Ion mobility mass spectrometry unveils conformational effects of drug lead EPI-001 on the intrinsically disordered N-terminal domain of the Androgen Receptor

Intrinsically disordered proteins (IDPs) are important drug targets as they are integral components of cell signalling networks. Accordingly, their dysregulation is highly implicated in a range of disease states. However, the conformational plasticity of IDPs renders them challenging to characterise both experimentally and theoretically, which is a major bottleneck in the development of small molecule drugs that bind to IDPs and alter how they behave. In relation to this, ion mobility mass spectrometry (IM-MS) is a useful tool in the investigation of IDPs, as it can reveal their conformational preferences when they are ionised from different conditions. It also has the potential to offer important insight to the drug discovery field as it can measure binding stoichiometry, and unveil conformational shifts of IDPs exerted by the binding of small drug-like molecules, which will affect their associated pharmacology. In the current study, we have used IM-MS to investigate the effect of drug lead EPI-001 on the intrinsically disordered N-terminal domain of the Androgen Receptor (AR-NTD). Despite structural heterogeneity rendering the NTD a challenging region of the protein to drug, this domain is responsible for most, if not all, of the transcriptional activity. We quantify the stoichiometry of EPI-001 binding to various constructs that correspond to functional domains of the AR-NTD, and show that it binds to separate constructs containing transactivation unit (TAU)-1 and TAU-5, respectively, and that 1-2 molecules bind to a larger construct containing both sequences. We also identify a conformational shift upon EPI-001 binding to the TAU-5 containing construct, and only to a much lesser extent with TAU-1. This work provides novel insight on the interactions of EPI-001 with the AR-NTD, and the structural alterations that it exerts, and positions IM-MS as an informative tool that will enhance the drugability of IDPs, leading to better therapies.