A new insight into purinergic pharmacology: Three fungal species as natural P2X7R antagonists.

P2X7 is a purinergic receptor involved in important physiological functions and pathological processes, such as inflammation, neurodegeneration, and pain. Despite its relevance, there is no selective antagonist useful in the treatment of diseases related to this receptor. In this context, research for a selective, safe, and potent antagonist compound that can be used in clinical therapy has been growing. In this work, we evaluated the potential antagonistic activity of three fungal extracts, namely, Vishniacozyma victoriae, Metschnikowia australis, and Ascomycota sp., which were discovered in a high-throughput screening campaign to search for new antagonists for P2X7R from natural products. First, the IC50 values of these fungal extracts were determined in J774.G8 (murine macrophage cell line) and U937 (human monocyte cell line) cells through dye uptake assays. The IC50 values of V. victoriae were 2.6 and 0.92 µg/mL, M. australis has IC50 values of 3.8 and 1.5 µg/mL, and Ascomycota sp. showed values of 2.1 and 0.67 µg/mL in J774.G8 and U937 cells, respectively. These extracts also significantly inhibited propidium iodide and Lucifer yellow uptake via P2X7R pore, P2X7R currents in electrophysiology, IL-1ß release, and the production of oxide nitric and reactive oxygen species. The extracts did not cause cytotoxicity within a period of 24 h. The results showed the promising antagonistic activity of these extracts toward P2X7R, thereby indicating that they can be future candidates for phytomedicines with potential clinical applicability.

New Insights in Purinergic Therapy: Novel Antagonists for Uridine 5'-Triphosphate-Activated P2Y Receptors from Brazilian Flora.

P2Y2 and P2Y4 receptors are physiologically activated by uridine 5'-triphosphate (UTP) and are widely expressed in many cell types in humans. P2Y2 plays an important role in inflammation and proliferation of tumor cells, which could be attenuated with the use of antagonists. However, little is known about the physiological functions related to P2Y4, due to the lack of selective ligands for these receptors. This can be solved through the search for novel compounds with antagonistic activity. The aim of this study was to discover new potential antagonist candidates for P2Y2 and P2Y4 receptors from natural products. We applied a calcium measurement methodology to identify new antagonist candidates for these receptors. First, we established optimal conditions for the calcium assay using J774.G8, a murine macrophage cell line, which expresses functional P2Y2 and P2Y4 receptors and then, we performed the screening of plant extracts at a cutoff concentration of 50 µg/mL. ATP and ionomycin, known intracellular calcium inductors, were used to stimulate cells. The calculated EC50 were 11 µM and 103 nM, respectively. These cells also responded to the UTP stimulation with an EC50 of 1.021 µM. Screening assays were performed and a total of 100 extracts from Brazilian plants were tested. Joannesia princeps Vell. (stem) and Peixotoa A. Juss (flower and leaf) extracts stood out due to their ability to inhibit UTP-induced responses without causing cytotoxicity, and presented an IC50 of 32.32, 14.99, and 12.98 µg/mL, respectively. Collectively, our results point to the discovery of potential antagonist candidates from Brazilian flora for UTP-activated receptors.

Brilliant Blue Dyes in Daily Food: How Could Purinergic System Be Affected?

Dyes were first obtained from the extraction of plant sources in the Neolithic period to produce dyed clothes. At the beginning of the 19th century, synthetic dyes were produced to color clothes on a large scale. Other applications for synthetic dyes include the pharmaceutical and food industries, which are important interference factors in our lives and health. Herein, we analyzed the possible implications of some dyes that are already described as antagonists of purinergic receptors, including special Brilliant Blue G and its derivative FD&C Blue No. 1. Purinergic receptor family is widely expressed in the body and is critical to relate to much cellular homeostasis maintenance as well as inflammation and cell death. In this review, we discuss previous studies and show purinergic signaling as an important issue to be aware of in food additives development and their correlations with the physiological functions.

An Improved Method for P2X7R Antagonist Screening.

ATP physiologically activates the P2X7 receptor (P2X7R), a member of the P2X ionotropic receptor family. When activated by high concentrations of ATP (i.e., at inflammation sites), this receptor is capable of forming a pore that allows molecules of up to 900 Da to pass through. This receptor is upregulated in several diseases, particularly leukemia, rheumatoid arthritis and Alzheimer's disease. A selective antagonist of this receptor could be useful in the treatment of P2X7R activation-related diseases. In the present study, we have evaluated several parameters using in vitro protocols to validate a high-throughput screening (HTS) method to identify P2X7R antagonists. We generated dose-response curves to determine the EC50 value of the known agonist ATP and the ICs50 values for the known antagonists Brilliant Blue G (BBG) and oxidized ATP (OATP). The values obtained were consistent with those found in the literature (0.7 ± 0.07 mM, 1.3-2.6 µM and 173-285 µM for ATP, BBG and OATP, respectively) [corrected].The Z-factor, an important statistical tool that can be used to validate the robustness and suitability of an HTS assay, was 0.635 for PI uptake and 0.867 for LY uptake. No inter-operator variation was observed, and the results obtained using our improved method were reproducible. Our data indicate that our assay is suitable for the selective and reliable evaluation of P2X7 activity in multiwell plates using spectrophotometry-based methodology. This method might improve the high-throughput screening of conventional chemical or natural product libraries for possible candidate P2X7R antagonist or agonist.

Natural Products as a Source for New Anti-Inflammatory and Analgesic Compounds through the Inhibition of Purinergic P2X Receptors.

Natural products have reemerged in traditional medicine as a potential source of new molecules or phytomedicines to help with health disorders. It has been established that members of the P2X subfamily, ATP-gated ion channels, are crucial to the inflammatory process and pain signalization. As such, several preclinical studies have demonstrated that P2X2R, P2X3R, P2X4R and P2X7R are promising pharmacological targets to control inflammatory and pain disorders. Several studies have indicated that natural products could be a good source of the new specific molecules needed for the treatment of diseases linked to inflammation and pain disorders through the regulation of these receptors. Herein, we discuss and give an overview of the applicability of natural products as a source to obtain P2X receptors (P2XR) selective antagonists for use in clinical treatment, which require further investigation.

In vitro sodium nitroprusside-mediated toxicity towards Leishmania amazonensis promastigotes and axenic amastigotes.

Leishmania parasites survive despite exposure to the toxic nitrosative oxidants during phagocytosis by the host cell. In this work, the authors investigated comparatively the resistance of Leishmania amazonensis promastigotes and axenic amastigotes to a relatively strong nitrosating agent that acts as a nitric oxide (NO) donor, sodium nitroprusside (SNP). Results demonstrate that SNP is able to decrease, in vitro, the number of L. amazonensis promastigotes and axenic amastigotes in a dose-dependent maner. Promastigotes, cultured in the presence of 0.25, 0.5, and 1 mmol L(-1) SNP for 24 h showed about 75% growth inhibition, and 97-100% when the cultures were treated with >2 mmol L(-1) SNP. In contrast, when axenic amastigotes were growing in the presence of 0.25-8 mM SNP added to the culture medium, 50% was the maximum of growth inhibition observed. Treated promastigotes presented reduced motility and became round in shape further confirming the leishmanicidal activity of SNP. On the other hand, axenic amastigotes, besides being much more resistant to SNP-mediated cytotoxicity, did not show marked morphological alteration when incubated for 24 h, until 8 mM concentrations of this nitrosating agent were used. The cytotoxicity toward L. amazonensis was attenuated by reduced glutathione (GSH), supporting the view that SNP-mediated toxicity triggered multiple oxidative mechanisms, including oxidation of thiols groups and metal-independent oxidation of biomolecules to free radical intermediates.

Effect of mesoionic 4-phenyl-5-(cinnamoyl)-1,3,4-thiadiazolium-2-phenylamine chloride derivative salts on the activities of the nitric oxide synthase and arginase of Leishmania amazonensis.

L-arginine is involved in the production of both nitric oxide (NO), mediated by nitric oxide synthase (NOS) and L-ornithine, by arginase activity. It is generally accepted that NO regulation occurs mainly at the transcriptional level of NOS. In a previous work we purported that there is evidence that Leishmania sp. can produce NO from L-arginine. An arginase activity in its gene sequence has also been reported in Leishmania parasites. In a search for intracellular targets as potential antileishmanicidal agents, such as the L-arginine metabolism, we used 1,3,4-thiadiazolium mesoionic compounds, that have been demonstrated to be cytotoxic to the Leishmania amazonensis, when compared to Pentamidine isethionate as a reference drug. Parasites were assayed in absence/presence of 4'- and 3'-methoxy mesoionic derivatives in order to verify the effect on NO production and arginase activity in L. amazonensis. The results indicated that the drugs reduce from 70 to 90% of the NO production by the parasite and act on a soluble nitric oxide synthase purified from L. amazonensis promastigotes and axenic amastigotes.

Nitric oxide synthase (NOS) characterization in Leishmania amazonensis axenic amastigotes.

BACKGROUND: Although Leishmania virulence may be modulated by environmental and genetic factors of their mammalian hosts and sand fly vectors, molecular determinants of Leishmania sp. are the key elements. This work evidences that Leishmania amazonensis axenic amastigotes produce comparatively more NO than infective promastigotes. METHODS: A soluble NOS was purified from L. amazonensis axenic amastigotes by affinity chromatography (2',5'-ADP-agarose), and on SDS-PAGE the enzyme migrates as a single protein band. RESULTS: The presence of a constitutive NOS was detected through immunofluorescence using antibody against neuronal NOS (nNOS) and in NADPH consumption assays. CONCLUSIONS: The present data show that NOS is prominent in axenic amastigote preparations, suggesting an association with the infectivity and/or an escaping mechanism of the parasite. The relationship between the NO-generating systems in the parasite and in their host cell warrants further investigation.

Recentes avanços da quimioterapia das leishmanioses: moléculas intracelulares como alvo de fármacos / Recent advances on leishmaniasis chemotherapy: intracelular molecules as a drug target

Leishmanioses são doenças causadas por protozoários do gênero Leishmania sp., que se apresentam na forma promastigota ou amastigota; os promastigotas infectam o inseto vetor e os amastigotas são as formas infectivas antes presentes em macrófagos humanos. O tratamento utilizado pela clínica tem se mostrado ineficaz; os fármacos utilizados se apresentam na forma injetável, o que dificulta o tratamento, já que o paciente, para ser tratado, deve ser internado para as aplicações - que são dolorosas - e assim muitos pacientes desistem do tratamento. Outra razão para a internação é a monitoração dos efeitos colaterais causados pelos fármacos...