Therapeutic potential of metal ions for COVID-19: insights from the papain-like protease of SARS-CoV-2.

Coronaviruses have been responsible for multiple challenging global pandemics, including coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Papain-like protease (PLpro), one of two cysteine proteases responsible for the maturation and infectivity of SARS-CoV-2, processes and liberates functional proteins from the viral polyproteins and cleaves ubiquitin and ISG15 modifications to inhibit innate immune sensing. Consequently, PLpro is an attractive target for developing COVID-19 therapies. PLpro contains a zinc-finger domain important for substrate binding and structural stability. However, the impact of metal ions on the activity and biophysical properties of SARS-CoV-2 PLpro has not been comprehensively studied. Here, we assessed the impacts of metal ions on the catalytic activity of PLpro. Zinc had the largest inhibitory effect on PLpro, followed by manganese. Calcium, magnesium, and iron had smaller or no effects on PLpro activity. EDTA at a concentration of 0.5 mM was essential for PLpro activity, likely by chelating trace metals that inhibit PLpro. IC50 values for ZnCl2, ZnSO4, and MnCl2 of 0.42 ± 0.02 mM, 0.35 ± 0.01 mM, and 2.6 ± 0.3 mM were obtained in the presence of 0.5 mM EDTA; in the absence of EDTA, the estimated IC50 of ZnCl2 was 14 µM. Tryptophan intrinsic fluorescence analysis confirmed the binding of zinc and manganese to PLpro, and differential scanning calorimetry revealed that zinc but not manganese reduced ΔHcal of PLpro. The results of this study provide a reference for further work targeting PLpro to prevent and treat COVID-19.

Baccharin and p-coumaric acid from green propolis mitigate inflammation by modulating the production of cytokines and eicosanoids.

ETHNOPHARMACOLOGICAL RELEVANCE: Green propolis is produced by Apis mellifera honeybees using Baccharis dracunculifolia D.C. (Asteraceae) as substrate. This Southern Brazilian native plant and green propolis have been used in traditional medicine to treat gastric diseases, inflammation and liver disorders. AIM OF THE STUDY: Investigate the effects of baccharin (Bac) or p-coumaric acid (pCA) isolated from B. dracunculifolia D.C. (Asteraceae) over the inflammation induced by lipopolysaccharide (LPS) in vivo. MATERIALS AND METHODS: Inflammation was induced by LPS injection into air-pouches in mice, which were subsequently treated with Bac or pCA. Lavage fluid was collected from air pouches for the quantification of cellular influx via microscopy, and quantification of inflammatory mediators via colorimetric methods, ELISA and liquid chromatography-tandem mass spectrometry (LC-MS/MS). RESULTS: LPS-induced inflammation increased cellular influx and increased the levels of parameters related to vascular permeability and edema formation, such as nitric oxide (NO) and protein extravasation. Moreover, LPS increased the levels of cytokines and eicosanoids in the air-pouches. Importantly, both Bac and pCA suppressed the infiltration of neutrophils, production of NO and protein extravasation. Notably, the compounds promote differential regulation of cytokine and eicosanoid production. CONCLUSIONS: Our results suggest that Bac from green propolis directly affects inflammation by inhibiting the production of cytokines and eicosanoids, while pCA may exert direct, but also indirect effects on inflammation by stimulating the production of regulatory effectors such as interkeukin-10 in vivo.

Green Propolis Compounds (Baccarin and p-Coumaric Acid) Show Beneficial Effects in Mice for Melanoma Induced by B16f10.

Background: Cutaneous melanoma is the most aggressive form of skin cancer, with the worst prognosis, and it affects a younger population than most cancers. The high metastatic index, in more advanced stages, and the high aggressiveness decrease the effectiveness of currently used therapies, such as surgical removal, radiotherapy, cryotherapy, and chemotherapy, used alone or in combination. Based on these disadvantages, research focused on alternative medicine offers great potential for therapeutic innovation. Medicinal plants represent a remarkable source of compounds for the treatment of various diseases. Methods: In this study, we investigated the tumoral behavior of melanoma under treatment with the compounds baccarin and p-coumaric acid, extracted from green propolis, in mice inoculated with B16F10 cells for 26 days. Results: A significant modulation in the number of inflammatory cells recruited to the tumor region and blood in the groups treated with the compounds was observed. In addition, a significant reduction in the amount of blood vessels and mitosis in the neoplastic area was noticed. Conclusions: Through our research, we confirmed that baccarin and coumaric acid, isolated substances from Brazilian green propolis, have a promising anticarcinogenic potential to be explored for the development of new antitumor agents, adhering to the trend of drugs with greater tolerance and biological effectiveness.

Oligomerization of the cysteinyl-rich oligopeptidase EP24.15 is triggered by S-glutathionylation.

Thimet oligopeptidase (EC 3.4.24.15; EP24.15) is a thiol-rich metallopeptidase ubiquitously distributed in mammalian tissues and involved in oligopeptide metabolism both within and outside cells. Fifteen Cys residues are present in the rat EP24.15 protein, seven are solvent accessible, and two are found inside the catalytic site cleft; no intraprotein disulfide is described. In the present investigation, we show that mammalian immunoprecipitated EP24.15 is S-glutathionylated. In vitro EP24.15 S-glutathionylation was demonstrated by the incubation of bacterial recombinant EP24.15 with oxidized glutathione concentration as low as 10 microM. The in vitro S-glutathionylation of EP24.15 was responsible for its oxidative oligomerization to dimer and trimer complexes. EP24.15 immunoprecipitated from cells submitted to oxidative challenge showed increased trimeric forms and decreased S-glutathionylation compared to immunoprecipitated protein from control cells. Our present data also show that EP24.15 maximal enzymatic activity is maintained by partial S-glutathionylation, a mechanism that apparently regulates the protein oligomerization. Present results raise the possibility of an unconventional property of protein S-glutathionylation, inducing oligomerization by interprotein thiol-disulfide exchange.

Oligomerization of the cysteinyl-rich oligopeptidase EP24.15 is triggered by S-glutathionylation

Thimet oligopeptidase (EC 3.4.24.15; EP24.15) is a thiol-rich metallopeptidase ubiquitously distributed in mammalian tissues and involved inoligopeptide metabolism both within and outside cells. Fifteen Cys residues are present in the rat EP24.15 protein, seven are solvent accessible, andtwo are found inside the catalytic site cleft; no intraprotein disulfide is described. In the present investigation, we show that mammalianimmunoprecipitated EP24.15 is S-glutathionylated. In vitro EP24.15 S-glutathionylation was demonstrated by the incubation of bacterialrecombinant EP24.15 with oxidized glutathione concentration as low as 10 ìM. The in vitro S-glutathionylation of EP24.15 was responsible for itsoxidative oligomerization to dimer and trimer complexes. EP24.15 immunoprecipitated from cells submitted to oxidative challenge showedincreased trimeric forms and decreased S-glutathionylation compared to immunoprecipitated protein from control cells. Our present data also showthat EP24.15 maximal enzymatic activity is maintained by partial S-glutathionylation, a mechanism that apparently regulates the proteinoligomerization. Present results raise the possibility of an unconventional property of protein S-glutathionylation, inducing oligomerization byinterprotein thiol–disulfide exchange.