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，并导致了各种治疗方法的发现。目前的建议包括四个研究目标，以建立一个更好地了解脂肪氧合酶的酶促机制，并发现和表征选择性底物。1）大豆脂肪氧合酶-1（SLO）的一般机制方案是很好的理解，但个别步骤的分子细节仍然不清楚。在目前的计划中，我们计划使用时间分辨晶体学，停流光谱和稳态动力学来探测活性位点基底，以确定SLO的分子机制（Fe（III-OH-）和氢键结合的分子机制。2）虽然我们对SLO的理解正在迅速成熟，我们对人血小板12-脂氧合酶（12-HLO）和人网织红细胞15-脂氧合酶（15-HLO）的了解仍然有限，尽管它们是治疗性治疗的靶点。因此，我们计划扩大我们的SLO的机制研究，包括人类酶，这将使我们能够回答的基本问题，如何做三个脂肪氧合酶之间的结构差异影响其功能？3)在过去的几年中，我们提出了在SLO和15-HLO中存在变构位点，这可能对脂氧合酶活性的调节至关重要，但其位置仍然未知。因此，我们提出了共结晶脂氧合酶与我们的变构抑制剂和突变的推定结合位点，以建立该网站的位置和结合的限制。我们还将采用抑制剂置换实验和停流荧光动力学来建立变构位点的酶作用及其对催化的影响。4）最后，我们将通过筛选海洋天然产物（MNP）文库（在过去24个月中从约600种提取物中筛选出12种抑制剂）来继续发现新型脂氧合酶抑制剂。我们将最终建立一个抑制剂库，将与生物化学和光谱方法进行研究，以确定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，其将选择性地抑制12-HLO或15-HLO并靶向催化或变构位点。

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.