MECHANISM OF PROMISCUOUS INHIBITION BY SMALL MOLECULE AGGREGATION

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 长期以来，科学界一直被非特异性小分子的存在所困扰，这些小分子抑制了它们假设靶点以外的蛋白质。 这些抑制剂的一个大的和不同的子集的行为可以通过一个共同的机制来解释：聚集。 在微摩尔浓度下，一些非特异性分子形成大的聚集体，抑制广泛的不相关的可溶性蛋白质。 从以前的研究中，我们知道，抑制的结果从蛋白质和聚集体的直接相互作用。 这种相互作用是可逆的，不会导致变性，但我们的理解程度到此为止。 与聚集体的结合如何导致抑制？聚集体是否结合到特定的蛋白质表面，或者它是非特异性的？ 结合是否抑制或诱导蛋白质的动力学运动？我将使用氘交换质谱法来解决这些问题。因为我们知道聚集体引起的变化比变性更微妙，所以我们有必要使用更灵敏的技术。 质谱联用酰胺氢的氘交换提供了某些区域如何暴露于溶剂的证据。 这使我们能够通过核心（或埋藏）质子的交换来检查表面质子的可及性和酶的整体动力学。 我们预测，聚集体通过改变酶的正常动态运动来抑制。 此外，我们将能够确定是否存在聚集体结合的特定表面。 有几种技术可以达到这种灵敏度，其中质谱法是最实用的。 这些研究将提供洞察混杂抑制聚集的机制，从而阐明小分子和它们的生物靶标之间的新的相互作用。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The scientific community has long been troubled by the presence of nonspecific small molecules that inhibit proteins other than their hypothesized targets. The behavior of a large and diverse subset of these inhibitors can be explained by a common mechanism: aggregation. At micromolar concentrations some nonspecific molecules form large aggregates that inhibit a wide range of unrelated soluble proteins. From previous studies we know that inhibition results from the direct interaction of protein and aggregates. This interaction is reversible and does not result in denaturation, but the extent of our understanding stops there. How does binding to the aggregate lead to inhibition? Does the aggregate bind to a particular protein surface or is it nonspecific? Does binding restrain or induce dynamic motion in the protein? I will use deuterium exchange mass spectrometry to address these questions.Since we know that aggregates induce changes subtler than denaturation, it is necessary that we use a more sensitive technique. Mass spectrometry coupled to deuterium exchange of amide hydrogens provides evidence of how exposed certain regions are to solvent. This allows us to examine the accessibility of surface protons and the overall dynamics of the enzyme through the exchange of core (or buried) protons. We predict that aggregates inhibit by altering the normal dynamic motions of the enzyme. Additionally, we will be able to determine whether there is a particular surface to which the aggregate binds. There are few techniques that allow this level of sensitivity and among them mass spectrometry is the most practical. These studies will provide insight into the mechanism of promiscuous inhibition by aggregation, thus elucidating a novel interaction between small molecules and their biological targets.