Biological Analysis of Synthetic alpha-helix mimetics

蛋白质之间的相互作用支配着生物体中发生的许多过程。选择性地破坏这些相互作用的能力是化学生物学的主要挑战。它们很难被容易制造的分子破坏，因为蛋白质很大，而且重要的非共价接触可能彼此距离很远。我们已经开发了一种抑制剂，用于一类相互作用，其中一种蛋白质的α -螺旋结合在另一种蛋白质的间隙中。这种相互作用是通过特定的氨基酸残基沿α -螺旋的一面投射而发生的。我们的抑制剂以模块化的方式构建，允许侧链的容易变化和快速形成一个大的化合物库。目前研究这些相互作用的技术依赖于复杂的分析，其中监测预结合配体的位移而不是实际的结合事件。该提案旨在开发一种新的测定方法来测量这些相互作用，该方法将使用抑制剂和蛋白质之间的荧光共振能量转移，两者都将被荧光标记。一旦抑制剂被结合，荧光标记就被带到近距离。激发其中一个荧光团将导致能量在一小段距离内转移到另一个荧光团。只有当结合发生时，才会导致发射光的波长发生移位。这允许直接测量结合事件，也适用于使用散装技术以及共聚焦显微镜进行体内测量。这项试验将使其他研究人员能够可视化蛋白质在细胞中的位置，并允许更快速地筛选活性化合物。这可能最终会产生一类新的药物，能够解决药物化学中的挑战性问题。

Protein-protein interactions govern many of the processes that occur in living organisms. The ability to disrupt these interactions selectively is a major challenge in chemical biology. They are hard to disrupt with easily made molecules, as proteins are large and the important non-covalent contacts can be distant from each other. We have developed an inhibitor for one class of these interactions where an alpha-helix of one protein binds in the cleft of another. Such interactions occur through specific amino-acid residues projected along one face of the alpha-helix. Our inhibitor is built in a modular fashion, allowing for easy variation of the side chains and rapid formation of a large library of compounds. Current techniques for the study of these interactions rely on a complex assay, where the displacement of a pre-bound ligand is monitored rather than the actual binding event. This proposal seeks to develop a new assay to measure these interactions that will use Fluorescent Resonant Energy Transfer between the inhibitor and the protein, both of which would be fluorescently labelled. Once the inhibitor is bound, the fluorescent labels are brought into close proximity. Excitation of one of the fluorophores will cause the energy to transfer to the other fluorophore over a small distance. This results in a shift in the wavelength of the emitted light, only when binding has occurred. This allows for direct measurement of the binding event, and is also amenable to in vivo measurements using both bulk techniques as well as confocal microscopy. This assay will enable other researchers to visualize the locations of proteins in cells as well as allow for a more rapid screen of active compounds. This could eventually result in a new class of pharmaceuticals able to tackle challenging problems in medicinal chemistry.