Generation of Reactive Oxygen Species Mediated by 1‑Hydroxyphenazine, a Virulence Factor of <i>Pseudomonas aeruginosa</i>

1-Hydroxyphenazine (1-HP) is a virulence factor produced by Pseudomonas aeruginosa. In this study, supercoiled plasmid DNA was employed as an analytical tool for the detection of ROS generation mediated by 1-HP. These assays provided evidence that 1-HP, in conjunction with NADPH alone or NADPH and the enzyme NADPH:cytochrome P450 reductase, mediated the production of superoxide radical under physiological conditions. Experiments with murine macrophage RAW264.7 cells and profluorescent ROS probes dichlorodihydrofluorescein or dihydroethidine provided preliminary evidence that 1-HP mediates the generation of intracellular oxidants. Generation of reactive oxygen species may contribute to the virulence properties of 1-HP in P. aeruginosa infections

Electrophilicity of Pyridazine-3-carbonitrile, Pyrimidine-2-carbonitrile, and Pyridine-carbonitrile Derivatives: A Chemical Model To Describe the Formation of Thiazoline Derivatives in Human Liver Microsomes

Certain aromatic nitriles are well-known inhibitors of cysteine proteases. The mode of action of these compounds involves the formation of a reversible or irreversible covalent bond between the nitrile and a thiol group in the active site of the enzyme. However, the reactivity of these aromatic nitrile-substituted heterocycles may lead inadvertently to nonspecific interactions with DNA, protein, glutathione, and other endogenous components, resulting in toxicity and complicating the use of these compounds as therapeutic agents. In the present study, the intrinsic reactivity and associated structure–property relationships of cathepsin K inhibitors featuring substituted pyridazines [6-phenylpyridazine-3-carbonitrile, 6-(4-fluorophenyl)­pyridazine-3-carbonitrile, 6-(4-methoxyphenyl)­pyridazine-3-carbonitrile, 6-<i>p</i>-tolylpyridazine-3-carbonitrile], pyrimidines [5-<i>p</i>-tolylpyrimidine-2-carbonitrile, 5-(4-fluorophenyl)­pyrimidine-2-carbonitrile], and pyridines [5-<i>p</i>-tolylpicolinonitrile and 5-(4-fluorophenyl)­picolinonitrile] were evaluated using a combination of computational and analytical approaches to establish correlations between electrophilicity and levels of metabolites that were formed in glutathione- and <i>N</i>-acetylcysteine-supplemented human liver microsomes. Metabolites that were characterized in this study featured substituted thiazolines that were formed following rearrangements of transient glutathione and <i>N</i>-acetylcysteine conjugates. Peptidases including γ-glutamyltranspeptidase were shown to catalyze the formation of these products, which were formed to lesser extents in the presence of the selective γ-glutamyltranspeptidase inhibitor acivicin and the nonspecific peptidase inhibitors phenylmethylsulfonyl fluoride and aprotinin. Of the chemical series mentioned above, the pyrimidine series was the most susceptible to metabolism to thiazoline-containing products, followed, in order, by the pyridazine and pyridine series. This trend was in keeping with the diminishing electrophilicity across these series, as demonstrated by <i>in silico</i> modeling. Hence, mechanistic insights gained from this study could be used to assist a medicinal chemistry campaign to design cysteine protease inhibitors that were less prone to the formation of covalent adducts

Coupling of an Acyl Migration Prodrug Strategy with Bio-activation To Improve Oral Delivery of the HIV‑1 Protease Inhibitor Atazanavir

HIV-1 protease inhibitors (PIs), which include atazanavir (ATV, <b>1</b>), remain important medicines to treat HIV-1 infection. However, they are characterized by poor oral bioavailability and a need for boosting with a pharmacokinetic enhancer, which results in additional drug–drug interactions that are sometimes difficult to manage. We investigated a chemo-activated, acyl migration-based prodrug design approach to improve the pharmacokinetic profile of <b>1</b> but failed to obtain improved oral bioavailability over dosing the parent drug in rats. This strategy was refined by conjugating the amine with a promoiety designed to undergo bio-activation, as a means of modulating the subsequent chemo-activation. This culminated in a lead prodrug that (1) yielded substantially better oral drug delivery of <b>1</b> when compared to the parent itself, the simple acyl migration-based prodrug, and the corresponding simple l-Val prodrug, (2) acted as a depot which resulted in a sustained release of the parent drug in vivo, and (3) offered the benefit of mitigating the pH-dependent absorption associated with <b>1</b>, thereby potentially reducing the risk of decreased bioavailability with concurrent use of stomach-acid-reducing drugs

Coupling of an Acyl Migration Prodrug Strategy with Bio-activation To Improve Oral Delivery of the HIV‑1 Protease Inhibitor Atazanavir

HIV-1 protease inhibitors (PIs), which include atazanavir (ATV, <b>1</b>), remain important medicines to treat HIV-1 infection. However, they are characterized by poor oral bioavailability and a need for boosting with a pharmacokinetic enhancer, which results in additional drug–drug interactions that are sometimes difficult to manage. We investigated a chemo-activated, acyl migration-based prodrug design approach to improve the pharmacokinetic profile of <b>1</b> but failed to obtain improved oral bioavailability over dosing the parent drug in rats. This strategy was refined by conjugating the amine with a promoiety designed to undergo bio-activation, as a means of modulating the subsequent chemo-activation. This culminated in a lead prodrug that (1) yielded substantially better oral drug delivery of <b>1</b> when compared to the parent itself, the simple acyl migration-based prodrug, and the corresponding simple l-Val prodrug, (2) acted as a depot which resulted in a sustained release of the parent drug in vivo, and (3) offered the benefit of mitigating the pH-dependent absorption associated with <b>1</b>, thereby potentially reducing the risk of decreased bioavailability with concurrent use of stomach-acid-reducing drugs