Mebendazole targets essential proteins in glucose metabolism leading gastric cancer cells to death.

Gastric cancer (GC) is among the most-diagnosed and deadly malignancies worldwide. Deregulation in cellular bioenergetics is a hallmark of cancer. Based on the importance of metabolic reprogramming for the development and cancer progression, inhibitors of cell metabolism have been studied as potential candidates for chemotherapy in oncology. Mebendazole (MBZ), an antihelminthic approved by FDA, has shown antitumoral activity against cancer cell lines. However, its potential in the modulation of tumoral metabolism remains unclear. Results evidenced that the antitumoral and cytotoxic mechanism of MBZ in GC cells is related to the modulation of the mRNA expression of glycolic targets SLC2A1, HK1, GAPDH, and LDHA. Moreover, in silico analysis has shown that these genes are overexpressed in GC samples, and this increase in expression is related to decreased overall survival rates. Molecular docking revealed that MBZ modifies the protein structure of these targets, which may lead to changes in their protein function. In vitro studies also showed that MBZ induces alterations in glucose uptake, LDH's enzymatic activity, and ATP production. Furthermore, MBZ induced morphologic and intracellular alterations typical of the apoptotic cell death pathway. Thus, this data indicated that the cytotoxic mechanism of MBZ is related to an initial modulation of the tumoral metabolism in the GC cell line. Altogether, our results provide more evidence about the antitumoral mechanism of action of MBZ towards GC cells and reveal metabolic reprogramming as a potential area in the discovery of new pharmacological targets for GC chemotherapy.

Vasorelaxant effect of Alpinia zerumbet's essential oil on rat resistance artery involves blocking of calcium mobilization.

Alpinia zerumbet is a plant from the Zingiberaceae family, popularly used for hypertension treatment. Several studies have demonstrated Alpinia zerumbet vasodilator effect on conductance vessels but not on resistance vessels. Thereby, the aim of this study was to verify the vasodilator effect of the essential oil of Alpinia zerumbet (EOAz) on isolated rat resistance arteries and characterize its mechanism of action. Therefore, the effect of EOAz (3 to 3000 µg/mL) was verified in second-order branches of the mesenteric artery (SOBMA) pre-contracted by KCl and U46619. To study the mechanism of action, the influence of several inhibitors (TEA, 4-AP, Glibenclamide, Atropine, L-NAME, ODQ and indomethacin) on the vasodilator effect of EOAz was evaluated. Some protocols were also performed aiming to study the effect of EOAz on Ca2+ influx and release from intracellular storage. Furthermore, the binding energy of the main constituents with calcium channels were evaluated by molecular docking. Results showed an endothelium-independent vasorelaxant effect of EOAz on SOBMA, and only ODQ and L-NAME produced significant alteration on its pEC50. Regarding the calcium assays, contraction reduction caused by incubation with EOAz was observed in all three protocols. Hence, our results suggest that EOAz has a vasodilator effect mediated by inhibition of Ca2+ influx and release from intracellular storage, as well as an activation of the NOS/sGC pathway.

New nitrosyl ruthenium complexes with combined activities for multiple cardiovascular disorders.

Nitrosyl ruthenium complexes are promising platforms for nitric oxide (NO) and nitroxyl (HNO) release, which exert their therapeutic application. In this context, we developed two polypyridinic compounds with the general formula cis-[Ru(NO)(bpy)2(L)]n+, where L is an imidazole derivative. These species were characterized by spectroscopic and electrochemical techniques, including XANES/EXAFS experiments, and further supported by DFT calculations. Interestingly, assays using selective probes evidenced that both complexes can release HNO on reaction with thiols. This finding was biologically validated by HIF-1α detection. The latter protein is related to angiogenesis and inflammation processes under hypoxic conditions, which is selectively destabilized by nitroxyl. These metal complexes also presented vasodilating properties using isolated rat aorta rings and demonstrated antioxidant properties in free radical scavenging experiments. Based on these results, the new nitrosyl ruthenium compounds showed promising characteristics as potential therapeutic agents for the treatment of cardiovascular conditions such as atherosclerosis, deserving further investigation.

Effects of cilostazol, a Phosphodiesterase-3 inhibitor, on kidney function and redox imbalance in acute kidney injury caused by Bothrops alternatus venom.

The mechanisms of pathogenesis of acute kidney injury (AKI) in snakebites is multifactorial and involves hemodynamic disturbances, with release of free radical causing cytotoxic effects. The phosphodiesterase-3 (PDE3) inhibitor, Cilostazol, has been reported to provide protection against renal oxidative stress. OBJECTIVE: We evaluated the protective effects of cilostazol against Bothrops alternatus snake venom (BaV)-induced nephrotoxicity. METHODS: Wistar rat kidneys (n = 6, 260-300 g) were isolated and perfused with Krebs-Henseleit solution containing 6 g/100 mL of bovine serum albumin. After 30 min, the kidneys were perfused with BaV to a final concentration of 1 and 3 µg/mL, and subsequently evaluated for perfusion pressure (PP), renal vascular resistance (RVR), urinary flow (UF), glomerular filtration rate (GFR), and percentage of electrolyte tubular sodium and chloride transport (%TNa+, %TCl-). Oxidative stress and renal histological analyses were performed. RESULTS: BaV caused a reduction in all the evaluated renal parameters (PP, RVR, GFR, UF, %TNa+, and %TCl-). Although only the effects on PP and UF were reversed with cilostazol treatment, the decrease in the malondialdehyde levels, without changes in glutathione levels, further reduced the venom-induced renal tissue changes. CONCLUSION: Our data suggest that PDE3 is involved in BaV-induced nephrotoxicity, as cilostazol administration significantly ameliorated these effects.

A fingerprint of plasma proteome alteration after local tissue damage induced by Bothrops leucurus snake venom in mice.

Bothrops spp. is responsible for about 70% of snakebites in Brazil, causing a diverse and complex pathophysiological condition. Bothrops leucurus is the main species of medical relevance found in the Atlantic coast in the Brazilian Northeast region. The pathophysiological effects involved B. leucurus snakebite as well as the organism's reaction in response to this envenoming, it has not been explored yet. Thus, edema was induced in mice paw using 1.2, 2.5, and 5.0 µg of B. leucurus venom, the percentage of edema was measured 30 min after injection and the blood plasma was collected and analyzed by shotgun proteomic strategy. We identified 80 common plasma proteins with differential abundance among the experimental groups and we can understand the early aspects of this snake envenomation, regardless of the suggestive severity of an ophidian accident. The results showed B. leucurus venom triggers a thromboinflammation scenario where family's proteins of the Serpins, Apolipoproteins, Complement factors and Component subunits, Cathepsins, Kinases, Oxidoreductases, Proteases inhibitors, Proteases, Collagens, Growth factors are related to inflammation, complement and coagulation systems, modulators platelets and neutrophils, lipid and retinoid metabolism, oxidative stress and tissue repair. Our findings set precedents for future studies in the area of early diagnosis and/or treatment of snakebites. SIGNIFICANCE: The physiopathological effects that the snake venoms can cause have been investigated through classical and reductionist tools, which allowed, so far, the identification of action mechanisms of individual components associated with specific tissue damage. The currently incomplete limitations of this knowledge must be expanded through new approaches, such as proteomics, which may represent a big leap in understanding the venom-modulated pathological process. The exploration of the complete protein set that suffer modifications by the simultaneous action of multiple toxins, provides a map of the establishment of physiopathological phenotypes, which favors the identification of multiple toxin targets, that may or may not act in synergy, as well as favoring the discovery of biomarkers and therapeutic targets for manifestations that are not neutralized by the antivenom.

Synthesis and potential vasorelaxant effect of a novel ruthenium-based nitro complex.

This study aimed to investigate the synthesis and potential vasodilator effect of a novel ruthenium complex, cis-[Ru(bpy)2(2-MIM)(NO2)]PF6 (bpy = 2,2'-bipyridine and 2-MIM = 2-methylimidazole) (FOR711A), containing an imidazole derivative via an in silico molecular docking model using ß1 H-NOX (Heme-nitric oxide/oxygen binding) domain proteins of reduced and oxidized soluble guanylate cyclase (sGC). In addition, pharmacokinetic properties in the human organism were predicted through computational simulations and the potential for acute irritation of FOR711A was also investigated in vitro using the hen's egg chorioallantoic membrane (HET-CAM). FOR711A interacted with sites of the ß1 H-NOX domain of reduced and oxidized sGC, demonstrating shorter bond distances to several residues and negative values of total energy. The predictive study revealed molar refractivity (RM): 127.65; Log Po/w = 1.29; topological polar surface area (TPSA): 86.26 Å2; molar mass (MM) = 541.55 g/mol; low solubility, high unsaturation index, high gastrointestinal absorption; toxicity class 4; failure to cross the blood-brain barrier and to react with cytochrome P450 (CYP) enzymes CYP1A2, CYP2C19, CYP2C9, CYP2D6 and CYP3A4. After the HET-CAM assay, the FOR711A complex was classified as non-irritant (N.I.) and its vasodilator effect was confirmed through greater evidence of blood vessels after the administration and ending of the observation period of 5 min. These results suggest that FOR711A presented a potential stimulator/activator effect of sGC via NO/sGC/cGMP. However, results indicate it needs a vehicle for oral administration.

Phosphodiesterase-5 inhibitor sildenafil attenuates kidney injury induced by Bothrops alternatus snake venom.

Acute kidney injury pathogenesis in envenoming by snakes is multifactorial and involves immunologic reactions, hemodynamic disturbances, and direct nephrotoxicity. Sildenafil (SFC), a phosphodiesterase 5 inhibitor, has been reported to protect against pathological kidney changes. OBJECTIVE: This study aimed to investigate the protective effect of sildenafil against Bothrops alternatus snake venom (BaV)-induced nephrotoxicity. METHODS: Kidneys from Wistar rats (n = 6, weighing 260-300 g) were isolated and divided into four groups: (1) perfused with a modified Krebs-Henseleit solution (MKHS) containing 6 g% of bovine serum albumin; (2) administered 3 µg/mL SFC; (3) perfused with 3 µg/mL BaV; and (4) administered SFC + BaV, both at 3 µg/mL. Subsequently, the perfusion pressure (PP), renal vascular resistance (RVR), urinary flow (UF), glomerular filtration rate (GFR), and percentage of electrolyte tubular sodium and chloride transport (%TNa+, %TCl-, respectively) were evaluated. The cyclic guanosine monophosphate (cGMP) levels were analyzed in the perfusate, and the kidneys were removed to perform oxidative stress and histopathological analyses. RESULTS: All renal parameters evaluated were reduced with BaV. In the SFC + BaV group, SFC restored PP to normal values and promoted a significant increase in %TNa+ and %TCl-. cGMP levels were increased in the SFC + BaV group. The oxidative stress biomarkers, malondialdehyde (MDA) and glutathione (GSH), were reduced by BaV. In the SFC + BaV group, a decrease in MDA without an increase in GSH was observed. These findings were confirmed by histological analysis, which showed improvement mainly in tubulis. CONCLUSION: Our data suggest the involvement of phosphodiesterase-5 and cGMP in BaV-induced nephrotoxicity since its effects were attenuated by the administration of SFC.

Bothrops pauloensis snake venom-derived Asp-49 and Lys-49 phospholipases A2 mediates acute kidney injury by oxidative stress and release of inflammatory cytokines.

The envenomation caused by the Bothrops pauloensis snake leads to severe local and systemic effects including acute kidney injury. In this study, we investigated the renal effects by phospholipases A2 (PLA2s), divided into two main subgroups, Asp-49 and Lys-49, isolated from the Bothrops pauloensis snake venom (BpV) in isolated rat kidney system. Both PLA2s (3 µg/mL), added alone to the perfusion system and analyzed for 120 min, had significant effects on isolated rat kidney. Asp-49 reduced Glomerular Filtration Rate (GFR) at 60, 90 and 120 min, and the percentage of total tubular sodium transport (%TNa+) and potassium transport (%TK+) at 120 min. Lys-49 increased Perfusion Pressure (PP) at 120 min and reduced GFR, %TNa+ and the percentage of total tubular chloride transport (%TCl-) at 60, 90 and 120 min. Cytokine release in the kidney tissues were increased with Asp-49 PLA2 (IL-10) and Lys-49 PLA2 (TNF-α, IL-1ß, IL-10). Both increased MPO activity. Asp-49 PLA2 decreased Glutathione (GSH) and increased nitrite levels, while Lys-49 PLA2 increased Malondialdehyde (MDA), GSH and nitrite levels. Histological analysis of the perfused kidneys revealed the presence of glomerular degeneration and atrophy, deposit of proteinaceous material in Bowman's space and intratubular with both PLA2s. These findings indicated that both PLA2s modified the functional parameters in an isolated perfused kidney model with increased oxidative stress and cytokine release. PLA2s are one of the components at high concentration in BpV and our results provide important knowledge about their involvement with the nephrotoxic mechanism.

In silico study of azithromycin, chloroquine and hydroxychloroquine and their potential mechanisms of action against SARS-CoV-2 infection.

Coronavirus disease 2019 (COVID-19) is a highly transmissible viral infection caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Clinical trials have reported improved outcomes resulting from an effective reduction or absence of viral load when patients were treated with chloroquine (CQ) or hydroxychloroquine (HCQ). In addition, the effects of these drugs were improved by simultaneous administration of azithromycin (AZM). The receptor-binding domain (RBD) of the SARS-CoV-2 spike (S) protein binds to the cell surface angiotensin-converting enzyme 2 (ACE2) receptor, allowing virus entry and replication in host cells. The viral main protease (Mpro) and host cathepsin L (CTSL) are among the proteolytic systems involved in SARS-CoV-2 S protein activation. Hence, molecular docking studies were performed to test the binding performance of these three drugs against four targets. The findings showed AZM affinity scores (ΔG) with strong interactions with ACE2, CTSL, Mpro and RBD. CQ affinity scores showed three low-energy results (less negative) with ACE2, CTSL and RBD, and a firm bond score with Mpro. For HCQ, two results (ACE2 and Mpro) were firmly bound to the receptors, however CTSL and RBD showed low interaction energies. The differences in better interactions and affinity between HCQ and CQ with ACE2 and Mpro were probably due to structural differences between the drugs. On other hand, AZM not only showed more negative (better) values in affinity, but also in the number of interactions in all targets. Nevertheless, further studies are needed to investigate the antiviral properties of these drugs against SARS-CoV-2.

Renal effects of venoms of Mexican coral snakes Micrurus browni and Micrurus laticollaris.

The Micrurus snake venoms mainly cause systemic complications, essentially neurotoxicity. Previous studies, however, have described that they are involved in the occurrence of acute kidney injury (AKI) in animal models. AKI pathogenesis in snakebites is multifactorial and involves immunological reactions, hemodynamic disturbances, and direct nephrotoxicity. The aim of this study was to compare the nephrotoxic effects of coral snake venoms from M. browni (MbV) and M. laticollaris (MlV) on the proximal tubular epithelial cell line (LLC-MK2) and isolated perfused kidney. Using an MTT assay, both venoms significantly reduced cell viability at higher concentrations (25-100 µg/mL). MlV (10 µg/mL) increased the perfusion pressure (PP) at 60, 90 and 120 min, while the MbV did it only at 90 and 120 min. Renal vascular resistance (RVR) decreased at 60 min and increased at 120 min with MbV, but decreased at 60, 90 and 120 min with MlV. Urinary flow (UF) alterations were not observed with MlV, but MbV elevated them at 90 and 120 min. Both venoms significantly decreased the glomerular filtration rate (GFR), %TNa+, %TK+ and %TCl- levels as of 60 min of perfusion. Oxidative stress analysis revealed that both venoms behaved similarly, reducing glutathione and increasing malondialdehyde levels. Kidney injury is not usually described in clinical cases of Micrurus snakebites. However, the potential for nephrotoxicity should be considered in the overall picture of envenomation.

Differences between renal effects of venom from two Bothrops jararaca populations from southeastern and southern Brazil.

Components from animal venoms may vary according to the snake's age, gender and region of origin. Recently, we performed a proteomic analysis of Bothrops jararaca venom from southern (BjSv) and southeastern (BjSEv) Brazil, showing differences in the venom composition, as well as its biological activity. To continue the study, we report in this short communication the different effects induced by the BjSEv and BjSv on isolated kidney and MDCK renal cells. BjSEv decreased perfusion pressure (PP) and renal vascular resistance (RVR) and increased urinary flow (UF) and glomerular filtration rate (GFR), while BjSv did not alter PP and RVR and reduced UF and GFR. Both types of venom, more expressively BjSEv, reduced %TNa+, %TK+ and %Cl-. In MDCK cells, the two types of venom showed cytotoxicity with IC50 of 1.22 µg/mL for BjSv and 1.18 µg/mL for BjSEv and caused different profiles of cell death, with BjSv being more necrotic. In conclusion, we suggest that BjSv is more nephrotoxic than BjSEv.