Table 2.
Mechanistic and Specificity Validation of Selected Catalytic Subunits of Nicotinamide Adenine Dinucleotide Phosphate Oxidase Inhibitors
| Antioxidant interference | Off-target effects examination | |||||
|---|---|---|---|---|---|---|
| Direct interaction with NOX complex | In vivo application route | Yes | No | Positive | Negative | |
| GKT136901 | + | p.o., i.v. | ONOO− | NO, O2•−, OH. | 135 proteins incl. CYP, channels, XOb; clinical Phase I completed | |
| GKT137831 | + | p.o., i.v. | ||||
| ML171 | No data published | No data published | O2•− (via XO), H2O2 | XO, CNS resident GPCRs, channels, and transportersb | ||
| VAS2870 | + (A) NOX2 | i.t. | O2•− (via XO), ONOO−, OH. | RyR1 receptor S-alkylation | eNOS, XO | |
| VAS3947 | + | O2•− (via XO) | ||||
| Celastrol | + | p.o likely | O2•− (via XO) | XO | ||
| Ebselen | + (A) NOX2 | p.o. | H2O2a | O2•− (via XO) | XO, clinical Phase II completed | |
| Perhexiline | + | p.o. | O2•− (via XO) | XO, approved drug | ||
| ACD084 | + | p.o. likely | O2•− (via mito) | Mitochondrial complex I | ||
| NOX2ds-tat | + (A) NOX1/2 | i.p. | O2•− | XO | ||
| NOXA1ds | + (A) NOX1 | No data published | O2•− (via XO) | XO | ||
Proof for a direct interference with the NADPH oxidase complex is suggested when the compound is active in cell-free assays (+). Cell-free NOX1 and NOX2 assays use isolated membranes of NOX1- or NOX2-expressing cells and the respective recombinant cytosolic binding partners, or they use cytosolic fractions containing these binding partners instead of recombinant proteins. Assembly of the active NADPH oxidase complexes was induced with LiDS, SDS, or arachidonic acid. Compounds marked with (A) were added before assembly of the active NADPH oxidase-1 or -2 complexes as indicated. They may not inhibit NOX activity when added after complex assembly. Inhibition of NOX4 and NOX5 that do not require external binding partners has been determined using isolated membranes of transfected cells or whole cell homogenates. Some compounds have been applied in vivo in animals or humans intravenously (i.v.), intraperitoneally (i.p.), per os (p.o.), or intrathecally (i.t.). For Celastrol and ACD084, oral application is likely, as whole plant extracts are used orally in case of Celastrol and ACD084 is found in edible plants. ROS-scavenging effects of the different compounds were determined by measuring the decay of applied radicals itself or by measuring the interference with ROS derived from xanthine oxidase as a measure of superoxide [O2•− (via XO)] scavenging. Ebselen did not interfere with XO but scavenged H2O2 at micromolar concentrationsa (IC50 ∼80 μM). Off-target effects were either examined systematically using commercially available librariesb or were found by accident. Successful completion of phase I clinical studies is indicated.
CNS, central nervous system; eNOS, endothelial nitric oxide synthase; GPCR, G-protein coupled receptor; LiDS, lithium dodecylsulfate; ROS, reactive oxygen species; SDS, sodium dodecyl sulfate.