous protein, that causes it to be broken down by the cell. The structure formed between a molecular glue, an E3 ligase, and the cancerous protein is called a ternary complex. The aim of this project was to use native mass spectrometry as a quick and simple method of identifying when these ternary complexes have formed.
There are a few reasons why we use native mass spectrometry rather than any other analytical method to analyse ternary complexes. The first is that the interaction between the molecular glue and the two proteins can be weak, and nMS is a “gentle” technique which allows us to maintain these weak interactions during analysis. Another reason is that ternary complexes are large and dynamic, which can cause other common techniques to struggle to resolve the data produced. Analysing one thing, or even two things by many analytical techniques is simple, but analysing three things comes with a plethora of challenges.
We demonstrated that native mass spectrometry is capable of determining the formation of ternary complexes. We were particularly interested in one type of E3 ligase that’s known as DCAF15. We found that DCAF15 self-associates, meaning it was observed binding to other copies of itself, and this behaviour had not been reported previously to our knowledge. While investigating this phenomenon, we identified that this DCAF15 self-association is reliant on salt concentration. When we analysed DCAF15 at low salt conditions, it mainly existed as a dimer and a trimer (two and three copies of itself bound together). When analysed at high salt conditions, it mainly existed as a monomer (a single copy). We then showed that ternary complexes can be formed at both low and high salt conditions, but at high salt conditions less ternary complex can be formed. This information allows for a better understanding of this E3 ligase, eventually leading to the development of more effective drugs.
This work was led by Cara Jackson who is a PhD student under the supervision of Rebecca Beveridge. It was part of a collaboration with TRIANA Biomedicines in Massachusetts.
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