NADPH-oxidases (NOXs) purposefully produce reactive-oxygen-species (ROS) and are found in most kingdoms of life. The seven human NOXs are each characterized by a specific expression profile and a fine regulation to spatio-temporally tune ROS concentration in cells and tissues. One of the best known roles for NOXs is in host protection against pathogens but ROS themselves are important second messengers involved in tissue regeneration and the modulation of pathways that induce and sustain cell proliferation. As such, NOXs are attractive pharmacological targets in immunomodulation, fibrosis and cancer. We have studied an extensive number of available NOX inhibitors, with the specific aim to identify bona fide ligands versus ROS-scavenging molecules. Accordingly, we have established a comprehensive platform of biochemical and biophysical assays. Most of the investigated small molecules revealed ROS-scavenging and/or assay-interfering properties to various degrees. A few compounds, however, were also demonstrated to directly engage one or more NOX enzymes. Diphenylene iodonium was found to react with the NOXs’ flavin and heme prosthetic groups to form stable adducts. We also discovered that two compounds, VAS2870 and VAS3947, inhibit NOXs through the covalent alkylation of a cysteine residue. Importantly, the amino acid involved in covalent binding was found to reside in the dehydrogenase domain, where the nicotinamide ring of NADPH is bound. This work can serve as a springboard to guide further development of bona fide ligands with either agonistic or antagonistic properties toward NOXs.

NADPH氧化酶（NOX）故意产生活性氧物种（ROS），在大多数生命王国中都有发现。七种人类NOX各自具有特定的表达特征和精细调节，可在时空上调节细胞和组织中的ROS浓度。NOX的最著名作用之一是对病原体的宿主保护，但是ROS本身是参与组织再生和诱导和维持细胞增殖的途径的调节的重要第二信使。因此，NOX在免疫调节，纤维化和癌症中是有吸引力的药理靶标。我们已经研究了许多可用的NOX抑制剂，其特定目的是鉴定真正的配体与清除ROS的分子。因此，我们建立了生物化学和生物物理测定的综合平台。大多数研究的小分子在不同程度上显示出ROS清除和/或检测干扰的特性。但是，还证明了一些化合物可直接与一种或多种NOX酶结合。发现二亚苯基碘鎓可与NOX的黄素和血红素假体基团反应形成稳定的加合物。我们还发现，两种化合物VAS2870和VAS3947通过半胱氨酸残基的共价烷基化抑制NOX。重要的是，发现参与共价结合的氨基酸位于脱氢酶结构域中，NADPH的烟酰胺环与该结构结合。这项工作可以作为指导善意进一步发展的跳板 对NOX具有激动或拮抗特性的配体。