Functional and inhibitory studies of human lipoxygenase

描述（由申请人提供）：本研究项目的总体目标是确定脂肪加氧酶识别底物和抑制剂的分子机制，并应用这些知识来理解脂肪加氧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及其在细胞生物学中的作用的基本问题仍然存在。我们拟采用动力学、光谱、量热、晶体学、计算机建模、抑制剂筛选和全细胞抑制剂测定等多方面方法，研究大豆15-LO-1和3种hLO（血小板12- hLO、网状细胞15-hLO-1和表皮15-hLO-2）的生物化学和生物学特性。第一个目的是确定LO结合底物的方式以及抑制剂和脂质体如何影响底物特异性。脂氧合酶与多种底物反应，包括花生四烯酸和亚油酸，产生具有多种功能的产物，但催化位点结合这些底物并将其转化为产物的方式尚不清楚。为此，我们提出的实验将定义底物如何结合，什么条件改变其底物特异性以及LO如何从脂质双分子层获得底物。第二个目的是确定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的结构理解主要局限于一些静态晶体结构，这些结构没有说明底物如何进入和停靠到催化位点，或者LO如何从脂质双分子层中招募底物。为此，我们将利用多种结构方法，如蛋白质水解、H/D交换、EPR自旋标记和晶体学，来探索LO的结构。具体来说，我们将研究高糖醛酸盐的结构是否与大豆和兔子的高糖醛酸盐晶体相匹配，底物或抑制剂结合后会发生什么结构变化，以及低糖醛酸盐底物-脂质复合物如何相互作用以实现催化。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活性位点模型，定义底物特异性效应，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强效和选择性抑制剂来完善我们的人体结构模型并改善我们的虚拟对接。将进行广泛的动力学研究，以评估这些化合物的抑制机制，无论是变构还是竞争性或还原性。这个特定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索LO特异性活性的调节，以及LO在前列腺癌和神经元细胞死亡（即中风）中的作用。

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.