Floriano P. Silva

Synthesis, Biological Activity, and Molecular Modeling Studies of 1H-1,2,3-Triazole Derivatives of Carbohydrates as α-Glucosidases Inhibitors

A class of drugs in use for treating type II diabetes mellitus (T2D), typified by the pseudotetrasaccharide acarbose, act by inhibiting the alpha-glucosidase activity present in pancreatic secretions and in the brush border of the small intestine. Herein, we report the synthesis of a series of 4-substituted 1,2,3-triazoles conjugated with sugars, including D-xylose, D-galactose, D-allose, and D-ribose. Compounds were screened for alpha-glucosidase inhibitory activity using yeast maltase (MAL12) as a model enzyme. Methyl-2,3-O-isopropylidene-beta-D-ribofuranosides, such as the 4-(1-cyclohexenyl)-1,2,3-triazole derivative, were among the most active compounds, showing up to 25-fold higher inhibitory potency than the complex oligosaccharide acarbose. Docking studies on a MAL12 homology model disclosed a binding mode consistent with a transition-state-mimicking mechanism. Finally, the actual pharmacological potential of this triazole series was demonstrated by the reduction of postprandial blood glucose levels in normal rats. These compounds could represent new chemical scaffolds for developing novel drugs against T2D.

Enzymatic and structural characterization of a basic phospholipase A2 from the sea anemone Condylactis gigantea

This work aimed at the isolation and structural/functional characterization of a phospholipase A(2) (CgPLA(2)) from the extract of the anemone Condylactis gigantea. CgPLA(2) was isolated with a high purity level through three chromatographic steps, showing pI 8.6 and molecular weights of 14,500 and 29,000 for the monomer and dimer, respectively. CgPLA(2) showed a high catalytic activity upon fluorescent phospholipids inducing no direct hemolytic activity. This enzyme, which is Ca(2+)-dependent, showed a lower stability against temperature and pH variations when compared with snake venom enzymes. The enzymatic activity was significantly reduced or completely abolished after chemical modification of CgPLA(2) with BPB. Its cDNA was then obtained, with 357 base pairs which codified for a mature protein of 119 amino acid residues. A comparative analysis of the primary structure of CgPLA(2) revealed 84%, 61%, 43% and 42% similarity to the PLA(2)s from Adamsia carciniopados, Nematostella vectensis, Vipera russelli russelli and Bothrops jararacussu, respectively.

Schistosoma mansoni infection causes oxidative stress and alters receptor for advanced glycation endproduct (RAGE) and tau levels in multiple organs in mice.

Schistosomiasis is a parasitic disease caused by trematode worms from the Schistosoma genus and is characterized by high rates of morbidity. The main organs affected in this pathology, such as liver, kidneys and spleen, are shifted to a pro-oxidant state in the course of the infection. Here, we compared oxidative stress parameters of liver, kidney and spleen with other organs affected by schistosomiasis - heart, brain cortex and lungs. The results demonstrated that mice infected with Schistosoma mansoni had altered non-enzymatic antioxidant status in lungs and brain, increased carbonyl levels in lungs, and a moderate level of oxidative stress in heart. A severe redox imbalance in liver and kidneys and decreased non-enzymatic antioxidant capacity in spleen were also observed. Superoxide dismutase and catalase activities were differently modulated in liver, kidney and heart, and we found that differences in Superoxide dismutase 2 and catalase protein content may be responsible for these differences. Lungs had decreased receptor for advanced glycation endproduct expression and the brain cortex presented altered tau expression and phosphorylation levels, suggesting important molecular changes in these tissues, as homeostasis of these proteins is widely associated with the normal function of their respective organs. We believe that these results demonstrate for the first time that changes in the redox profile and expression of tissue-specific proteins of organs such as heart, lungs and brain are observed in early stages of S. mansoni infection.

Kinetics Studies on the Inhibition Mechanism of Pancreatic α-Amylase by Glycoconjugated 1H-1,2,3-Triazoles: A New Class of Inhibitors with Hypoglycemiant Activity

Glycoconjugated 1H‐1,2,3‐triazoles (GCTs) comprise a new class of glycosidase inhibitors that are under investigation as promising therapeutic agents for a variety of diseases, including type 2 diabetes mellitus. However, few kinetics studies have been performed to clarify the mode of inhibition of GCTs with their target glycosidases. Our group has previously shown that some methyl‐β‐D‐ribofuranosyl‐1H‐1,2,3‐triazoles that inhibit bakers yeast maltase were also able to reduce post‐prandial glucose levels in normal rats. We hypothesized that this hypoglycemiant activity was attributable to inhibition of mammalian α‐glucosidases involved in sugar metabolism, such as pancreatic α‐amylase. Hence, the aim of this work was to test a series of 26 GCTs on porcine pancreatic α‐amylase (PPA) and to characterize their inhibition mechanisms. Six GCTs, all ribofuranosyl‐derived GCTs, significantly inhibited PPA, with IC50 values in the middle to high micromolar range. Our results also demonstrated that ribofuranosyl‐derived GCTs are reversible, noncompetitive inhibitors when using 2‐chloro‐4‐nitrophenyl‐α‐D‐maltotrioside as a substrate. E/ES affinity ratios (α) ranged from 0.3 to 1.1, with the majority of ribofuranosyl‐derived GCTs preferentially forming stable ternary ESI complexes. Competition assays with acarbose showed that ribofuranosyl‐derived GCTs bind to PPA in a mutually exclusive fashion. The data presented here show that pancreatic α‐amylase is one of the possible molecular targets in the pharmacological activity of ribofuranosyl‐derived GCTs. Our results also provide important mechanistic insight that can be of major help to develop this new class of synthetic small molecules into more potent compounds with anti‐diabetic activity through rational drug design.

Sequence analysis of NAT2 gene in Brazilians: identification of undescribed single nucleotide polymorphisms and molecular modeling of the N-acetyltransferase 2 protein structure

N-Acetyltransferase 2 (NAT2) metabolizes a variety of xenobiotics that includes many drugs, chemicals and carcinogens. This enzyme is genetically variable in human populations and polymorphisms in the NAT2 gene have been associated with drug toxicity and efficacy as well as cancer susceptibility. Here, we have focused on the identification of NAT2 variants in Brazilian individuals from two different regions, Rio de Janeiro and Goiás, by direct sequencing, and on the characterization of new haplotypes after cloning and re-sequencing. Upon analysis of DNA samples from 404 individuals, six new SNPs (c.29T>C, c.152G>T, c.203G>A, c.228C>T, c.458C>T and c.600A>G) and seven new NAT2 alleles were identified with different frequencies in Rio de Janeiro and Goiás. All new SNPs were found as singletons (observed only once in 808 genes) and were confirmed by three independent technical replicates. Molecular modeling and structural analysis suggested that p.Gly51Val variant may have an important effect on substrate recognition by NAT2. We also observed that amino acid change p.Cys68Tyr would affect acetylating activity due to the resulting geometric restrictions and incompatibility of the functional group in the Tyr side chain with the admitted chemical mechanism for catalysis by NATs. Moreover, other variants, such like p.Thr153Ile, p.Thr193Met, p.Pro228Leu and p.Val280Met, may lead to the presence of hydrophobic residues on NAT2 surface involved in protein aggregation and/or targeted degradation. Finally, the new alleles NAT2*6H and NAT2*5N, which showed the highest frequency in the Brazilian populations considered in this study, may code for a slow activity. Functional studies are needed to clarify the mechanisms by which new SNPs interfere with acetylation.

The Comparative Genomics and Phylogenomics of Leishmania amazonensis Parasite

Leishmaniasis is an infectious disease caused by Leishmania species. Leishmania amazonensis is a New World Leishmania species belonging to the Mexicana complex, which is able to cause all types of leishmaniasis infections. The L. amazonensis reference strain MHOM/BR/1973/M2269 was sequenced identifying 8,802 codifying sequences (CDS), most of them of hypothetical function. Comparative analysis using six Leishmania species showed a core set of 7,016 orthologs. L. amazonensis and Leishmania mexicana share the largest number of distinct orthologs, while Leishmania braziliensis presented the largest number of inparalogs. Additionally, phylogenomic analysis confirmed the taxonomic position for L. amazonensis within the “Mexicana complex”, reinforcing understanding of the split of New and Old World Leishmania. Potential non-homologous isofunctional enzymes (NISE) were identified between L. amazonensis and Homo sapiens that could provide new drug targets for development.

Compound profiling and 3D-QSAR studies of hydrazone derivatives with activity against intracellular Trypanosoma cruzi.

Chagas disease is a tropical disease caused by the parasite Trypanosoma cruzi, which is endemic in Central and South America. Few treatments are available with effectiveness limited to the early (acute) stage of disease, significant toxicity and widespread drug resistance. In this work we report the outcome of a HTS-ready assay chemical library screen to identify novel, nontoxic, small-molecule inhibitors of T. cruzi. We have selected 50 compounds that possess hydrazone as a common group. The compounds were screened using recombinant T. cruzi (Tulahuen strain) expressing beta-galactosidase. A 3D quantitative structure-activity relationship (QSAR) analysis was performed using descriptors calculated from comparative molecular field analysis (CoMFA). Our findings show that of the fifty selected hydrazones, compounds LpQM-19, 28 and 31 displayed the highest activity against T. cruzi, leading to a selectivity index (SI) of 20-fold. The 3D-QSAR analysis indicates that a particular electrostatic arrangement, where electron-deficient atoms are aligned along the molecule main axis positively correlates with compound biological activity. These results provide new candidate molecules for the development of treatments against Chagas disease.

Insights into cytochrome bc1 complex binding mode of antimalarial 2-hydroxy-1,4-naphthoquinones through molecular modelling

BACKGROUND Malaria persists as a major public health problem. Atovaquone is a drug that inhibits the respiratory chain of Plasmodium falciparum, but with serious limitations like known resistance, low bioavailability and high plasma protein binding. OBJECTIVES The aim of this work was to perform molecular modelling studies of 2-hydroxy-1,4-naphthoquinones analogues of atovaquone on the Qo site of P. falciparum cytochrome bc 1 complex (Pfbc1) to suggest structural modifications that could improve their antimalarial activity. METHODS We have built the homology model of the cytochrome b (CYB) and Rieske iron-sulfur protein (ISP) subunits from Pfbc1 and performed the molecular docking of 41 2-hydroxy-1,4-naphthoquinones with known in vitro antimalarial activity and predicted to act on this target. FINDINGS Results suggest that large hydrophobic R2 substituents may be important for filling the deep hydrophobic Qo site pocket. Moreover, our analysis indicates that the H-donor 2-hydroxyl group may not be crucial for efficient binding and inhibition of Pfbc1 by these atovaquone analogues. The C1 carbonyl group (H-acceptor) is more frequently involved in the important hydrogen bonding interaction with His152 of the Rieske ISP subunit. MAIN CONCLUSIONS Additional interactions involving residues such as Ile258 and residues required for efficient catalysis (e.g., Glu261) could be explored in drug design to avoid development of drug resistance by the parasite.

Synthesis, cytotoxicity and mechanistic evaluation of 4-oxoquinoline-3-carboxamide derivatives: finding new potential anticancer drugs.

As part of a continuing search for new potential anticancer candidates, we describe the synthesis, cytotoxicity and mechanistic evaluation of a series of 4-oxoquinoline-3-carboxamide derivatives as novel anticancer agents. The inhibitory activity of compounds 10–18 was determined against three cancer cell lines using the MTT colorimetric assay. The screening revealed that derivatives 16b and 17b exhibited significant cytotoxic activity against the gastric cancer cell line but was not active against a normal cell line, in contrast to doxorubicin, a standard chemotherapeutic drug in clinical use. Interestingly, no hemolytical activity was observed when the toxicity of 16b and 17b was tested against blood cells. The in silico and in vitro mechanistic evaluation indicated the potential of 16b as a lead for the development of novel anticancer agents against gastric cancer cells.

Pleural Tuberculosis Diagnosis Based On Artificial Neural Networks Models

The diagnosis of pleural tuberculosis (pTB) is still a challenge, because the de facto stan- dard diagnostic test (histopathological examination) and all classical diagnostic tests require thoracen- tesis, which is an invasive procedure and incurs on increased costs and risks. Innovative approaches which take in account only clinical features are thus highly relevant to provide less invasive diagnos- tic tools for pTB. This paper proposes the investigation of non-linear models using artificial neural networks to develop a diagnosis support tool based solely on the clinical variables available without the need to perform thoracentesis exam. Models based on multilayer perceptrons achieved 84.7% (95%CI, 82.4% to 87.1%) of diagnostic accuracy versus 82.5% (95%CI, 80.2% to 84.8%) obtained through histopathological examination. Moreover, the sensitivity of the models was again tested, but only on patients that were diagnosed with pTB based only on clinical grounds, as classical testing did not yield positive results; the resulting estimated sensitivity indicates that the models can exploit the underlying clinical features even in pTB cases undetected by classical testing, but the low number of patients in this situation poses a limitation to this analysis. Keywords - Artificial neural networks, pleural tuberculosis, diagnosis support, nonlinear models. Resumo - O diagnostico da tuberculose pleural (pTB) ainda se apresenta como um desafio, pois o teste diagnostico tido como padrao de facto (exame histopatologico) e todos os testes diagnosticos classicos requerem a realizacao da toracocentese, procedimento invasivo que incorre em custos e riscos elevados. Abordagens inovadoras que levem em conta somente aspectos clinicos sao portanto muito relevantes para prover ferramentas para um diagnostico menos invasivo da pTB. Este artigo propoe a investigacao de modelos nao-lineares utilizando redes neurais artificiais para desenvolver uma ferramenta de suporte ao diagnostico baseada tao somente no processamento de variaveis clinicas disponiveis sem a realizacao da toracocentese. Perceptrons multicamadas alcancaram 84.7% (95%CI, 82.4% to 87.1%) de acuracia no diagnostico, versus 82.5% (95%CI, 80.2% to 84.8%) obtidos pelo exame histopatologico. Adicionalmente, a sensibilidade dos modelos foi novamente avaliada, mas somente em pacientes cujo diagnostico de pTB se deu apenas com base no acompanhamento clinico, nao tendo obtido resultado positivo em nenhum dos testes classicos; a sensibilidade estimada desta maneira indica que os modelos sao capazes de extrair caracteristicas relevantes dos dados clinicos, mesmo em casos em que a pTB nao e detectada pelos testes usuais, mas o pequeno numero de pa- cientes nesta situacao se apresenta como uma limitacao para esta analise.