Native mass spectrometry (nMS) allows the interrogation of proteins, protein complexes and their binding partners as noncovalent interactions are maintained within the instrument. Hence, nMS has been used to investigate the non-traditional drug modalities that are PROteolysis TArgeting Chimeras (PROTACs) and nanodisc membrane mimetics that are used to stabilise and solubilise membrane proteins.
PROTACs facilitate target degradation as a therapeutic mechanism in contrast to canonical inhibition of a protein target. These bifunctional molecules simultaneously bind the target protein and an E3-ubqiuitin ligase, bringing the two proteins into close spatial proximity to promote target ubiquitinylation and degradation via the cell’s endogenous degradation pathways. nMS was utilised to study the ternary complexes promoted by previously reported PROTAC GNE-987 with targets bromodomains 1 and 2 from Brd4 and the E3-Ubiquitin Ligase Von Hippel Lindau (VHL). nMS confirmed the stoichiometry of the ternary complexes, allowed single measurement monitoring of all species present at equilibrium, and allowed quantification of ternary complex formation that revealed higher ternary complex was formed, potentially through increased complex affinity or stability, when GNE-987 engaged bromodomain 1 compared to bromodomain 2.1 This highlights nMS as a direct screening method to measure ternary complexes for PROTAC development.
nMS has also been established as a technique to study membrane proteins, a highly relevant therapeutic target class that are often challenging due to poor solubility and stability. Nanodiscs, discoidal membrane bilayers that are encircled by amphipathic proteins, are one method of stabilising and solubilising membrane proteins for introduction into the MS, with the aim to allow for fragment screening against membrane proteins. Detection of an intact empty nanodisc by nMS is a first crucial step towards the visualisation of membrane protein embedded nanodiscs. Furthermore, fragment screens against empty nanodiscs are being pursued to identify fragments capable of binding nanodiscs, as opposed to the embedded membrane proteins. These experiments aim to establish methodologies for performing fragment screens against membrane proteins of interest by nMS.