Functional and Inhibitory Studies of Human Lipoxygenase

描述（由申请人提供）：脂氧合酶是哮喘、心脏病和癌症的关键生物学参与者。它们是药物靶点，并导致各种治疗方法的发现。目前的建议包括四个研究目标，以建立一个更好的理解脂氧合酶的酶促机制，并发现和表征选择性抑制剂。1)大豆脂氧合酶- 1 （SLO）的一般机制已被了解，但各个步骤的分子细节尚不清楚。在目前的建议中，我们计划利用时间分辨晶体学，停止流动光谱和稳态动力学来确定SLO的分子机制，以探测活性位点碱（Fe(III-OH-)）和氢键重排的分子机制。2)尽管我们对SLO的认识正在迅速成熟，但我们对人血小板12-脂氧合酶（12-HLO）和人网织细胞15-脂氧合酶（15-HLO）的认识仍然有限，尽管它们是治疗性治疗的靶点。因此，我们计划将SLO的机制研究扩展到包括人类酶，这将使我们能够回答基本问题；三种脂氧合酶的结构差异如何影响它们的功能？3)在过去的几年中，我们已经提出在SLO和15-HLO中存在一个变构位点，这可能对脂氧合酶活性的调节至关重要，但其位置尚不清楚。因此，我们建议将脂氧合酶与我们的变构抑制剂共结晶，并使假定的结合位点发生突变，以确定该位点的位置和结合约束。我们还将采用抑制剂置换实验和停流荧光动力学来确定变构位点的酶促作用及其对催化的影响。4)最后，我们将继续通过筛选海洋天然产物（MNP）文库（过去24个月从约600种提取物中筛选出12种抑制剂）来发现新的脂氧合酶抑制剂。我们将最终建立一个抑制剂文库，将用生化和光谱方法来研究化合物如何结合和抑制脂氧合酶。这将使我们能够绘制结构趋势并设计下一代抑制剂，这些抑制剂将选择性地抑制12-HLO或15-HLO，并针对催化或变构位点。

DESCRIPTION (provided by applicant): Lipoxygenases are implicated as key biological players in asthma, heart disease and cancer. They are pharmaceutical targets and have lead to the discovery of various therapeutic treatments. The current proposal encompasses four aims of study in order to establish a better understanding of the enzymatic mechanism for lipoxygenase and to discover and characterize selective inhibitors.1) The general mechanistic scheme for soybean lipoxygenase- 1 (SLO) is well understood but the molecular details of the individual steps remain unclear. In the current proposal, we plan to determine the molecular mechanism of SLO using time-resolved crystallography, stopped-flow spectroscopy and steady-state kinetics to probe the active site base (Fe(III-OH-) and the molecular mechanism for hydrogen bond rearrangement.2) Although our understanding of SLO is maturing rapidly, our knowledge of the human platelet 12-lipoxygenase (12-HLO) and human reticulocyte 1 5-lipoxygenase (15-HLO) remains limited, even though they are the targets for therapeutic treatment. We therefore plan to extend our mechanistic studies of SLO to include the human enzymes which will allow us to answer the fundamental question; how do the structural differences between the three lipoxygenases affect their function?3) Over the past several years, we have proposed the presence of an allosteric site in both SLO and 15-HLO, which could be critical to the regulation of lipoxygenase activity, however its location remains unknown. We have therefore proposed to co-crystallize lipoxygenase with our allosteric inhibitors and mutate the putative binding site to establish the location and binding constraints of the site. We shall also employ inhibitor displacement experiments and stopped-flow fluorescence kinetics to establish the enzymatic role of the allosteric site and its effect on catalysis.4) Finally, we shall continue to discover novel lipoxygenase inhibitors by screening a marine natural products (MNP) library (12 inhibitors from about 6OO extracts in the last 24 months). We will eventually establish an inhibitor library that will be investigated with biochemical and spectroscopic methods to determine how the compounds bind and inhibit lipoxygenase. This will allow us to draw structural trends and design the next generation of inhibitors, which will selectively inhibit 12-HLO or 15-HLO and target either the catalytic or allosteric sites.