High Throughput and Virtual Screening for Human 12-LO, 15-LO-1, and 15-LO-2 Inhib

描述（由申请人提供）：高通量筛选（HTS）是早期药物发现的主要方法，尚未完全应用于所有人类脂氧合酶（hLO）同工酶，这是许多人类疾病进展中的关键酶。我们目前已经发现了20多种针对人类脂氧合酶的新型抑制剂，主要是利用人工筛选，但最近开发了一种高通量测定方法来提高发现率。我们现在希望将这种HTS技术应用于NIH DPISMR中大约73,000种化合物，以确定我们的三种LO同工酶（12- hLO， 15-hLO-1和15-hLO-2）的特异性抑制剂。我们发现的特定抑制剂将针对特定细胞系进行筛选，以确定它们对前列腺癌细胞和神经细胞（作为缺血模型）的体内活性。与HTS并行，NIH无需额外费用，我们将把完整的HTS库与我们的三种脂氧合酶同源模型（12-hLO， 15-hLO-1和15-hLO-2）对接，并将对接结果与HTS试验发现的结果进行比较。这种比较将使我们既可以改进我们的计算机方法，又可以帮助我们从结构上解释我们将获得的HTS数据。目前，我们已经对47个化合物类黄酮文库和NCI 3104化合物Mech./Div./Nat.Prod显示了该筛选的准确性和可靠性。图书馆。此外，美国国立卫生研究院化学基因组学中心（NCGC）的Anton Simeonov已经成功地用Kalypsys机器人筛选了LOPAC文库，并证明可以准确地找到已知的LO抑制剂。最后，Matt Jacobson教授的建模预测已经从早期的筛选中产生了抑制剂。所有这些数据表明，这些目标不仅可行，而且极有可能成功。脂氧合酶（LO）广泛分布于动植物界，在这些生物的生物学中起着核心作用。在植物中，它们参与发芽和衰老。在人体组织中，有三种主要的人脂氧合酶，5-、12-和15-LO，它们的主要酶学差异在于它们在花生四烯酸（AA）上的位置特异性。脂氧合酶的产物是激素的前体，如白三烯和脂毒素，它们被认为是多种炎症性疾病和癌症的关键信号分子。目前有强有力的证据表明，如果科学家能够发现针对每种特定脂氧合酶同工酶的有效抑制剂，这些抑制剂可能成为有效的细胞探针和治疗LO的方法。该批准申请建议对血小板12-LO、网织红细胞15- lo -1和表皮15- LO-2进行这种治疗。

DESCRIPTION (provided by applicant): High-throughput screening (HTS) is a major method in early drug discovery and has yet to be fully applied to all human lipoxygenase (hLO) isozymes, critical enzymes in the progression of a number of human diseases. We have currently discovered over 20 novel inhibitors against human lipoxygenase primarily utilizing manual screening but have recently developed a high-throughput assay to increase the rate of discovery. We would now like to apply this HTS technology to the approximately 73,000 compounds at the NIH DPISMR to identify specific inhibitors to each of our three LO isozymes (12- hLO, 15-hLO-1 and 15-hLO-2). The specific inhibitors we discover will then be screened against specific cell lines to determine their in-vivo activity against prostate cancer cells and neuronal cells (as ischemia models). In parallel with the HTS, and at no extra cost to the NIH, we will dock the full HTS library against our three lipoxygenase homology models (12-hLO, 15-hLO-1 and 15-hLO-2) and compare the docking hits to those found by the HTS assay. This comparison will allow us to both improve our in silico methods and help us structurally interpret the HTS data we will obtain. Currently, we have shown both the accuracy and the reliability of this screen against a 47 compound flavonoid library and the NCI 3104 compound Mech./Div./Nat.Prod. library. In addition, Anton Simeonov, at NIH Chemical Genomics Center (NCGC), has successfully screened the LOPAC library with the Kalypsys robot and shown known LO inhibitors can be accurately found. Finally, modeling predictions with Prof. Matt Jacobson have already produced inhibitors from earlier screens. All of this data suggests that these aims are not only feasible but highly likely to be successful. Lipoxygenases (LO) are widely distributed throughout the plant and animal kingdoms and play a central role in the biology of these organisms. In plants they are involved in germination and senescence. In human tissue, there are three major human lipoxygenases, 5-, 12-, and 15-LO, whose primary enzymatic difference lies in their positional specificity on arachidonic acid (AA). The products of lipoxygenase are the precursors of hormones, such as leukotrienes and lipoxins, which have been implicated as critical signaling molecules in a variety of inflammatory diseases and cancers. There is currently strong evidence that if scientists can discover potent inhibitors against each specific lipoxygenase isozyme, these may become effective cellular probes and therapeutics against LO. This grant submission proposes to do exactly this for platlet 12-LO, reticulocyte 15-LO-1 and epidermal 15- LO-2.