Fluorescent probes for monitoring myeloperoxidase-derived hypochlorous acid: a comparative study

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.