Xanthine oxidase (XO) is a flavoprotein catalysing the oxidation of hypoxanthine to xanthine and then to uric acid, while simultaneously producing reactive oxygen species. Altered functions of XO may lead to severe pathological diseases, including gout-causing hyperuricemia and oxidative damage of tissues. These findings prompted research studies aimed at targeting the activity of this crucial enzyme. During the course of a virtual screening study aimed at the discovery of novel inhibitors targeting another oxidoreductase, superoxide dismutase, we identified four compounds with non-purine-like structures, namely ALS-1, -8, -15 and -28, that were capable of causing direct inhibition of XO. The kinetic studies of their inhibition mechanism allowed a definition of these compounds as competitive inhibitors of XO. The most potent molecule was ALS-28 (Ki 2.7 ± 1.5 µM), followed by ALS-8 (Ki 4.5 ± 1.5 µM) and by the less potent ALS-15 (Ki 23 ± 9 µM) and ALS-1 (Ki 41 ± 14 µM). Docking studies shed light on the molecular basis of the inhibitory activity of ALS-28, which hinders the enzyme cavity channel for substrate entry consistently with the competitive mechanism observed in kinetic studies. Moreover, the structural features emerging from the docked poses of ALS-8, -15 and -1 may explain the lower inhibition power with respect to ALS-28. All these structurally unrelated compounds represent valuable candidates for further elaboration into promising lead compounds.

Glutathione (GSH) biosynthesis occurs through two ATP-dependent reactions, usually involving distinct enzymes; in the second step of this process, catalysed by glutathione synthetase (GshB), GSH is formed from γ-glutamylcysteine and glycine. A recombinant form of GshB from the cold-adapted source Pseudoalteromonas haloplanktis (rPhGshB) was purified and characterised. The enzyme formed a disulfide adduct with β-mercaptoethanol, when purified in the presence of this reducing agent. The homotetrameric form of rPhGshB observed at high protein concentration disassembled into two homodimers at low concentration. A new method for directly determining the rPhGshB activity was developed, based on [γ-³²P]ATP hydrolysis coupled to the GSH synthesis. The ATPase activity required the presence of both γ-glutamylcysteine and glycine and its optimum was reached in the 7.4–8.6 pH range; a divalent cation was absolutely required for the activity, whereas monovalent cations were dispensable. rPhGshB was active at low temperatures and had a similar affinity for ATP (K_m 0.26 mM) and γ-glutamylcysteine (K_m 0.25 mM); a lower affinity was measured for glycine (K_m 0.75 mM). The oxidised form of glutathione (GSSG) acted as an irreversible inhibitor of rPhGshB (K_i 10.7 mM) and formed disulfide adducts with the enzyme. rPhGshB displayed a great temperature-dependent increase in its activity with an unusually high value of energy of activation (75 kJ mol⁻¹) for a psychrophilic enzyme. The enzyme was moderately thermostable, its half inactivation temperature being 50.5 °C after 10 min exposure. The energy of activation of the heat inactivation process was 208 kJ mol⁻¹. To our knowledge, this is the first contribution to the characterization of a GshB from cold-adapted sources.

The microaerophile Streptococcus mutans, the main microaerophile responsible for the development of dental plaque, has a single cambialistic superoxide dismutase (SmSOD) for its protection against reactive oxygen species. In order to discover novel inhibitors of SmSOD, possibly interfering with the biofilm formation by this pathogen, a virtual screening study was realised using the available 3D-structure of SmSOD. Among the selected molecules, compound ALS-31 was capable of inhibiting SmSOD with an IC₅₀ value of 159 µM. Its inhibition power was affected by the Fe/Mn ratio in the active site of SmSOD. Furthermore, ALS-31 also inhibited the activity of other SODs. Gel-filtration of SmSOD in the presence of ALS-31 showed that the compound provoked the dissociation of the SmSOD homodimer in two monomers, thus compromising the catalytic activity of the enzyme. A docking model, showing the binding mode of ALS-31 at the dimer interface of SmSOD, is presented. Cell viability of the fibroblast cell line BJ5-ta was not affected up to 100 µM ALS-31. A preliminary lead optimization program allowed the identification of one derivative, ALS-31-9, endowed with a 2.5-fold improved inhibition power. Interestingly, below this concentration, planktonic growth and biofilm formation of S. mutans cultures were inhibited by ALS-31, and even more by its derivative, thus opening the perspective of future drug design studies to fight against dental caries.

The dual phosphatases CDC25 are involved in cell cycle regulation and overexpressed in many tumours, including melanoma. CDC25 is a promising target for discovering anticancer drugs, and several studies focussed on characterisation of quinonoid CDC25 inhibitors, frequently causing undesired side toxic effects. Previous work described an optimisation of the inhibition properties by naphthylphenylamine (NPA) derivatives of NSC28620, a nonquinonoid CDC25 inhibitor. Now, the CDC25B•inhibitor interaction was investigated through fluorescence studies, shedding light on the different inhibition mechanism exerted by NPA derivatives. Among the molecular processes, mediating the specific and high cytotoxicity of one NPA derivative in melanoma cells, we observed decrease of phosphoAkt, increase of p53, reduction of CDC25 forms, cytochrome c cytosolic translocation and increase of caspase activity, that lead to the activation of an apoptotic programme. A basic knowledge on CDC25 inhibitors is relevant for discovering potent bioactive molecules, to be used as anticancer agents against the highly aggressive melanoma. Graphical Abstract

Gastric cancer remains a leading cause of cancer-related mortality worldwide. Citrus fruits are rich in polyphenols, exerting antioxidant and chemo-preventive activities, and lemon peel represents a valuable source of such bioactive compounds. Previous studies showed that Citrus limon peel extracts (LPE) inhibited the activity of some enzymes of the antioxidant system and reduced the interleukin-6-dependent invasiveness of gastric and colon cancer cells. In the present study, we have investigated the effects of LPE on the human gastric adenocarcinoma AGS and MKN-28 cells and on the activity of a crucial redox enzyme, catalase (CAT). Indeed, LPE significantly reduced the cell viability and clonogenic potential of the gastric cancer cells and induced morphological changes indicative of cytotoxicity. Moreover, LPE modulated the intracellular redox homeostasis by decreasing levels of the hydrogen peroxide-related reactive oxygen species (ROS) while increasing those of superoxide anions and decreasing levels of superoxide dismutases (SODs). Western blotting analysis revealed that LPE downregulated CAT, SOD-1, SOD-2, and monoamine oxidase A (MAO-A) protein expression level in both cell lines. Finally, the extract inhibited CAT activity in a dose-dependent manner (IC₅₀ = 0.008 ± 0.003 mg/mL; K_i = 0.012 ± 0.002 mg/mL). These findings indicate that LPE exerts cytotoxic and redox-modulating effects through the inhibition of antioxidant enzymes and the alteration of ROS balance. Therefore, the agro-industrial by-product LPE could be considered as a promising natural source of polyphenolic compounds with potential applications in the prevention and therapy of gastric cancer.