Dysfunctional reactive oxygen species (ROS) signaling is considered an important disease mechanism. Therapeutically, non-selective scavenging of ROS by antioxidants, however, has failed in multiple clinical trials to provide patient benefit. Instead, pharmacological modulation of disease-relevant, enzymatic sources of ROS appears to be an alternative, more promising and meanwhile successfully validated approach. With respect to targets, the family of NADPH oxidases (NOX) stands out as main and dedicated ROS sources. Validation of the different NOX isoforms has been mainly through genetically modified rodent models and is lagging behind in other species. It is unclear whether the different NOX isoforms are sufficiently distinct to allow selective pharmacological modulation. Here we show for five widely used NOX inhibitors that isoform selectivity can be achieved, although individual compound specificity is as yet insufficient. NOX1 was most potently (IC50) targeted by ML171 (0.1 μM); NOX2, by VAS2870 (0.7 μM); NOX4, by M13 (0.01 μM) and NOX5, by ML090 (0.01 μM). In addition, some non-specific antioxidant and assay artefacts may limit the interpretation of data, which included, surprisingly, the clinically advanced NOX inhibitor, GKT136901. In a human ischemic blood-brain barrier hyperpermeability model where genetic target validation is not an option, we provide proof-of-principle that pharmacological target validation for different NOX isoforms is possible by applying an inhibitor panel at IC50 concentrations. Moreover, our findings encourage further lead optimization and development efforts for isoform-selective NOX inhibitors in different indications.

中文翻译：

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异构形式选择性NADPH氧化酶抑制剂面板，用于药理靶标验证。

功能失活的活性氧（ROS）信号被认为是重要的疾病机制。在治疗上，通过抗氧化剂非选择性清除ROS在多项临床试验中未能为患者带来益处。取而代之的是，与疾病相关的酶促ROS的药理学调节似乎是一种替代的，更有希望的方法，同时也已成功验证了该方法。就靶标而言，NADPH氧化酶（NOX）家族是主要和专用ROS来源。主要通过基因改造的啮齿动物模型来验证不同的NOX异构体，而在其他物种中则滞后。尚不清楚不同的NOX亚型是否足够不同以允许选择性药理调节。在这里，我们显示了五种广泛使用的NOX抑制剂，尽管单个化合物的特异性仍不够，但可以实现同工型选择性。ML171（0.1μM）最有效地靶向了NOX1（IC50）；NOX2，通过VAS2870（0.7μM）；M13（0.01μM）的NOX4和ML090（0.01μM）的NOX5。此外，某些非特异性抗氧化剂和分析方法可能会限制数据的解释，令人惊讶的是，这些数据包括临床上先进的NOX抑制剂GKT136901。在人类缺血性血脑屏障通透性过高的模型中，无法进行遗传靶标验证，我们提供的原理证明，通过在IC50浓度下应用抑制剂组，可以对不同的NOX异构体进行药理学靶标验证。而且，