Abstract：
MPO-derived oxidants including HOCl contribute to tissue damage and the initiation and propagation of inflammatory diseases. The search for small molecule inhibitors of myeloperoxidase, as molecular tools and potential drugs, requires the application of high throughput screening assays based on monitoring the activity of myeloperoxidase. In this study, we have compared three classes of fluorescent probes for monitoring myeloperoxidase-derived hypochlorous acid, including boronate-, aminophenyl- and thiol-based fluorogenic probes and we show that all three classes of probes are suitable for this purpose. However, probes based on the coumarin fluorophore turned out to be not reliable indicators of the inhibitors\' potency. We have also determined the rate constants of the reaction between HOCl and the probes and they are equal to 1.8 × 104 M-1s-1 for coumarin boronic acid (CBA), 1.1 × 104 M-1s-1 for fluorescein based boronic acid (FLBA), 3.1 × 104 M-1s-1 for 7-(p-aminophenyl)-coumarin (APC), 1.6 × 104 M-1s-1 for 3\'-(p-aminophenyl)-fluorescein (APF), and 1 × 107 M-1s-1 for 4-thiomorpholino-7-nitrobenz-2-oxa-1,3-diazole (NBD-TM). The high reaction rate constant of NBD-TM with HOCl makes this probe the most reliable tool to monitor HOCl formation in the presence of compounds showing HOCl-scavenging activity.
摘要：
包括HOCl的MPO衍生的氧化剂有助于组织损伤以及炎性疾病的引发和传播。寻找髓过氧化物酶的小分子抑制剂，作为分子工具和潜在的药物，需要应用基于监测髓过氧化物酶活性的高通量筛选试验。在这项研究中,我们比较了三类荧光探针监测髓过氧化物酶衍生的次氯酸，包括硼酸盐-，基于氨基苯基和巯基的荧光探针，我们表明所有三类探针都适用于此目的。然而,基于香豆素荧光团的探针被证明不是抑制剂效力的可靠指标。我们还确定了HOCl与探针之间反应的速率常数，它们等于香豆素硼酸（CBA）的1.8×104M-1s-1，1.1×104M-1s-1用于荧光素基硼酸（FLBA），7-（对氨基苯基）香豆素（APC）的3.1×104M-1s-1，1.6×104M-1s-1，用于3'-（对氨基苯基）-荧光素（APF），和1×107M-1s-1的4-硫代吗啉-7-硝基苯-2-氧杂-1,3-二唑（NBD-TM）。NBD-TM与HOCl的高反应速率常数使该探针成为在显示HOCl清除活性的化合物存在下监测HOCl形成的最可靠工具。
