Chemistry and Biochemistry Department, University of California, Santa Cruz, CA 95064, USA.
Bioorg Med Chem. 2013 Jul 1;21(13):3894-9. doi: 10.1016/j.bmc.2013.04.016. Epub 2013 Apr 18.
Understanding the mode of action for lipoxygenase (LOX) inhibitors is critical to determining their efficacy in the cell. The pseudoperoxidase assay is an important tool for establishing if a LOX inhibitor is reductive in nature, however, there have been difficulties identifying the proper conditions for each of the many human LOX isozymes. In the current paper, both the 234 nM decomposition (UV) and iron-xylenol orange (XO) assays are shown to be effective methods of detecting pseudoperoxidase activity for 5-LOX, 12-LOX, 15-LOX-1 and 15-LOX-2, but only if 13-(S)-HPODE is used as the hydroperoxide substrate. The AA products, 12-(S)-HPETE and 15-(S)-HPETE, are not consistent hydroperoxide substrates since they undergo a competing transformation to the di-HETE products. Utilizing the above conditions, the selective 12-LOX and 15-LOX-1 inhibitors, probes for diabetes, stroke and asthma, are characterized for their inhibitory nature. Interestingly, ascorbic acid also supports the pseudoperoxidase assay, suggesting that it may have a role in maintaining the inactive ferrous form of LOX in the cell. In addition, it is observed that nordihydroguaiaretic acid (NDGA), a known reductive LOX inhibitor, appears to generate radical species during the pseudoperoxidase assay, which are potent inhibitors against the human LOX isozymes, producing a negative pseudoperoxidase result. Therefore, inhibitors that do not support the pseudoperoxidase assay with the human LOX isozymes, should also be investigated for rapid inactivation, to clarify the negative pseudoperoxidase result.
了解脂氧合酶 (LOX) 抑制剂的作用模式对于确定其在细胞中的疗效至关重要。假过氧化物酶测定法是确定 LOX 抑制剂是否具有还原性质的重要工具，但是，对于许多人类 LOX 同工酶中的每一种，都存在确定适当条件的困难。在当前的论文中，均显示 234 nM 分解（UV）和铁-二甲氧基苯酚（XO）测定法是检测 5-LOX、12-LOX、15-LOX-1 和 15-LOX-2 的假过氧化物酶活性的有效方法，但是前提是使用 13-（S）-HPODE 作为氢过氧化物底物。AA 产物 12-（S）-HPETE 和 15-（S）-HPETE 不是一致的氢过氧化物底物，因为它们会转化为竞争产物二-HETE。利用上述条件，可对选择性 12-LOX 和 15-LOX-1 抑制剂（糖尿病、中风和哮喘的探针）进行抑制性质的特征分析。有趣的是，抗坏血酸也支持假过氧化物酶测定法，表明它可能在维持细胞中 LOX 的非活性亚铁形式方面发挥作用。此外，还观察到，一种已知的还原型 LOX 抑制剂，去甲二氢愈创木酸（NDGA），在假过氧化物酶测定法中似乎会产生自由基，这些自由基是针对人类 LOX 同工酶的有效抑制剂，产生负的假过氧化物酶结果。因此，对于那些不支持人类 LOX 同工酶的假过氧化物酶测定法的抑制剂，也应该进行快速失活的研究，以阐明负的假过氧化物酶结果。
