Ahmed Ikhlas M M, Rofe Adam, Henry Martyn C, West Eric, Jamieson Craig, McEwan Iain J, Beveridge Rebecca
Department of Pure and Applied Chemistry, University of Strathclyde, Glasgow, UK.
Institute of Medical Sciences, School of Medicine, Medical Sciences and Nutrition, University of Aberdeen, Aberdeen, UK.
Protein Sci. 2025 Jan;34(1):e5254. doi: 10.1002/pro.5254.
Intrinsically disordered proteins (IDPs) are important drug targets as they are key actors within cell signaling networks. However, the conformational plasticity of IDPs renders them challenging to characterize, which is a bottleneck in developing small molecule drugs that bind to IDPs and modulate their behavior. In relation to this, ion mobility mass spectrometry (IM-MS) is a useful tool to investigate IDPs, as it can reveal their conformational preferences. It can also offer important insights in drug discovery, as it can measure binding stoichiometry and unveil conformational shifts of IDPs exerted by the binding of small drug-like molecules. Herein, we have used IM-MS to investigate the effect of drug lead EPI-001 on the 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 harbors most, if not all, of the transcriptional activity. We quantify the stoichiometry of EPI-001 binding to various constructs corresponding to functional domains of 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, and to a much lesser extent with TAU-1 containing constructs. 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 tractability of IDPs, potentially leading to better therapies.
内在无序蛋白（IDPs）是重要的药物靶点，因为它们是细胞信号网络中的关键角色。然而，IDPs的构象可塑性使其难以表征，这是开发与IDPs结合并调节其行为的小分子药物的一个瓶颈。与此相关的是，离子淌度质谱（IM-MS）是研究IDPs的有用工具，因为它可以揭示它们的构象偏好。它还可以在药物发现中提供重要见解，因为它可以测量结合化学计量并揭示由类药物小分子结合引起的IDPs的构象变化。在此，我们使用IM-MS研究了先导药物EPI-001对雄激素受体无序N端结构域（AR-NTD）的影响。尽管结构异质性使NTD成为蛋白质中一个难以药物作用的区域，但该结构域承载了大部分（如果不是全部）转录活性。我们量化了EPI-001与对应于AR-NTD功能结构域的各种构建体的结合化学计量，并表明它分别与包含反式激活单元（TAU）-1和TAU-5的不同构建体结合，并且1-2个分子与包含这两个序列的更大构建体结合。我们还确定了EPI-001与TAU-5结合时的构象变化，而与包含TAU-1的构建体结合时的构象变化程度要小得多。这项工作为EPI-001与AR-NTD的相互作用及其引起的结构改变提供了新的见解，并将IM-MS定位为一种信息丰富的工具，将提高IDPs的可处理性，有可能带来更好的治疗方法。
