In silico mapping of essential residues in the catalytic domain of PDE5 responsible for stabilization of its commercial inhibitors

Phosphodiesterase type 5 (PDE5) is an important enzyme associated with the hydrolysis of cyclic guanosine monophosphate (cGMP) to guanosine monophosphate (GMP). Due to the relevant role of second messenger cGMP as a mediator in many physiological processes, efforts have been converged to find a safe pharmacological approach, seeking a specific, selective and potent inhibitor of the PDE5 enzyme. There are five commercial drugs with potential for clinical use: tadalafil, sildenafil, avanafil, udenafil and vardenafil. Here, we applied molecular modeling to obtain different profiles of protein-ligand interactions by adopting distinct PDE5 structures, specifically PDBid:1XOZ and two extracted from molecular dynamics (MD) simulations. The results generated by molecular docking showed several possibilities for inhibitor interactions with the catalytic pocket. Tadalafil, sildenafil and vardenafil were clearly stabilized by Gln817 via a well-oriented hydrogen bond. Another set of different interactions, such as polar, hydrophobic, pi-stacking, metal-ligand and electrostatic, were responsible for accommodating avanafil and udenafil. All of the ligands are discussed in detail with consideration of the distinct protein structures, and a profile of the probability of residue-ligand contact is suggested, with the most frequently observed being: Tyr612, His613, Ser661, Thr723, Asp724, Asp764, Leu765, Val782 and Phe786. The molecular interactions displayed herein confirm findings achieved by previous authors and also present new contacts. In addition, the discussion can help researchers obtain a molecular basis for planning new selective PDE5 inhibitors, as well as explain an inhibitor's experimental assays by considering the specific interactions occurring at the catalytic site874CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP470374/2013-62010/16947-9; 2013/05475-7; 2013/08293-7; 2013/22360-9; 2017/26687-3; 2017/02201-

Characterization and optimization of oil microcapsules from Attalea phalerata Mart. for the preservation of bioactive compounds

This study aimed microencapsulating Attalea phalerata Mart. oil, containing high carotenoid and phenolic compounds content, with Arabic gum and gelatin, using the complex coacervation method. The yield, efficiency, morphology of microcapsules and content of phenolic compounds, carotenoids and antioxidant activity in different processes conditions (concentration of the filling, temperature and agitation speed) were evaluated. The results showed 88% of yield, efficiency up to 70% and a characteristic size of microcapsules. The amount of carotenoids was high in crude oil (394.84 µg of carotenoids/g oil) and the microencapsulation tests showed amounts of 19.19 to 166.40 µg of carotenoids/g oil. The phenolic compounds in the crude oil were 20.73 mg GAE/g sample and the microencapsulation tests showed amounts of 3.17 to 15.16 mg GAE/g oil. The values of bioactive compounds influenced in the antioxidant activity though ABTS•+ method with values of 161.70 µM trolox/g oil to crude oil and 7.70 and 159.54 µM trolox/g oil for microcapsules tests

In silico, in vitro and in vivo evaluation of candidate compounds for phosphodiesterase 5 inhibitors

Orientadores: Gilberto De Nucci, Fabíola Taufic Mónica IglesiasTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências MédicasResumo: A terapêutica farmacológica dos inibidores da fosfodiesterase tipo 5 (PDE5) é considerada como tratamento de primeira linha para a disfunção erétil. Adicionalmente, são utilizados para o tratamento da hipertensão pulmonar idiopática e hiperplasia prostática benigna. Todos os inibidores de PDE5 têm semelhanças estruturais com o monofosfato de guanosina cíclico (GMPc) e competem pela ligação no sítio catalítico. A seletividade e afinidade dos inibidores de PDE5 são determinadas pelo contato direto entre certos resíduos do domínio catalítico, e entender como ocorrem as interações moleculares pode ajudar no desenvolvimento de novos fármacos. Desse modo, o presente trabalho pretendeu avaliar uma série de novos derivados de 6,7-dihidro-3H-oxazolo[3,4-a] pirazina-5,8-diona in silico e in vitro sobre a proteína alvo PDE5, bem como avaliar a biodisponibilidade das novas moléculas. Neste trabalho, usamos como ferramenta de estudo a docagem molecular e a simulação de dinâmica molecular para analisar as interações das novas moléculas com a PDE5, além de utilizar métodos in vitro para estudar os efeitos dos compostos em células, plaquetas e tecidos humanos e por fim investigar os parâmetros farmacocinéticos. Os resultados de docagem e dinâmica molecular sugerem que os resíduos dos bolsões (bolsão-Q, bolsão-H e região L), que formam o domínio catalítico da PDE5A são orientados para criar uma rede de interações que acomodam os inibidores da forma mais estável, dependendo das características moleculares, estruturais e amostragem conformacional. Ademais, nas células de carcinoma de colón ou T84 os compostos promoveram o aumento dos níveis de GMPc sem efeito citotóxico. Nas plaquetas os compostos foram capazes de inibir a agregação após estímulo com o doador de óxido nítrico (nitroprussiato de sódio) e aumentar os níveis e GMPc e AMPc com melhores resultados na presença do composto BL-106. Complementarmente, mostramos que os compostos BL-106, BL-220, BL-230 e BL-236 foram capazes de inibir a enzima fosfodiesterase 5A. A atividade do BL-106 foi investigada no corpo cavernoso humano e de coelho e em ureter humano mostrando relaxamento com valores de pEC50 de 6.48 ± 0.20 (coelho), 7.14 ± 0.30 (humano) e 5.88 ± 0.38 (humano). Finalmente, espera-se que os resultados apresentados nesta tese auxiliem no entendimento das interações dos novos compostos com a PDE5. Fornecendo embasamento químico para a compreensão da ação destes compostos em nível biológico e que a descoberta destes possam abrir uma nova alternativa para o desenvolvimento de outros inibidoresAbstract: The pharmacological therapy with phosphodiesterase type 5 (PDE5) inhibitors is considered a first line of treatment for erectile dysfunction. In addition, they are used for the treatment of idiopathic pulmonary hypertension and benign prostatic hyperplasia. All PDE5 inhibitors have structural similarities with cyclic guanosine monophosphate (cGMP) and compete for the bond in the catalytic site. The selectivity and affinity of PDE5 inhibitors are determined by the direct contact between certain residues of the catalytic domain, and understanding how the molecular interactions occur may help in the development of new drugs. This study, therefore, sought to assess a series of new 6.7-dihydro-3H-oxazolo[3.4] pyrazine-5,8-dione derivatives through in silico and in vitro studies on the target protein PDE5, in addition to evaluating the bioavailability of the new molecules. In this study, we used the molecular docking and molecular dynamics simulation tools to analyze the interactions of the new molecules with PDE5, in addition to using in vitro methods to study the effects of the compounds on cells, platelets and human tissue, and finally, to investigate the pharmacokinetic parameters. The molecular docking and dynamics results suggest that residues of binding sites (binding site-Q, binding site-H and region L), which form the catalytic domain of PDE5A, are oriented to create a network of interactions that can accommodate the inhibitors in a more stable way, depending on the molecular, structural and conformational sampling characteristics. Furthermore, the compounds promoted increased levels of cGMP in the colon carcinoma or T84 cells without cytotoxic effect. In the platelets, the compounds were able to inhibit aggregation after stimulation with the nitric oxide donor (sodium nitroprusside) and to increase the cGMP and cAMP levels with better results in the presence of the BL-106 compound. In addition, we showed that the BL-106, Bl-220, Bl-230 and BL-236 compounds were able to inhibit the phosphodiesterase 5A enzyme. The activity of Bl-106 was investigated in the corpus cavernosum of a human and rabbit and in a human ureter, showing relaxation with pEC50 values of 6.48 ± 0.20 (rabbit), 7.14 ± 0.30 (human) and 5.88 ± 0.38 (human). Finally, it is expected that the results presented in this thesis may assist in understanding the interactions of new compounds with PDE5, providing a foundation for understanding the action of these compounds on a biological level, and that the discovery of these mechanisms may open up new alternatives for the development of other inhibitorsDoutoradoFarmacologiaDoutora em Farmacologia01-P-3371/2017CAPE

Q817G mutation in phosphodiesterase type 5 : conformational analysis and dissociation profile of the inhibitor tadalafil

Phosphodiesterase type 5 (PDE-5) is an important enzyme involved in the hydrolysis of cyclic guanosine monophosphate (cGMP) to guanosine monophosphate (GMP). The inhibition of this protein leads to the accumulation of cGMP in cells with various biological and therapeutic effects. Several PDE-5 inhibitors exist, with Tadalafil being one of the most commonly studied and used in clinical therapy. In this study, we applied Molecular Dynamics simulations coupled to the ABF (Adaptive Biasing Force) method to study the effect of the mutation on the Gln817 residue (Q817G). The results of the free energy profiles made clear that the affinity of the inhibitor for PDE-5 is dependent on the amino acid residue Gln817. The hydrogen bond made between the side chain of glutamine and the indole ring of Tadalafil results in the stabilization of the ligand in the catalytic site. Despite the prominent role of this interaction, it is important to highlight the contribution of other residues of the catalytic domain for the stabilization of the compound, due to the set of polar, hydrophobic and electrostatic interactions performed by specific amino acid residues934419429CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQCOORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP470374/2013-6não tem2010/16947-9; 2013/05475-7; 2013/08293-7; 2013/22

Nota científica: Conservação pós-colheita de laranjas Champagne (Citrus reticulata × Citrus sinensis)

Objetivou-se avaliar os efeitos de diferentes recobrimentos e temperaturas de armazenamento na conservação de laranjas Champagne. Após refrigeração e sanitização, os frutos foram submetidos aos seguintes tratamentos: 1) controle, sem recobrimento; 2) frutos recobertos com fécula de mandioca a 2% (m/v); 3) frutos recobertos com fécula de mandioca a 4% (m/v); 4) embalagem com filme de PVC esticável; 5) embalagem em saco plástico de polietileno de baixa densidade. Estes frutos foram, em seguida, armazenados em três diferentes temperaturas: ambiente (25ºC / 70 ± 5% UR) e em câmara fria a 3ºC e a 8ºC, com 85 ± 5% UR. Determinou-se a perda de massa pelos frutos; no suco, foram determinados o pH e os sólidos solúveis (SS), a acidez titulável, os açúcares totais e o ácido ascórbico. As amostragens foram realizadas a cada 4 dias, durante 24 dias, nos frutos armazenados em condição ambiente, e a cada 10 dias, durante 60 dias de armazenamento, nos frutos mantidos em câmara fria. Os frutos armazenados a 3ºC apresentaram menor perda de massa. Os frutos do controle e os revestidos com fécula, independentemente da temperatura de estocagem, tiveram perdas maiores do que aqueles embalados em filme de PVC e polietileno. A acidez apresentou redução ao longo do armazenamento em todas as temperaturas estudadas e sem diferenças entre os revestimentos/embalagens. Os tratamentos e o período de armazenamento, independentemente da temperatura, não influenciaram nos resultados de pH, teores de SS, açúcares totais e ácido ascórbico. Os frutos refrigerados a 3ºC mantiveram a qualidade por até 60 dias, desde que acondicionados com polietileno e PVC, enquanto que, sob a temperatura ambiente, a qualidade dos frutos embalados com estes filmes foi mantida por até 20 dias. Frutos embalados com o filme de polietileno apresentaram sinais de podridão e odor estranho a partir do 20º dia, quando armazenados a 25ºC, e a partir do 50º dia, quando armazenados a 8ºC

Campomanesia adamantium peel extract in antidiarrheal activity: the ability of Inhibition of heat-stable enterotoxin by polyphenols

Campomanesia adamantium (Myrtaceae) is a medicinal plant distributed in Brazilian Cerrado. Different parts of this plant are used in popular medicine for treatment of several diseases like fever, diarrhea, hypercholesterolemia and rheumatism. The aim of this work was to evaluate the inhibition of heat-stable enterotoxin type A (STa) by gallic acid present in the peel of C. adamantium fruit and assays to assess the antidiarrheal activity, anti-inflammatory and cytotoxic properties of peel extract using the T84 cell line model. The possible inhibition exerted by the gallic acid of the peel extract on the STa peptide was inferred by molecular dynamics simulations. The antidiarrheal effects were investigated measuring cGMP accumulation in cells after stimulation by STa toxin and antibacterial activity was assessed. The anti-inflammatory activity was assessed by inhibition of COX-1 and COX-2. MTT and LDH assays were used to evaluate any possible cytotoxic action while the CyQUANT test was used to investigate the effect on cell proliferation. A representation showing how the possible interactions between STa and the gallic acid of the extract might reduce the action of the enterotoxin is presented. C. adamantium peel extract significantly decreased the levels of cGMP in T84 cells. However, no effect on the species of microorganisms was observed. The extract also inhibited COX-1 (IC50 255.70 +/- 0.04 ng/mL) and COX-2 (IC50 569.50 +/- 0.11 ng/mL) enzymes. Cytotoxicity assay have shown significant changes in cells treated with the extract, which inhibited the cell proliferation until 72 hours of treatment. Direct interactions of phenolic compounds present in the extract with the STa toxin may limit its activity. Curative effect in the diarrhea treatment and its anti-inflammatory action is based on the pharmacological properties, mechanism of action of the C. adamantium peel extract, and no toxic effects of the peel extract presented on this work1110CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP470374/2013-62010/16947-9; 2013/05475-7; 2013/08293-

Campomanesia adamantium Peel Extract in Antidiarrheal Activity: The Ability of Inhibition of Heat-Stable Enterotoxin by Polyphenols.

Campomanesia adamantium (Myrtaceae) is a medicinal plant distributed in Brazilian Cerrado. Different parts of this plant are used in popular medicine for treatment of several diseases like fever, diarrhea, hypercholesterolemia and rheumatism. The aim of this work was to evaluate the inhibition of heat-stable enterotoxin type A (STa) by gallic acid present in the peel of C. adamantium fruit and assays to assess the antidiarrheal activity, anti-inflammatory and cytotoxic properties of peel extract using the T84 cell line model. The possible inhibition exerted by the gallic acid of the peel extract on the STa peptide was inferred by molecular dynamics simulations. The antidiarrheal effects were investigated measuring cGMP accumulation in cells after stimulation by STa toxin and antibacterial activity was assessed. The anti-inflammatory activity was assessed by inhibition of COX-1 and COX-2. MTT and LDH assays were used to evaluate any possible cytotoxic action while the CyQUANT test was used to investigate the effect on cell proliferation. A representation showing how the possible interactions between STa and the gallic acid of the extract might reduce the action of the enterotoxin is presented. C. adamantium peel extract significantly decreased the levels of cGMP in T84 cells. However, no effect on the species of microorganisms was observed. The extract also inhibited COX-1 (IC50 255.70 ± 0.04 ng/mL) and COX-2 (IC50 569.50 ± 0.11 ng/mL) enzymes. Cytotoxicity assay have shown significant changes in cells treated with the extract, which inhibited the cell proliferation until 72 hours of treatment. Direct interactions of phenolic compounds present in the extract with the STa toxin may limit its activity. Curative effect in the diarrhea treatment and its anti-inflammatory action is based on the pharmacological properties, mechanism of action of the C. adamantium peel extract, and no toxic effects of the peel extract presented on this work

Cytotoxic, genotoxic and mutagenic evaluation of Alibertia edulis (rich.) a. Rich. ex DC: an indigenous species from Brazil

Tea leaves of Alibertia edulis is popularly used in folk medicine. However, studies on the genotoxicity of this plant are not available. We aimed to investigate the in vivo and in vitro cytotoxic, genotoxic and mutagenic potentials of the aqueous extract of A. edulis leaves (AEAE). Antioxidant assays, the Artemia salina test, MTT in human platelets, micronucleus in bone marrow and comet in peripheral blood were performed. Animals received four different doses of the AEAE by oral gavage for 30 days. Saline and cyclophosphamide were used as controls. The AEAE exhibited a maximal inhibition at 100 and 250 µg/mL, according to the ABTS and DPPH methods, respectively. The A. salina assay showed that the AEAE presented some toxicity at doses of 100, 250 and 500 μg/mL. Through the MTT assay, the AEAE showed no toxic effects on human platelets during the incubation period. The alkaline comet assay showed that all doses of the AEAE were statistically similar to the negative control group since they did not induce any significant increase of the overall number of damaged cells nor the severity of the cell damage. In the micronucleous assay, results demonstrate that the AEAE did not increase the production of micronucleated polychromatic erythrocytes and was statistically similar to the negative control. The four doses of the plant extract did not affect the production of new erythrocytes and were statistically similar to the negative control groups. Furthermore, the AEAE demonstrated no cytotoxicity, genotoxicity and mutagenicity at the doses tested in rats432200207CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQCOORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESNão temNão temThis work was supported by CAPES, FUNDECT and CNP