Next Generation Helix Mimetics - Probes for Target Selectivity

蛋白质是身体的机器，它们相互作用的方式对它们的正常运作至关重要。这些蛋白-蛋白相互作用（PPIs）不仅控制细胞中的关键过程，而且还可能参与疾病的发展。例如，在许多癌症中，通常用于调节细胞存活或死亡的ppi被利用来促进肿瘤生长。因此，人们已经付出了相当大的努力，试图防止这些有害的相互作用的发生。然而，以ppi为目标并不简单。蛋白质通常通过非常大的、通常是平坦的表面相互作用。这使得更小的药物分子很难破坏/抑制这些相互作用。克服这一挑战的一个策略是，以这样一种方式构建分子，使其模仿PPI中涉及的一种蛋白质的相互作用表面的关键特征。通过这种方式，分子可以复制原生PPI中发生的相同的相互作用，取代其他相互作用的伙伴，现在可以自由地执行其正常的生物功能。这种模仿策略已经成功地用于抑制癌细胞中有害的PPIs。然而，目前尚不清楚的是，除了它们的预定目标外，这些被称为模拟物的分子能与多少蛋白质相互作用。如果这些分子要在药物开发过程中取得进一步进展，了解这种选择性特征是至关重要的。在这个项目中，我们将描述针对相同PPI的一系列领先模拟物的选择性概况。这不仅将提供关于任何有问题的偏离目标的关键信息，而且还将突出可能提高其效力的任何有益的附加目标。这项研究还将提供领先的模拟物的第一次全面比较，以使最有希望的候选物的开发得到优先考虑。我们将通过向模仿者的结构中添加两个附加功能来实现这一点。首先，我们将添加一个化学基团，当被紫外光激活时，它将在模拟物和与之相互作用的任何蛋白质之间形成永久的连接。其次，我们将添加一个分子鱼钩，使我们能够仅从细胞内复杂的蛋白质混合物中分离出与模拟物相关的蛋白质。然后，我们将通过它们的质量识别与每个模拟物结合的所有蛋白质，并使用替代的单蛋白聚焦方法验证结果。该项目不仅将大大提高我们对复杂生物环境中主要模拟分子活性的理解，而且还将为未来模拟物的靶谱表征提供一般方法，从而准确预测其完整的生物活性，为建立蛋白质模拟物作为治疗方法的潜力提供重要的一步。

Proteins are the machines of the body and the way they interact with one another is critical for their correct functioning. These protein-protein interactions (PPIs) not only control key processes in cells but can also be involved in the development of disease. For example, in many cancers PPIs which are normally used to regulate whether a cell lives or dies are exploited to enable tumour growth. Therefore, considerable effort has been placed on trying to prevent these harmful interactions from occurring. However, targeting PPIs is not straightforward. Proteins commonly interact with one another using very large, and often mostly flat, surfaces. This makes it challenging for a much smaller drug molecule to disrupt/inhibit these interactions. One strategy to overcome this challenge is to construct molecules in such a way that they mimic a key feature of the interacting surface of one of the proteins involved in the PPI. This way the molecule can replicate the same interactions which occur in the native PPI in place of the other interacting partner which is now free to carry out its normal biological function. This strategy of mimicry has been used successfully to inhibit harmful PPIs in cancer cells. However, what is not currently clear is how many proteins these molecules, known as mimetics, can interact with aside from their intended target. It is critical to understand this selectivity profile if these molecules are to progress further in the drug development process.In this project, we will characterise the selectivity profile of a series of leading mimetics targeted against the same PPI. This will not only provide critical information on any problematic off-targets but also will highlight any beneficial additional targets that may enhance their effectiveness. This study will also provide the first comprehensive comparison of leading mimetics to enable development of the most promising candidates to be prioritised. We will achieve this by adding two additional features to structures of the mimetics. Firstly, we will add a chemical group which, when activated by UV light, will form a permanent linkage between the mimetic and any protein it is interacting with. Secondly, we will add a molecular fishing hook which will enable us to isolate only the proteins that are linked to the mimetic from the complex mixture of proteins inside a cell. We will then identify all the proteins bound to each mimetic by their mass and validate the results using alternative, single protein focused methods.This project will not only allow significantly enhance our understanding of the activity of leading mimetic molecules in complex biological environments but it will also provide access to general methods for the target profile characterisation of future mimetics allowing their complete biological activity to be accurately predicted providing a vital step forward in establishing the potential of protein mimetics as therapeutics.