P2X7 receptors are ligand-gated ion channels activated by ATP and play a significant role in cellular immunity. These receptors are considered as a potential therapeutic target for the treatment of multiple inflammatory diseases. In the present work, using spectrofluorimetry, spectrophotometry, Western blotting and ELISA approaches, the ability of 1,4-naphthoquinone thioglucoside derivatives, compounds U-286 and U-548, to inhibit inflammation induced by ATP/LPS in RAW 264.7 cells via P2X7 receptors was demonstrated. It has been established that the selected compounds were able to inhibit ATP-induced calcium influx and the production of reactive oxygen species, and they also exhibited pronounced antioxidant activity in mouse brain homogenate. In addition, compounds U-286 and U-548 decreased the LPS-induced activity of the COX-2 enzyme, the release of pro-inflammatory cytokines TNF-α and IL-1β in RAW 264.7 cells, and significantly protected macrophage cells against the toxic effects of ATP and LPS. This study highlights the use of 1,4-naphthoquinones as promising purinergic P2X7 receptor antagonists with anti-inflammatory activity. Based on the data obtained, studied synthetic 1,4-NQs can be considered as potential scaffolds for the development of new anti-inflammatory and analgesic drugs.

A series of new tetracyclic oxathiine-fused quinone-thioglycoside conjugates based on biologically active 1,4-naphthoquinones and 1-mercapto derivatives of per-O-acetyl d-glucose, d-galactose, d-xylose, and l-arabinose have been synthesized, characterized, and evaluated for their cytotoxic and antimicrobial activities. Six tetracyclic conjugates bearing a hydroxyl group in naphthoquinone core showed high cytotoxic activity with EC₅₀ values in the range of 0.3 to 0.9 μM for various types of cancer and normal cells and no hemolytic activity up to 25 μM. The antimicrobial activity of conjugates was screened against Gram-positive bacteria (Staphylococcus aureus, Bacillus cereus), Gram-negative bacteria (Pseudomonas aeruginosa and Escherichia coli), and fungus Candida albicans by the agar diffusion method. The most effective juglone conjugates with d-xylose or l-arabinose moiety and hydroxyl group at C-7 position of naphthoquinone core at concentration 10 µg/well showed antimicrobial activity comparable with antibiotics vancomicin and gentamicin against Gram-positive bacteria strains. In liquid media, juglone-arabinosidic tetracycles showed highest activity with MIC 6.25 µM. Thus, a positive effect of heterocyclization with mercaptosugars on cytotoxic and antimicrobial activity for group of 1,4-naphthoquinones was shown.

Hyperglycemia may contribute to the progression of carcinomas by triggering epithelial-to-mesenchymal transition (EMT). Some proteostasis systems are involved in metastasis; in this paper, we sought to explore the mechanism of Hsp70 chaperone in EMT. We showed that knockdown of Hsp70 reduced cell migration capacity concomitantly with levels of mRNA of the Slug, Snail, and Twist markers of EMT, in colon cancer cells incubated in high glucose medium. Conversely, treatment of cells with Hsp70 inducer U-133 were found to elevate cell motility, along with the other EMT markers. To prove that inhibiting Hsp70 may reduce EMT efficiency, we treated cells with a CL-43 inhibitor of the HSF1 transcription factor, which lowered Hsp70 and HSF1 content in the control and induced EMT in carcinoma cells. Importantly, CL-43 reduced migration capacity, EMT-linked transcription factors, and increased content of epithelial marker E-cadherin in colon cancer cells of three lines, including one derived from a clinical sample. To prove that Hsp70 chaperone should be targeted when inhibiting the EMT pathway, we treated cancer cells with 2-phenylethynesulfonamide (PES) and demonstrated that the compound inhibited substrate-binding capacity of Hsp70. Furthermore, PES suppressed EMT features, cell motility, and expression of specific transcription factors. In conclusion, the Hsp70 chaperone machine efficiently protects mechanisms of the EMT, and the safe inhibitors of the chaperone are needed to hamper metastasis at its initial stage.

Targeted screening using the MTT cell viability test with a mini-library of natural and synthetic 1,4-naphthoquinones and their derivatives was performed in order to increase the survival of Neuro-2a neuroblastoma cells in in vitro paraquat and 6-hydroxydopamine models of Parkinson’s disease. As a result, 10 compounds were selected that could protect neuronal cells from the cytotoxic effects of both paraquat and 6-hydroxydopamine. The five most active compounds at low concentrations were found to significantly protect the activity of nonspecific esterase from the inhibitory effects of neurotoxins, defend cell biomembranes from lytic destruction in the presence of paraquat and 6-hydroxydopamine, and normalize the cell cycle. The protective effects of these compounds are associated with the suppression of oxidative stress, decreased expression of reactive oxygen species and nitric oxide formation in cells and normalization of mitochondrial function, and restoration of the mitochondrial membrane potential altered by neurotoxins. It was suggested that the neuroprotective activity of the studied 1,4-NQs is attributable to their pronounced antioxidant and free radical scavenging activity and their ability to reduce the amount of reactive oxygen species formed by paraquat and 6-hydroxydopamine action on neuronal cells. The significant correlation between the neuroprotective properties of 1,4-naphthoquinones and Quantitative Structure–Activity Relationship descriptors describing the physicochemical properties of these compounds means that the hydrophobicity, polarity, charge, and shape of the molecules can be of decisive importance in determining the biological activity of studied substances.

The MTS cell viability test was used to screen a mini library of natural and synthetic 1,4-naphthoquinone derivatives (1,4-NQs) from marine sources. This screening identified two highly effective compounds, U-443 and U-573, which showed potential in protecting Neuro-2a neuroblastoma cells from the toxic effects of rotenone in an in vitro model of neurotoxicity. The selected 1,4-NQs demonstrated the capability to reduce oxidative stress by decreasing the levels of reactive oxygen species (ROS) and nitric oxide (NO) in Neuro-2a neuroblastoma cells and RAW 264.7 macrophage cells and displayed significant antioxidant properties in mouse brain homogenate. Normal mitochondrial function was restored and the mitochondrial membrane potential was also regained by 1,4-NQs after exposure to neurotoxins. Furthermore, at low concentrations, these compounds were found to significantly reduce levels of proinflammatory cytokines TNF and IL-1β and notably inhibit the activity of cyclooxygenase-2 (COX-2) in RAW 264.7 macrophages. The results of docking studies showed that the 1,4-NQs were bound to the active site of COX-2, analogically to a known inhibitor of this enzyme, SC-558. Both substances significantly improved the behavioral changes in female CD1 mice with rotenone-induced early stage of Parkinson’s disease (PD) in vivo. It is proposed that the 1,4-NQs, U-443 and U-573, can protect neurons and microglia through their potent anti-ROS and anti-inflammatory activities.