RESUMO
Trichomonas vaginalis, a flagellated and anaerobic protozoan, is a causative agent of trichomoniasis. This disease is among the world's most common non-viral sexually transmitted infection. A single class drug, nitroimidazoles, is currently available for the trichomoniasis treatment. However, resistant isolates have been identified from unsuccessfully treated patients. Thus, there is a great challenge for a discovery of innovative anti-T. vaginalis agents. As part of our ongoing search for antiprotozoal chalcones, we designed and synthesized a series of 21 phenolic chalcones, which were evaluated against T. vaginalis trophozoites. Structure-activity relationship indicated hydroxyl group plays a role key in antiprotozoal activity. 4'-Hydroxychalcone (4HC) was the most active compound (IC50 = 27.5 µM) and selected for detailed bioassays. In vitro and in vivo evaluations demonstrated 4HC was not toxic against human erythrocytes and Galleria mellonella larvae. Trophozoites of T. vaginalis were treated with 4HC and did not present significant reactive oxygen species (ROS) accumulation. However, compound 4HC was able to increase ROS accumulation in neutrophils coincubated with T. vaginalis. qRT-PCR Experiments indicated that 4HC did not affect the expression of pyruvate:ferredoxin oxidoreductase (PFOR) and ß-tubulin genes. In silico simulations, using purine nucleoside phosphorylase of T. vaginalis (TvPNP), corroborated 4HC as a promising ligand. Compound 4HC was able to establish interactions with residues D21, G20, M180, R28, R87 and T90 through hydrophobic interactions, π-donor hydrogen bond and hydrogen bonds. Altogether, these results open new avenues for phenolic chalcones to combat trichomoniasis, a parasitic neglected infection.
Assuntos
Antiprotozoários , Chalconas , Tricomoníase , Trichomonas vaginalis , Humanos , Trichomonas vaginalis/metabolismo , Chalconas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Tricomoníase/tratamento farmacológico , Tricomoníase/parasitologia , Antiprotozoários/metabolismo , Fenóis/metabolismoRESUMO
Screening of compounds comprising 8-substituted guanine revealed that 8-aminoguanosine and 8-aminoguanine cause diuresis/natriuresis/glucosuria, yet decrease potassium excretion. Subsequent investigations demonstrated that 8-aminoguanosine's effects are mediated by its metabolite 8-aminoguanine. The mechanism by which 8-aminoguanine causes diuresis/natriuresis/glucosuria involves inhibition of PNPase (purine nucleoside phosphorylase), which increases renal interstitial inosine levels. Additional evidence suggests that inosine, via indirect or direct adenosine A2B receptor activation, increases renal medullary blood flow which enhances renal excretory function. Likely, 8-aminoguanine has pleiotropic actions that also alter renal excretory function. Indeed, the antikaliuretic effects of 8-aminoguanine are independent of PNPase inhibition. 8-Aminoguanine is an endogenous molecule; nitrosative stress leads to production of biomolecules containing 8-nitroguanine moieties. Degradation of these biomolecules releases 8-nitroguanosine and 8-nitro-2'-deoxyguanosine which are converted to 8-aminoguanine. Also, guanosine and guanine per se may contribute to 8-aminoguanine formation. 8-Aminoinosine, 8-aminohypoxanthine, and 8-aminoxanthine likewise induce diuresis/natriuresis/glucosuria, yet do not reduce potassium excretion. Thus, there are several pharmacologically active 8-aminopurines with nuanced effects on renal excretory function. Chronic treatment with 8-aminoguanine attenuates hypertension in deoxycorticosterone/salt rats, prevents strokes, and increases lifespan in Dahl salt-sensitive rats on a high salt diet and attenuates the metabolic syndrome in rats; 8-aminoguanosine retards progression of pulmonary hypertension in rats and anemia and organ damage in sickle cell mice. 8-Aminoguanine reverses age-associated lower urinary tract dysfunction and retinal degeneration. 8-Aminopurines represent a new class of agents (and potentially endogenous factors) that have beneficial effects on the cardiovascular system and kidneys and may turn back the clock in age-associated diseases.
Assuntos
Sistema Cardiovascular , Guanina , Ratos , Camundongos , Animais , Ratos Endogâmicos Dahl , Guanina/metabolismo , Guanina/farmacologia , Natriurese , Sistema Cardiovascular/metabolismo , Potássio , Inosina/farmacologiaRESUMO
Polygala boliviensis is found in the Brazilian semiarid region. This specie is little chemically and biologically studied. Polygala spp. have different metabolites, especially coumarins. Studies indicate that coumarins have antimalarial potential, denoting the importance of researching new active compounds from plants, since the resistance of Plasmodium strains to conventional therapy has increased. The present study aimed to evaluate the antiplasmodial activity of auraptene and poligalen against a chloroquine-resistant strain of Plasmodium falciparum. Coumarins were isolated from P. boliviensis by open column chromatography and identified by Nuclear Magnetic Resonance Spectroscopy. A cytotoxicity assay was carried out using MTT test, and the in vitro antiplasmodial activity was evaluated using the W2 strain. The antiplasmodial activity results found were IC50=0.171 ± 0.016 for auraptene and 0.164 ± 0.012 for poligalen; the selectivity indexes were 78.71 and 609.76, respectively. Inverse virtual screening in the BRAMMT database by OCTOPUS 1.2 was applied to coumarins to find potential P. falciparum targets and showed higher affinity energy of auraptene for purine nucleoside phosphorylase (PfPNP) and of poligalen for dihydroorotate dehydrogenase (PfDHODH). Molecular Dynamics studies (MD and MM-GBSA) approach were applied to calculate binding energies against selected P. falciparum targets and showed that all coumarins were stable at the binding site during simulations. Furthermore, energies were favorable for complexation. This is the first report of auraptene in P. boliviensis species and of in vitro antiplasmodial activity of auraptene and poligalen. In silico studies indicated that the mechanism of action of coumarins is the inhibition of PfPNP and PfDHODH.Communicated by Ramaswamy H. Sarma.
Assuntos
Antimaláricos , Plasmodium , Polygala , Antimaláricos/farmacologia , Antimaláricos/química , Plasmodium falciparum , Extratos Vegetais/química , Cumarínicos/farmacologiaRESUMO
BACKGROUND: The endogenous purine 8-aminoguanine induces diuresis/natriuresis/glucosuria by inhibiting PNPase (purine nucleoside phosphorylase); however, mechanistic details are unknown. METHODS: Here, we further explored in rats 8-aminoguanine's effects on renal excretory function by combining studies using intravenous 8-aminoguanine, intrarenal artery infusions of PNPase substrates (inosine and guanosine), renal microdialysis, mass spectrometry, selective adenosine receptor ligands, adenosine receptor knockout rats, laser doppler blood flow analysis, cultured renal microvascular smooth muscle cells, HEK293 cells expressing A2B receptors and homogeneous time resolved fluorescence assay for adenylyl cyclase activity. RESULTS: Intravenous 8-aminoguanine caused diuresis/natriuresis/glucosuria and increased renal microdialysate levels of inosine and guanosine. Intrarenal inosine, but not guanosine, exerted diuretic/natriuretic/glucosuric effects. In 8-aminoguanine-pretreated rats, intrarenal inosine did not induce additional diuresis/natriuresis/glucosuria. 8-Aminoguanine did not induce diuresis/natriuresis/glucosuria in A2B-receptor knockout rats, yet did so in A1- and A2A-receptor knockout rats. Inosine's effects on renal excretory function were abolished in A2B knockout rats. Intrarenal BAY 60-6583 (A2B agonist) induced diuresis/natriuresis/glucosuria and increased medullary blood flow. 8-Aminoguanine increased medullary blood flow, a response blocked by pharmacological inhibition of A2B, but not A2A, receptors. In HEK293 cells expressing A2B receptors, inosine activated adenylyl cyclase, and this was abolished by MRS 1754 (A2B antagonist). In renal microvascular smooth muscle cells, 8-aminoguanine and forodesine (PNPase inhibitor) increased inosine and 3',5'-cAMP; however, in cells from A2B knockout rats, 8-aminoguanine and forodesine did not augment 3',5'-cAMP yet increased inosine. CONCLUSIONS: 8-Aminoguanine induces diuresis/natriuresis/glucosuria by increasing renal interstitial levels of inosine which, via A2B receptor activation, increases renal excretory function, perhaps in part by increasing medullary blood flow.
Assuntos
Adenilil Ciclases , Diurese , Ratos , Humanos , Animais , Adenilil Ciclases/farmacologia , Células HEK293 , Diuréticos/farmacologia , Natriurese , Receptores Purinérgicos P1 , Inosina/farmacologiaRESUMO
Human purine nucleoside phosphorylase (HsPNP) catalyzes reversible phosphorolysis of nucleosides and deoxynucleosides in the purine cascade. HsPNP has been a target on behalf of the development of new leads for the treatment of a variety of T-cell mediated disorders. Several studies on the HsPNP are focused on the identification of effective, safe, and selective inhibitors. Therefore, this study describes the development of direct, simple, reliable, and inexpensive enzymatic assays to screen HsPNP inhibitors. Initially, HsPNP was covalently immobilized on the surface of magnetic particles (MPs). Due to the versatility of the MPs as solid support for enzyme immobilization, two different methods to monitor the enzyme activity are presented. Firstly, the activity of HsPNP-MPs was assessed offline by HPLC-DAD quantifying the formed hypoxanthine. Then, HsPNP-MPs were trapped in a peek tube, furnishing a microreactor which was inserted on-flow in an HPLC-DAD system to monitor the enzyme activity by the hypoxanthine quantification. Kinetic assays provided KMapp values for the inosine substrate of 488.2 ± 49.1 and 1084 ± 111 µM for the offline and on-flow assays, respectively. For the first time, kinetic studies for Pi as substrate using the HsPNP-MPs exhibits a Michaelis-Menten kinetic, yielding KMapp values for offline and on-flow of 521.2 ± 62.9 µM and 601 ± 66.5 µM, respectively. Inhibition studies conducted with a fourth generation immucillin derivative (DI4G) were employed as proof of concept to validate the use of the HsPNP-MPs assays for screening purposes. Additionally, a small library containing 11 compounds was used to assess the selectivity of the developed assays. The results showed that both presented assays can be applied to selectively recognizing and characterizing HsPNP inhibitors. Particularly, the on-flow method exhibited a high throughput and performance because of its automation and represents an easy and practical approach to reuse the HsPNP-MPs. Besides, this novel enzyme activity assay model can be further applied to other biological targets.
Assuntos
Fenômenos Magnéticos , Purina-Núcleosídeo Fosforilase , Inibidores Enzimáticos/farmacologia , Humanos , Cinética , Nucleosídeos de Purina , Purina-Núcleosídeo Fosforilase/metabolismoRESUMO
Several Schistosoma species cause Schistosomiasis, an endemic disease in 78 countries that is ranked second amongst the parasitic diseases in terms of its socioeconomic impact and human health importance. The drug recommended for treatment by the WHO is praziquantel (PZQ), but there are concerns associated with PZQ, such as the lack of information about its exact mechanism of action, its high price, its effectiveness - which is limited to the parasite's adult form - and reports of resistance. The parasites lack the de novo purine pathway, rendering them dependent on the purine salvage pathway or host purine bases for nucleotide synthesis. Thus, the Schistosoma purine salvage pathway is an attractive target for the development of necessary and selective new drugs. In this study, the purine nucleotide phosphorylase II (PNP2), a new isoform of PNP1, was submitted to a high-throughput fragment-based hit discovery using a crystallographic screening strategy. PNP2 was crystallized and crystals were soaked with 827 fragments, a subset of the Maybridge 1000 library. X-ray diffraction data was collected and structures were solved. Out of 827-screened fragments we have obtained a total of 19 fragments that show binding to PNP2. Fourteen of these fragments bind to the active site of PNP2, while five were observed in three other sites. Here we present the first fragment screening against PNP2.
Assuntos
Descoberta de Drogas/métodos , Purina-Núcleosídeo Fosforilase/química , Purina-Núcleosídeo Fosforilase/metabolismo , Piridinas/metabolismo , Pirimidinas/metabolismo , Schistosoma mansoni/enzimologia , Tiazóis/metabolismo , Animais , Domínio Catalítico , Cristalização , Cristalografia por Raios X/métodos , Dimetil Sulfóxido/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Modelos Moleculares , Conformação Proteica em alfa-Hélice , Purina-Núcleosídeo Fosforilase/genética , Esquistossomose mansoni/tratamento farmacológico , Esquistossomose mansoni/parasitologiaRESUMO
Malaria, a tropical parasitic disease caused by Plasmodium spp., continues to place a heavy social burden, with almost 200 million cases and more than 580,000 deaths per year. Plasmodium falciparum purine nucleoside phosphorylase (PfPNP) can be targeted for antimalarial drug design since its inhibition kills malaria parasites both in vitro and in vivo. Although the currently known inhibitors of PfPNP, immucillins, are orally available and of low toxicity to animals and humans, to the best of our knowledge, none of these compounds has entered clinical trials for the treatment of malaria. Using a pharmacophore-based virtual screening coupled to a consensual molecular docking approach, we identified 59 potential PfPNP inhibitors that are predicted to be orally absorbed in a Caco-2 cell model. Although most of these compounds are predicted to have high plasma protein binding levels, poor water solubility (except for compound 25) and CYP3A4 metabolic stability (except for 4, 7 and 8), four structures (4, 7, 8 and 25) remain as potential leads because of their plausible interaction with a specific hydrophobic pocket of PfPNP, which would confer them higher selectivity for PfPNP over human PNP. Additionally, both predicted Gibbs free energies for binding and molecular dynamics suggest that compound 4 may form a more stable complex with PfPNP than 5[Formula: see text]-methylthio-immucillin-H, a potent and selective inhibitor of PfPNP.
Assuntos
Antimaláricos/química , Antimaláricos/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Plasmodium falciparum/enzimologia , Purina-Núcleosídeo Fosforilase/antagonistas & inibidores , Células CACO-2 , Simulação por Computador , Bases de Dados de Produtos Farmacêuticos , Humanos , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Plasmodium falciparum/efeitos dos fármacos , Proteínas de Protozoários/antagonistas & inibidores , Purinas/metabolismoRESUMO
The aim of this work was to optimize the preparation of a capillary human purine nucleoside phosphorylase (HsPNP) immobilized enzyme reactor (IMER) for characterization and affinity screening studies of new inhibitors by frontal affinity chromatography coupled to mass spectrometry (FAC-MS). For this purpose two monolithic supports, a Chromolith Speed Rod (0.1mm I.D.×5cm) and a methacrylate-based monolithic epoxy polymeric capillary column (0.25mm I.D.×5cm) with epoxy reactive groups were considered and compared to an IMER previously developed using an open fused silica capillary. Each HsPNP-IMER was characterized in terms of catalytic activity using Inosine as standard substrate. Furthermore, they were also explored for affinity ranking experiments. Kd determination was carried out with the based fused silica HsPNP-IMER and the results are herein discussed.