Chemical synthesis of a reported p47phox/p22phox inhibitor and characterization of its instability and irreproducible activity

Abstract

The nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2) multi-subunit complex is a highly abundant and central source of reactive oxygen species. NOX2 is a key enzyme of the innate immune system involved in antibacterial response, but excessive NOX2 activity is involved in oxidative stress and inflammation in many diseases. Inhibition of NOX2 has great potential as a therapeutic strategy. An intriguing pharmacological approach for inhibiting NOX2 is to target the p47phox subunit and thereby block the protein-protein interaction with p22phox, whereby assembling and activation of NOX2 is prevented. However, the shallow binding pocket of p47phox makes it difficult to develop drug-like p47phox/p22phox inhibitors. Recently, the small molecule LMH001 was reported to inhibit the p47phox/p22phox interaction, reduce endothelial NOX2 activity, and protect mice from angiotensin II-induced vascular oxidative stress. These noteworthy results could have significant impact on the field of NOX2 pharmacology, as specific and efficient inhibitors are scarce. Here, we synthesized and tested LMH001 to have it available as a positive control. We established a robust synthetic route for providing LMH001, but subsequently we experienced that LMH001 is chemically unstable in aqueous buffer. In addition, neither LMH001 nor its breakdown products were able to inhibit the p47phox/p22phox interaction in a non-cellular fluorescence polarization assay. However, LHM001 was a weak inhibitor of NOX2 in a functional cell assay, but with same low potency as one of its breakdown products. These findings question the activity and suggested mechanism of LMH001 and constitute important information for other researchers interested in chemical probes for studying NOX2 biology.

Targeting NOX2 with Bivalent Small-Molecule p47phox–p22phox Inhibitors

“…The recombinant His-tagged human p47phox SH3A–B (p47phox 151–286 ) (UniProtKB P14598) was cloned into pRSET A vector and expressed in Escherichia coli BL21 (DE3) pLysS followed by purification by column chromatography, as previously described. , …”

“…The synthesized compounds were tested for their ability to inhibit the interaction between human p47phox SH3A‑B (human His-tagged p47phox 151–286 ) and the peptide probe Cy5-p22phox 149–162 ; sequence: Ac–C­(Cy5)-KQPPSNPPPRPPAE-NH 2 ; Cy5: cyanine 5) as described previously. , First, a saturation assay was carried out to determine the binding affinity ( K d value) between Cy5-p22phox 149–162 and p47phox SH3A‑B in 1 × HBST buffer (50 mM Hepes, 150 mM NaCl and 0.005% Tween 20, pH = 7.4) using black flat-bottom 384-well plates (Corning Life Sciences, NY) and a total volume of 30 μL/well. Increasing concentrations of p47phox SH3A‑B were added to a fixed concentration (5 nM) of Cy5-p22phox 149–162 in a final DMSO concentration of 2%.…”

“…White solid (0.10 g, 34%). 1 (17). This previously unreported compound was synthesized according to general procedure A using 1,17-dibromo-3,6,9,12,15-pentaoxaheptadecane (0.10 g, 0.25 mmol), 81c (0.15 g, 0.55 mmol), and Cs 2 CO 3 (0.26 g, 0.80 mmol).…”

“…24 However, ebselen is nonspecific in its action and even shown to induce aggregation of p47phox via a covalent mechanism, and LMH001 is chemically very unstable. 17,25 CPP11G (or CPP11H) and C6 have not been shown to directly bind p47phox using biophysical experiments, but this has been demonstrated for two other small-molecule series: Garsi et al recently reported some peptide-derived triproline mimetics developed by structure-based drug discovery, which showed binding to p47phox by surface plasmon resonance (SPR). 26 Before that, we identified 2-aminoquinoline (1) as a fragment hit toward p47phox SH3A−B by fragment-based screening using fluorescence polarization (FP), thermal shift assay (TSA), and SPR.…”

NADPH Oxidases: From Molecular Mechanisms to Current Inhibitors

“…In addition, 19 reduced hypertension and aortic wall inflammation in a mouse model of AngII-induced vascular oxidative stress . However, a following study by Bach and co-workers raised concerns about this compound as a NOX2 inhibitor . Indeed, 19 was shown to be hydrolyzed in standard aqueous buffer and was not able to inhibit the p47 phox /p22 phox interaction.…”