Pharmacological characterization of the seven human NOX isoforms and their inhibitors.

BACKGROUND: NADPH oxidases (NOX) are a family of flavoenzymes that catalyze the formation of superoxide anion radical (O2•-) and/or hydrogen peroxide (H2O2). As major oxidant generators, NOX are associated with oxidative damage in numerous diseases and represent promising drug targets for several pathologies. Various small molecule NOX inhibitors are used in the literature, but their pharmacological characterization is often incomplete in terms of potency, specificity and mode of action. EXPERIMENTAL APPROACH: We used cell lines expressing high levels of human NOX isoforms (NOX1-5, DUOX1 and 2) to detect NOX-derived O2•- or H2O2 using a variety of specific probes. NOX inhibitory activity of diphenylene iodonium (DPI), apocynin, diapocynin, ebselen, GKT136901 and VAS2870 was tested on NOX isoforms in cellular and membrane assays. Additional assays were used to identify potential off target effects, such as antioxidant activity, interference with assays or acute cytotoxicity. KEY RESULTS: Cells expressing active NOX isoforms formed O2•-, except for DUOX1 and 2, and in all cases activation of NOX isoforms was associated with the detection of extracellular H2O2. Among all molecules tested, DPI elicited dose-dependent inhibition of all isoforms in all assays, however all other molecules tested displayed interesting pharmacological characteristics, but did not meet criteria for bona fide NOX inhibitors. CONCLUSION: Our findings indicate that experimental results obtained with widely used NOX inhibitors must be carefully interpreted and highlight the challenge of developing reliable pharmacological inhibitors of these key molecular targets.

Isolation and Synthesis of Laxaphycin B-Type Peptides: A Case Study and Clues to Their Biosynthesis.

The laxaphyci's B family constitutes a group of five related cyclic lipopeptides isolated from diverse cyanobacteria from all around the world. This group shares a typical structure of 12 amino acids from the l and d series, some of them hydroxylated at the beta position, and all containing a rare beta-amino decanoic acid. Nevertheless, they can be differentiated due to slight variations in the composition of their amino acids, but the configuration of their alpha carbon remains conserved. Here, we provide the synthesis and characterization of new laxaphycin B-type peptides. In doing so we discuss how the synthesis of laxaphycin B and analogues was developed. We also isolate minor acyclic laxaphycins B, which are considered clues to their biosynthesis.

A subset of N-substituted phenothiazines inhibits NADPH oxidases.

NADPH oxidases (NOXs) constitute a family of enzymes generating reactive oxygen species (ROS) and are increasingly recognized as interesting drug targets. Here we investigated the effects of 10 phenothiazine compounds on NOX activity using an extensive panel of assays to measure production of ROS (Amplex red, WST-1, MCLA) and oxygen consumption. Striking differences between highly similar phenothiazines were observed. Two phenothiazines without N-substitution, including ML171, did not inhibit NOX enzymes, but showed assay interference. Introduction of an aliphatic amine chain on the N atom of the phenothiazine B ring (promazine) conferred inhibitory activity toward NOX2, NOX4, and NOX5 but not NOX1 and NOX3. Addition of an electron-attracting substituent in position 2 of the C ring extended the inhibitory activity to NOX1 and NOX3, with thioridazine being the most potent inhibitor. In contrast, the presence of a methylsulfoxide group at the same position (mesoridazine) entirely abolished NOX-inhibitory activity. A cell-free NOX2 assay suggested that inhibition by N-substituted phenothiazines was not due to competition with NADPH. A functional implication of NOX-inhibitory activity of thioridazine was demonstrated by its ability to block redox-dependent myofibroblast differentiation. Our results demonstrate that NOX-inhibitory activity is not a common feature of all antipsychotic phenothiazines and that substitution on the B-ring nitrogen is crucial for the activity, whereas that on the second position of the C ring modulates it. Our findings contribute to a better understanding of NOX pharmacology and might pave the path to discovery of more potent and selective NOX inhibitors.

Synthesis of new steroidal inhibitors of P-glycoprotein-mediated multidrug resistance and biological evaluation on K562/R7 erythroleukemia cells.

A simple route for improving the potency of progesterone as a modulator of P-gp-mediated multidrug resistance was established by esterification or etherification of hydroxylated 5α/ß-pregnane-3,20-dione or 5ß-cholan-3-one precursors. X-ray crystallography of representative 7α-, 11α-, and 17α-(2'R/S)-O-tetrahydropyranyl ether diastereoisomers revealed different combinations of axial-equatorial configurations of the anomeric oxygen. Substantial stimulation of accumulation and chemosensitization was observed on K562/R7 erythroleukemia cells resistant to doxorubicin, especially using 7α,11α-O-disubstituted derivatives of 5α/ß-pregnane-3,20-dione, among which the 5ß-H-7α-benzoyloxy-11α-(2'R)-O-tetrahydropyranyl ether 22a revealed promising properties (accumulation index 2.9, IC50 0.5 µM versus 1.2 and 10.6 µM for progesterone), slightly overcoming those of verapamil and cyclosporin A. Several 7α,12α-O-disubstituted derivatives of 5ß-cholan-3-one proved even more active, especially the 7α-O-methoxymethyl-12α-benzoate 56 (accumulation index 3.8, IC50 0.2 µM). The panel of modulating effects from different O-substitutions at a same position suggests a structural influence of the substituent completing a simple protection against stimulating effects of hydroxyl groups on P-gp-mediated transport.

First total synthesis and stereochemical revision of laxaphycin B and its extension to lyngbyacyclamide A.

The first total synthesis of laxaphycin B was accomplished through stepwise automated Solid Phase Peptide Synthesis (SPPS), leading to the structural revision of its stereochemistry especially with regard to the configuration of one of the two 3-hydroxyleucines of this cyclic dodecapeptide of marine origin. The analogous Lyngbyacyclamide A was obtained by an extension of this synthesis.

NADPH oxidase NOX2 defines a new antagonistic role for reactive oxygen species and cAMP/PKA in the regulation of insulin secretion.

In insulin-secreting cells, expression of NADPH oxidase (NOX), a potent source of ROS, has been reported, along with controversial findings regarding its function. Here, the role of NOXs was investigated: first by expression and cellular localization in mouse and human pancreatic islets, and then by functional studies in islets isolated from Nox isoform-specific knockout mice. Both human and mouse ß-cells express NOX, in particular NOX2. With use of Nox isoform-specific knockout mice, functional analysis revealed Nox2 as the predominant isoform. In human islets, NOX2 colocalized with both insulin granules and endosome/lysosome membranes. Nox2-deficient islets stimulated with 22.8 mmol/L glucose exhibited potentiation of insulin release compared with controls, an effect confirmed with in vitro knockdown of Nox2. The enhanced secretory function in Nox2-deficient islets was associated with both lower superoxide levels and elevated cAMP concentrations. In control islets, GLP-1 and other cAMP inducers suppressed glucose-induced ROS production similarly to Nox2 deficiency. Inhibiting cAMP-dependent protein kinase reduced the secretory response in Nox2-null islets, although not in control islets. This study ascribes a new role for NOX2 in pancreatic ß-cells as negative modulator of the secretory response, reducing cAMP/PKA signaling secondary to ROS generation. Results also show reciprocal inhibition between the cAMP/PKA pathway and ROS.

Progesterone-adenine hybrids as bivalent inhibitors of P-glycoprotein-mediated multidrug efflux: design, synthesis, characterization and biological evaluation.

Bivalent ligands were designed on the basis of the described close proximity of the ATP-site and the putative steroid-binding site of P-glycoprotein (ABCB1). The syntheses of 19 progesterone-adenine hybrids are described. Their abilities to inhibit P-glycoprotein-mediated daunorubicin efflux in K562/R7 human leukemic cells overexpressing P-glycoprotein were evaluated versus progesterone. The hybrid with a hexamethylene linker chain showed the best inhibitory potency. The efficiency of these progesterone-adenine hybrids depends on two main factors: (i) the nature of the linker and (ii) its attachment point on the steroid skeleton.

Solvent-dependent oxidations of 5- and 6-azaindoles to trioxopyrrolopyridines and functionalised azaindoles.

A regioselective synthesis of 4,7-dimethoxy 5- and 6-azaindoles 2 has been achieved, based on the appropriate choice of ortho-directing or ortho-repulsing groups in the formylation of a pyridine ring. Studies on the regioselectivity of the formylation step and on the preparation of azidoacrylate intermediates 4 are described in this paper. The reactivity of the 5- and 6-azaindole structures towards BBr3-mediated selective monodemethylation and oxidative demethylation reactions were also investigated. The regioselectivity of the deprotection was confirmed using a chemical approach. Oxidation reactions were then carried out on either dimethoxy- or hydroxymethoxyazaindoles, in different solvents, using [bis(trifluoroacetoxy)iodo]benzene. In acetonitrile-water, trioxopyrrolopyridines 12 were obtained, whereas the formation of functionalised azaindoles 17 was observed in acetonitrile-methanol. The tautomeric structure of the trioxopyrrolopyridines was proved by X-ray diffraction analysis.