Functional and inhibitory studies of human lipoxygenase

描述(申请人提供)：本研究计划的主要目标是确定脂氧合酶识别底物和抑制物的分子机制，并将这一知识应用于了解脂氧合酶在细胞生物学和人类疾病中的作用。人类脂氧合酶(HLO)同工酶是重要的治疗靶点，因为它们与许多人类疾病有关，然而，关于它们的生物化学和它们在细胞生物学中的作用仍然存在基本问题。本文拟从动力学、光谱、量热、结晶学、计算机模拟、抑制剂筛选和全细胞抑制剂分析等多方面研究大豆15-LO-1和三种HLO：血小板12-HLO、网织红细胞15-HLO-1和表皮15-HLO-2的生化和生物学性质。第一个目标是确定LO与底物结合的方式以及抑制剂和脂质体如何影响底物特异性。脂肪氧合酶与包括花生四烯酸和亚油酸在内的各种底物反应，产生具有广泛功能的产物，但催化部位如何不同地结合这些底物并将它们转化为产物仍不清楚。为此，我们提出了一些实验，这些实验将定义底物是如何结合的，什么条件改变了它的底物专一性，以及LO如何从脂质双层中获得底物。第二个目标是确定LO的溶液结构以及抑制剂和脂质体如何影响变化。我们目前对LO的结构理解很大程度上局限于一些静态的晶体结构，对底物如何进入并对接到催化位置，或者LO如何从脂质双层中招募底物没有任何影响。为此，我们将利用各种结构方法，如蛋白质分解、H/D交换、EPR自旋标记和结晶学，来探索LO的结构。具体地说，我们将调查HLOS的结构是否与结晶大豆和兔LOS的结构匹配，底物或抑制剂结合时发生了什么结构变化，以及LO-底物-脂质复合体如何相互作用实现催化。第三个目标是利用我们发现的抑制剂来优化我们的虚拟筛选，完善人类LO活性部位模型，定义底物特异性效应，并探索LO在人类疾病中的细胞作用。我们建议利用我们之前从高通量筛选中发现的有效和选择性的抑制剂，来完善我们的人体结构模型，并改进我们的虚拟对接。将进行广泛的动力学研究，以评估这些化合物的抑制机制，无论是变构的、竞争的或还原的。这一特定的抑制剂家族将构成一个工具箱，使我们能够通过变构结合来探讨LO的特定活性的调节，以及LO在前列腺癌和神经细胞死亡(即中风)中的作用。 脂氧合酶(LO)是一种与多种人类疾病有关的关键酶，如癌症、中风和心脏病。这项应用的目的是发现和表征LO的抑制剂，希望了解更多关于LO的生化和细胞机制，并开发可能的治疗方法。

DESCRIPTION (provided by applicant): The overarching goal of this research program is to determine the molecular mechanism of both substrate and inhibitor recognition by lipoxygenase and apply this knowledge to understanding the role of lipoxygenase in cellular biology and human disease. Human lipoxygenase (hLO) isozymes are critical therapeutic targets because they are involved in numerous human diseases and yet, fundamental questions remain regarding their biochemistry and their role in cellular biology. We propose to investigate both the biochemical and biological properties of soybean 15-LO-1 and three hLOs, platelet 12- hLO, reticulocyte 15-hLO-1 and epidermal 15-hLO-2, using a multi-faceted approach, including kinetics, spectroscopy, calorimetry, crystallography, computer modeling, inhibitor screening and whole cell inhibitor assays. The first aim is to determine the manner in which LO binds substrate and how inhibitors and liposomes affect the substrate specificity. Lipoxygenases react with a variety of substrates, including arachidonic acid and linoleic acid, producing products with a wide range of functions but the manner in which the catalytic site differentially binds these substrates and converts them to products remains unclear. In this aim, we propose experiments which will define how the substrate is bound, what conditions change its substrate specificity and how LO obtains its substrate from the lipid bilayer. The second aim is to determine the solution structures of LO and how inhibitors and liposomes affect change. Our current structural understanding of LO is largely limited to a few static crystal structures that say nothing of how the substrate enters and docks to the catalytic site, or how LO recruits substrate from the lipid bilayer. In this aim, we shall utilize a variety of structural methods, such as proteolysis, H/D exchange, EPR spin labeling, and crystallography, to probe the structure of LO. Specifically, we will investigate whether the structures of the hLOs match that of the crystallized soybean and rabbit LOs, what structural changes occur upon substrate or inhibitor binding and how the LO-substrate-lipid complex interacts to achieve catalysis. The third aim is to utilize our discovered inhibitors to optimize our virtual screen, perfect human LO active site models, define the substrate specificity effect, and probe the cellular role of LO in human disease. We propose to utilize our previously discovered potent and selective inhibitors from our high throughput screen, to perfect our human structural model and improve our virtual docking. Extensive kinetic studies will be performed to assess the inhibitory mechanism of these compounds, be it allosteric or competitive or reductive. This family of specific inhibitors will then constitute a tool box which will allow us to probe the regulation of LO's specific activity via allosteric binding, and the role of LO in both prostate cancer and neuronal cell death (i.e. stroke). Lipoxygenase (LO) is a critical enzyme involved in numerous human diseases, such as cancer, stroke and heart disease. The goal of this application is to discover and characterize inhibitors to LO with the hope of learning more about its biochemical and cellular mechanism and developing possible therapeutics.