Novel Probes for Sirtuins: A Chemical Biology Approach

项目总结 Sirtuins，也被称为III类HDAC，消耗化学计量剂量的烟酰胺腺嘌呤二核苷酸 (NAD+)从赖氨酸残基中去除乙酰基，并产生烟酰胺和O-乙酰-ADP-核糖。这 耐人寻味的一类酶被认为与调节各种细胞活动有关，也被认为是 建议调节卡路里限制(CR)的有益效果。Sirtuins一直受到 作为治疗目标的学术界和制药业。 然而，由于相互矛盾的结果，关于sirtuin生物学的争论在过去几年也达到了顶峰。 来自不同的研究小组。这在一定程度上是因为最近发现了这些酶，而 Sirtuins和其他蛋白质之间错综复杂的相互作用环使它们的特征 很难。其中一项艰巨的任务是将sirtuin活性与疾病发病机制联系起来。电流分子 生物学和蛋白质组学技术报告的是蛋白质丰度，而不是活性sirtuin含量。创新型 人们迫切需要能够直接探测sirtuins功能状态的化学工具。 这项赠款申请建议采取高度综合的方法来询问sirtuins的功能状态 在复杂的生物样本中。我们的初步结果证明了该策略的可行性。我们有 获得了一套功能强大的化学探针，能够在模型中评估sirtuins的活性含量 系统。在这个提案中，我们计划合成基于活性的化学探针库。脚手架 旨在提高选择性和贴标效率。在标记方面得分较高的探针 重组sirtuins将接受全细胞裂解产物的分析。在复杂的天然蛋白质组中，探针 应有选择地“突出”有效的sirtuin成分。结合基于质谱学的蛋白质组学 分析认为，这一策略将揭示sirtuins在不同生理和病理条件下的功能图谱。 条件。这将提供有关酶活性异常如何导致疾病进展的信息。 此外，细胞渗透性探针也将用于细胞成像研究。它将使 同时检测功能状态和定位，使Sirtuin功能的直接分析成为可能 对细胞和环境提示的反应。

PROJECT SUMMARY Sirtuins, also called Class III HDACs, consume stoichiometric amounts of nicotinamide adenine dinucleotide (NAD+) to remove acetyl group from lysine residues and to produce nicotinamide and O-acetyl-ADP-ribose. This intriguing class of enzymes has been implicated in regulating various cellular events and has also been suggested to mediate the beneficial effects of calorie restriction (CR). Sirtuins have been intensely pursued by academia and pharmaceutical industry as therapeutic targets. However, controversies on sirtuin biology also peaked during the last few years because of conflicting results from different research groups. This is partly because these enzymes have been discovered recently, and the intricate interaction loops between sirtuins and other proteins make the characterization of them extremely difficult. One of the daunting tasks is to correlate sirtuin activity to disease pathogenesis. Current molecular biology and proteomics techniques report protein abundance rather than active sirtuin content. Innovative chemical tools that can directly probe the functional state of sirtuins are desperately needed. This grant application proposes to take a highly integrative approach to interrogate the functional state of sirtuins in complex biological samples. Our preliminary results demonstrate the feasibility of this strategy. We have obtained a set of powerful chemical probes that are capable of assessing the active content of sirtuins in model systems. In this proposal we plan to synthesize focused libraries of activity-based chemical probes. The scaffolds are designed for enhanced selectivity and labeling efficiency. The probes that score favorably in labeling of recombinant sirtuins will be subjected to the profiling of whole cell lysate. In complex native proteome, the probe should selectively “highlight” the active sirtuin components. Combined with mass spectrometry based proteomics analysis, this strategy should unveil the functional profile of sirtuins under different physiological and pathological conditions. This will provide information on how abnormal enzyme activity will contribute to disease progression. Furthermore, cell permeable probes will also be employed in cellular imaging study. It will enable the simultaneous detection of functional state and localization and empower the direct analysis of sirtuin function in response to cellular and environmental cues.