RESUMEN
BACKGROUND: Antiplasmodial activities of angiotensin II and its analogues have been extensively investigated in Plasmodium gallinaceum and Plasmodium falciparum parasite species. Due to its vasoconstrictor property angiotensin II cannot be used as an anti-malarial drug. METHODS: This work presents the solid-phase syntheses and liquid chromatography and mass spectrometry characterization of ten linear peptides related to angiotensin II against mature P. gallinaceum sporozoites and erythrocyte invasion by P. falciparum. Conformational analyses were performed by circular dichroism. IC50 assays were performed to identify the ideal concentration used on the biological tests and haemolytical erythrocytic assays were made to verify the viability of the biological experiments. The contractile responses of the analogues were made to evaluate if they are promising candidates to be applied as antiplasmodial drugs. RESULTS: The results indicate two short-peptides constituted by hydrophobic residues (5 and 6) with antiplasmodial activity in these models, 89 and 94 % of biological activity against P. gallinaceum sporozoite, respectively, and around 50 % of activity against P. falciparum. Circular dichroism spectra suggested that all the peptides adopted ß-turn conformation in different solutions, except peptide 3. Besides the biological assays IC50, the haemolysis assays and contractile response activities were applied for peptides 5 and 6, which did not present expressive results. CONCLUSIONS: The hydrophobic portion and the arginine, tyrosine, proline, and phenylalanine, when present on peptide primary sequence, tend to increase the antiplasmodial activity. This class of peptides can be explored, as anti-malarial drugs, after in vivo model tests. Graphical abstract: The most active peptide presented 94 % activity on P. gallinaceum sporozoites and 53 % inhibited P. falciparum ring forms invasion.
Asunto(s)
Angiotensina II/análogos & derivados , Angiotensina II/farmacología , Antimaláricos/farmacología , Productos Biológicos/farmacología , Péptidos/farmacología , Plasmodium falciparum/efectos de los fármacos , Plasmodium gallinaceum/efectos de los fármacos , Aedes/parasitología , Angiotensina II/efectos adversos , Animales , Antimaláricos/efectos adversos , Antimaláricos/síntesis química , Productos Biológicos/síntesis química , Pollos/parasitología , Cromatografía Liquida , Eritrocitos/parasitología , Hemólisis , Concentración 50 Inhibidora , Espectrometría de Masas , Ratones Endogámicos C57BL , Pruebas de Sensibilidad Microbiana , Contracción Muscular/efectos de los fármacos , Péptidos/síntesis química , Estómago/efectos de los fármacosRESUMEN
In this work, the metabolism of adenosine by isolated BLM associated-enzymes and the implications of this process for the cAMP-signaling pathway are investigated. Inosine was identified as the major metabolic product, suggesting the presence of adenosine deaminase (ADA) activity in the BLM. This was confirmed by immunoblotting and ADA-specific enzyme assay. Implications for the enzymatic deamination of adenosine on the receptor-modulated cAMP-signaling pathway were also investigated. We observed that inosine induced a 2-fold increase in [(35)S] GTPgammaS binding to the BLM and it was inhibited by 10(-6)M DPCPX, an A(1) receptor-selective antagonist. Inosine (10(-7)M) inhibited protein kinase A activity in a DPCPX-sensitive manner. Molecular association between ADA and G(alphai-3) protein-coupled A(1) receptor was demonstrated by co-immunoprecipitation assay. These data show that adenosine is deaminated by A(1) receptor-associated ADA to inosine, which in turn modulates PKA in the BLM through A(1) receptor-mediated inhibition of adenylyl cyclase.
Asunto(s)
Adenosina/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Inosina/metabolismo , Túbulos Renales Proximales/metabolismo , Antagonistas del Receptor de Adenosina A1 , Adenosina Desaminasa/metabolismo , Animales , Membrana Celular/metabolismo , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Técnicas In Vitro , Inosina/farmacología , Túbulos Renales Proximales/efectos de los fármacos , Receptor de Adenosina A1/metabolismo , Transducción de Señal/efectos de los fármacos , Porcinos , Xantinas/farmacologíaRESUMEN
Ecto-phosphatase activities of Giardia lamblia were characterized in intact cells, which are able to hydrolyze the artificial substrate p-nitrophenylphosphate (p-NPP) to p-nitrophenol (p-NP) at a rate of 8.4+/-0.8 nmol p-NP/h/10(7) cells. The ecto-phosphatase activities were inhibited at high pH as well as by classical inhibitors of acid phosphatases, such as sodium fluoride and sodium molybdate and by inorganic phosphate, the final product of the reaction. Experiments using a classical inhibitor of phosphotyrosine phosphatase, sodium orthovanadate, also showed that the ecto-phosphatase activity was inhibited in a dose-dependent manner. Different phosphorylated amino acids were used as substrates for the G. lamblia ecto-phosphatase activities the highest rate of phosphate release was achieved using phosphotyrosine. Not only p-NPP hydrolysis but also phosphotyrosine hydrolysis was inhibited by sodium orthovanadate. Phosphotyrosine but not phospho-serine or phospho-threonine inhibited the p-nitrophenylphosphatase activity. We also observed a positive correlation between the ecto-phosphatase activity and the capacity to encystation of G. lamblia trophozoites.