HIV - recentes avanços na pesquisa de fármacos

The development of new antiretroviral drugs is a dynamic process that is continuously fueled by identification of new molecular targets and new compounds for know targets. The current available drugs can be classified into five categories: nucleoside analogues reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, protease inhibitors, integrase inhibitors and entry inhibitors (fusion inhibitors and CCR5 antagonist). In addition, the maturation inhibitors may be considered as potential target for chemotherapeutic intervention. This review presents some anti-HIV agents that have already gone through the advance development process for final approval for the treatment of AIDS.

Isolamento de polímeros da parede celular de Saccharomyces cerevisiae e avaliação da atividade antioxidante da manana-proteína isolada

Yeast cell wall contains polymers glucan and mannan-protein that have received much attention with respect to their biological activities. Conventional isolation process involving treatments with hot alkali and acids cause degradation of these polymers. The aim of this paper was to study a low-degrading process for the isolation of glucan and mannan-protein from S. cerevisiae cell wall comprising physic and enzymatic treatments. Yeast cell glucan was obtained in a purity of 87.4% and a yield of 33.7%. The isolated mannan-protein presented antioxidant activity that was increased after thirty minutes of protease treatment. Antioxidant activity was determined by β-carotene/linoleate model system.

Emprego de metodologia enzimática na obtenção de extratos protéicos de farinha de trigo

An enzymatic method was used for obtaining protein extracts from wheat flour using an alkaline protease. Some parameters were evaluated aiming the optimization of this method: temperature (40-50 ºC); time (2-5 h); physical treatment of the sample (no treatment, ultra-turrax/16,000 rpm/5 min and ultrasound/120 W/10 min); enzyme:substrate ratio (E:S) of 5:100 - 10:100 and concentration of wheat flour (1:3, 1:5 and 1:10 w/v). The results showed that the best condition for protein extraction was that using the sample concentration of 1:3 (w/v), ultra-turrax, E:S of 10:100, at 40 ºC, 2 h, having reached an extraction yield of 88.53%.

Utilização de resíduos agroindustriais para a produção de proteases pelo termofílico Bacillus sp em fermentação submersa: otimização do meio de cultura usando a técnica de planejamento experimental

Thermophilic Bacillus sp. SMIA-2, produced protease when grown on apple pectic, whey protein and corn step liquor medium, whose concentration was varied from 3 to 10 gL-1, according to the central composite design 2³. The experiments were conducted in shaker, at 50 °C, 150 rpm and initial pH 6.5. The results revealed that the culture medium affected both, cell growth and enzyme production. After graphical and numerical optimization procedure, the enzyme production reached its maximum value at 30 h fermentation, reaching, approximately, 70 U protein mg-1, suggesting that this process was partially associated to the growth.

Proteases de Leishmania: novos alvos para o desenvolvimento racional de fármacos

Leishmania causes tegumental and visceral diseases called leishmaniasis. Disease control is possible interrupting the transmission cycle, but HIV co-infection, chemotheraphy toxicity and lack of a vaccine are paramount difficulties. So, is necessary to study new Leishmania molecules and investigate the possibility to develop rational drugs using these molecules as targets. Leishmania express many peptidases during their life, and cysteine are the most abundant protease and many inhibitors were developed but failed to kill parasites. On the other hand, inhibitors of serine proteases killed promastigotes, indicating the possibility of these enzymes to be important targets in the development of anti-Leishmania drugs.

Constituintes químicos das cascas do caule de Vochysia thyrsoidea Pohl. (Vochysiaceae) e avaliação das atividades citotóxica e inibitória frente as catepsinas B e K

A new flavonoid, catechin-3-O-(3"-O-trans-cinnamoyl)-α-rhamnopyranoside, along with known compounds, catechin-3-O-α-rhamnopyranoside, 3-oxo-urs-12-en-28-oic acid, 2,4,6-trimethoxybenzoic acid, 2-butyl-D-fructofuranoside and 1-butyl-D-fructofuranoside, has been isolated from the stem bark of V. thyrsoidea. These compounds were assayed for inhibition of protease activity (cathepsins B and K) and against cancer cell lines. Catechin-3-O-(3"-O-trans-cinnamoyl)-α-rhamnopyranoside showed moderate inhibitory activity (IC50 = 62.02 µM) against cathepsin B while 2-butyl-D-fructofuranoside was the most potent against a strain of CNS (SF-295) and human leukemia (HL-60) with IC50 = 36.80 µM and IC50 = 25.37 µM, respectively.

Proteases virais: importantes alvos terapêuticos de compostos peptideomiméticos

Proteases catalyze the hydrolysis of peptide bonds of proteins and peptides to produce smaller peptides and free amino acids. These enzymes are involved in physiologic processes such as blood coagulation and cellular death, and are related to life cycle of several viruses, such as hepatitis C, dengue, and AIDS. These features make most of proteases very important therapeutic targets for new pharmaceutical compounds. The development of peptidemimetics with improved pharmacokinetic properties is driving extensive research in the field of viral protease inhibitors. The present paper aims to highlight the design and synthesis of peptidemimetics that are able to inhibit viral proteases related to hepatitis C, dengue, and AIDS.

Aplicações de enzimas na síntese e na modificação de polímeros

Enzymes are biological catalysts that offer great potential for use in the synthesis and modification of polymers, being more specific and greener than chemical catalysts. In this work, enzymes from the classes of hydrolases (lipase, cutinase and protease) and of oxidoreductases (horseradish peroxidase, manganese peroxidase and laccase) were identified as the main biocatalysts responsible for the synthesis of polymers. Biocatalysis can potentially be part of the life cycle of several polymers, including polyesters, polyurethanes, polycarbonates, polyamides, functionalized polysaccharides and polystyrene, allowing the synthesis of specialty macromolecules for fine applications and with higher added-value than commodity polymers.

Especificidade de Pyrenophora avenae aos tecidos da semente de Avena sativa e sua atividade enzimática

Manchas nos grãos de aveia (Avena sativa) é limitante à sua comercialização por tornar o produto escuro e não permitir seu uso pela indústria alimentícia. A localização do micélio de Pyrenophora avenae nos grãos de aveia e sua atividade enzimática podem esclarecer a causa das manchas. O objetivo deste trabalho foi determinar a localização de P. avenae, na cariopse de aveia, avaliar a sua atividade enzimática e seu efeito sobre proteínas e lipídios dos grãos de aveia. A localização do micélio nos tecidos da cariopse foi determinada após hidratação e cortes da mesma, seguido da análise dos tecidos sob lupa e microscópio. Para avaliação da atividade enzimática foram utilizados 18 isolados de P. avenae obtidos das principais regiões produtoras de aveia do Brasil, avaliando-os quanto às suas atividades amilolítica, proteolítica e lipolítica, sendo realizada por plaqueamento das estruturas vegetativas em meio sólido específico para as enzimas testadas. As determinações do percentual de proteínas e lipídios foram obtidas pelos métodos de Kjeldahl e Bligh & Dyer, respectivamente. O micélio de P. avenae é a principal causa da mancha nos grãos de aveia, localizando-se nos três tecidos do pericarpo. O fitopatógeno apresenta boa atividade enzimática para lipase e protease porém insignificante para a amilase. Os grãos de aveia manchados e sadios não diferiram nos teores de proteínas e de lipídios. Esses teores foram mais elevados nos tecidos superficiais do pericarpo e aleurona independente da presença ou não de manchas, justificando o crescimento superficial de P. avenae sobre os grãos de aveia.

Caracterização enzimática e patogenicidade cruzada de Colletotrichum spp. associados a doenças de pós-colheita

A antracnose, causada por Colletotrichum spp., pode ocasionar grandes perdas a nível de campo e em pós-colheita sobre diversas culturas e seus produtos. O presente trabalho teve por objetivos testar a patogenicidade cruzada de isolados de C. gloeosporioides do caju (Anacardium occidentale) (CAJ), manga (Mangifera indica) (MG), mamão (Carica papaya) (MM), maracujá (Passiflora edulis) (MR) e C. musae da banana (Musa spp.) (BAJ); avaliar a produção de enzimas extracelulares (amilolítica, celulolítica, lipolítica e proteolítica) produzidas pelos isolados em substratos sólidos específicos; e detectar padrões eletroforéticos de proteínas totais e isoenzimas (alfa-esterase, beta-esterase, fosfatase ácida e leucina aminopeptidase). Na análise da patogenicidade cruzada, todos os isolados de Colletotrichum spp. induziram lesões necróticas, deprimidas sobre os frutos, exceto em maracujá que foi suscetível tão somente ao isolado MR. Quanto à produção de enzimas extracelulares hidrolíticas, os isolados de C. gloeosporioides produziram amilase, lipase, protease e celulase, sendo que esta última enzima não foi detectada em C. musae. Com relação à análise eletroforética de proteínas totais e isoenzimas, os isolados apresentaram variações no número e posição das bandas no gel de poliacrilamida em todos os sistemas, com exceção de leucina aminopeptidase, onde bandas monomórficas foram formadas, sem variação na intensidade e pouca variação na mobilidade relativa.

Inibidor de tripsina em raízes de Eucalyptus urophylla

As raízes de eucalipto (Eucalyptus urophylla) podem estar associadas a fungos como Pisolithus tinctorius, formando uma simbiose conhecida como ectomicorriza, mas também podem estar colonizadas por fungos patogênicos, como Rhizoctonia solani, agente causal do tombamento de plantas em viveiros. O objetivo deste trabalho foi verificar a presença de atividade inibitória de tripsina, uma serino-protease, em raízes de E. urophylla e a atividade de tripsina em filtrados desses fungos. Alíquotas de extrato protéico bruto de raízes de E. urophylla e frações protéicas parcialmente purificadas por cromatografia de exclusão molecular, do tipo Sephacryl S-100-HR, foram testadas para atividade inibitória de tripsina. Proteínas do extrato ou das frações, quando incubadas com o substrato BAPNA (a-benzoil-arginina-p-nitroanilida) e tripsina comercial na presença de tampão Tris-HCl 0,1 M (pH 8,0), resultou em atividade de inibidor de tripsina ao redor de 80%. Filtrados de meios de cultura de P. tinctorius e R. solani foram parcialmente purificados em cromatografia de exclusão molecular, porém atividade de tripsina sobre o substrato BAPNA não foi verificada em nenhuma das frações. Portanto, não foi possível estabelecer uma correlação direta entre o inibidor da planta e proteases dos fungos. Os resultados apresentados abrem novas perspectivas para o estudo dessas proteínas nas interações entre patógenos e simbiontes para espécies de eucalipto.

Efeito do Soil-borne wheat mosaic virus sobre o metabolismo de cinco genótipos de trigo com diferentes níveis de resistência à doença

Com o objetivo de conhecer as alterações metabólicas promovidas pelo Soil-borne wheat mosaic virus (SBWMV), um dos vírus economicamente mais importantes da cultura do trigo (Triticum aestivum), foram analisados os níveis de proteínas solúveis e determinadas as atividades da peroxidase e da protease em quatro cultivares (BRS Guabiju, BRS 194, BRS 179, BR 23) e uma linhagem (PF 980524) de trigo com diferentes níveis de resistência ao vírus. Os dados obtidos foram submetidos à análise de variância, comparando-se as médias, pelo Teste de Duncan a 5%. Os níveis de proteínas solúveis foram mais elevados nas plantas sem sintomas, enquanto que as atividades da peroxidase e da protease foram maiores em plantas com sintoma de mosaico do que em plantas assintomáticas. Além disso, pode-se constatar que quanto maior a suscetibilidade do genótipo, maior o nível de atividade da protease. Estes resultados são promissores para estudos de inibição da protease para controle de viroses.

New mechanisms regulating human Th1 and Th2 cell differentiation

Selective development of human T helper (Th) cells into functionally distinct Th1 and Th2 subtypes plays an essential role in the host immune response towards pathogens. However, abnormal function or differentiation of these cells can lead to development of various autoimmune diseases as well as asthma and allergy. Therefore, identification of key factors and the molecular mechanisms mediating Th1 and Th2 cell differentiation is important for understanding the molecular mechanisms of these diseases. The goal of this study was to identify novel factors involved in the regulation of Th1 and Th2 differentiation processes. A new method was optimized for enrichment of transiently transfected resting human primary T lymphocytes, that allowed the study of the influence of genes of interest in human Th1/Th2 cell differentiation and other primary Th cell functions. Functional characterization of PRELI, a novel activation-induced protein in human Th cells, identified it as a mitochondrial protein involved in the regulation of Th cell differentiation and apoptosis. By influencing the intracellular redox state, PRELI induces mitochondrial apoptosis pathway and downregulates STAT6 and Th2 differentiation. The data suggested that Calpain, an oxidative stress induced cysteine protease, is involved as a mediator in PRELI-induced downregulation of STAT6. PIM serine/threonine-specific kinases were identified as new regulators of human Th1 cell differentiation. PIM1 and PIM2 kinases were shown to be preferentially expressed in Th1 cells as compared to Th2 cells. RNA interference studies showed that PIM kinases enhance the production of IFN, the hallmark cytokine produced by Th1 cells. They also induce the expression of the key Th1-driving factor T-bet and the IL-12 signaling pathway during early phases of Th1 cell differentiation. Taken together, new regulators of human T helper cell differentiation were identified in this study, which provides new insights into the signaling mechanisms controlling the selective activation of human Th cell subsets.

Study of complexes of cadmium with some L-amino acids and vitamin-C by voltammetric technique

Voltammetric technique was used to study the binary and ternary complexes of cadmium with L-amino acids and vitamin-C (L-ascorbic acid) at pH =7.30 ± 0.01, µ = 1.0M KNO3 at 25ºC and 35ºC. Cd (II) formed 1:1:1, 1:1:2 and 1:2:1 complexes with L-lysine, L-ornithine, L-threonine, L-serine, L-phenylglycine, L-phenylalanine, L-glutamic acid and L-aspartic acid used as primary ligands and L-ascorbic acid used as secondary ligand. The trend of stability constant of complexes was L-lysine < L-ornithine < L-threonine < L-serine < L-phenylglycine < L-phenylalanine < L-glutamic acid < L-aspartic acid which can be explained on the basis of size, basicity and steric hindrance of ligands. The values of stability constant (log β) varied from 2.23 to11.33 confirm that these drugs i.e. L-amino acids or in combination with L-ascorbic acid or their complexes could be used against Cd (II) toxicity. The study has been carried out at 35ºC also to determine the thermodynamic parameters such as enthalpy change (ΔH), Free energy change (ΔG) and entropy change (ΔS) respectively.

Differentiation of Colletotrichum gloeosporioides isolates by using total proteins and esterase electrophoretic patterns and extracellular enzymes production

Isolates of Colletotrichum gloeosporioides (ISO-1, ISO-2, ISO-3, ISO-4, ISO-5 and ISO-6), the causal agent of anthracnose disease on mango fruits, were characterized by electrophoretic patterns of total proteins and esterase in polyacrylamida gel, and also, by production of extracellular enzymes on specific solid substrate. The electrophoretic analysis showed variation in number, intensity of coloration and position of the bands in the gel at each studied system tested. In contrast to the monomorphic behavior to total proteins, high esterase polymorfism was observed indicating difference among isolates. All isolates showed the activity of extracellular enzymes such as amylase, lipase, and protease with some variation among them. The proteolitic activity seemed to be more accentuated than the two other enzymes studied.