RESUMO
Background: Apple (Malus domestica) is a fruit commonly associated with allergic oral symptoms in the Mexican pediatric population; however, knowledge of its allergenic proteins is limited. This information is crucial as sensitization frequencies to specific allergens can vary among different populations. The main allergic symptomatology before apple ingestion derives from primary sensitizations induced by pollen, promoting cross-reactivity with the main allergenic protein of apple. Therefore, this study aims to identify new potential sensitizing proteins to apple using immunoproteomic techniques. Methods: We collected serum samples from 14 pediatric patients with confirmed immunoglobulin E (IgE)-mediated apple allergy and used these samples to assess immunoreactivity to apple protein extracts through 2D-western blot assays. The spots corresponding to the 2D-SDS-PAGE were analyzed using nanoLC-MS/MS. Results: We identified 11 non-redundant proteins, including Mal d 2 and Mal d 1, the latter showing a high frequency of sensitization (79%) in our patients, and being considered the main apple allergenic protein. The remaining identified proteins have not been previously described as apple allergens in the International Union of Immunological Societies databases. However, three of these may be categorized as pan-allergens. Conclusions: This study shows evidence that the repertoire of apple allergens in the Mexican population could differ from those reported internationally, highlighting the importance of studies in different countries to improve the certainty of allergy diagnosis and allow the implementation of precision medicine.
Assuntos
Alérgenos , Hipersensibilidade Alimentar , Imunoglobulina E , Malus , Proteínas de Plantas , Proteômica , Humanos , Malus/imunologia , Malus/química , Criança , México , Feminino , Masculino , Hipersensibilidade Alimentar/imunologia , Alérgenos/imunologia , Proteômica/métodos , Proteínas de Plantas/imunologia , Imunoglobulina E/imunologia , Imunoglobulina E/sangue , Adolescente , Pré-Escolar , Frutas/imunologia , Frutas/química , Espectrometria de Massas em TandemRESUMO
Leishmaniasis is a disease caused by parasites of the Leishmania genus that affects 98 countries worldwide, 2 million of new cases occur each year and more than 350 million people are at risk. The use of the actual treatments is limited due to toxicity concerns and the apparition of resistance strains. Therefore, there is an urgent necessity to find new drugs for the treatment of this disease. In this context, enzymes from the polyamine biosynthesis pathway, such as arginase, have been considered a good target. In the present work, a chemical library of benzimidazole derivatives was studied performing computational, enzyme kinetics, biological activity, and cytotoxic effect characterization, as well as in silico ADME-Tox predictions, to find new inhibitors for arginase from Leishmania mexicana (LmARG). The results show that the two most potent inhibitors (compounds 1 and 2) have an I50 values of 52 µM and 82 µM, respectively. Moreover, assays with human arginase 1 (HsARG) show that both compounds are selective for LmARG. According to molecular dynamics simulation studies these inhibitors interact with important residues for enzyme catalysis. Biological activity assays demonstrate that both compounds have activity against promastigote and amastigote, and low cytotoxic effect in murine macrophages. Finally, in silico prediction of their ADME-Tox properties suggest that these inhibitors support the characteristics to be considered drug candidates. Altogether, the results reported in our study suggest that the benzimidazole derivatives are an excellent starting point for design new drugs against leishmanisis.
Assuntos
Antiprotozoários/farmacologia , Arginase/antagonistas & inibidores , Benzimidazóis/farmacologia , Leishmania mexicana/efeitos dos fármacos , Proteínas de Protozoários/antagonistas & inibidores , Animais , Antiprotozoários/química , Arginase/metabolismo , Benzimidazóis/química , Linhagem Celular , Descoberta de Drogas , Humanos , Leishmania mexicana/enzimologia , Leishmania mexicana/fisiologia , Leishmaniose Cutânea/tratamento farmacológico , Camundongos , Modelos Moleculares , Proteínas de Protozoários/metabolismoRESUMO
Protein-tyrosine phosphatase 1B (PTP1B) is a negative regulator of insulin signaling pathway and has been validated as a therapeutic target for type 2 diabetes. A wide variety of scaffolds have been included in the structure of PTP1B inhibitors, one of them is the benzimidazole nucleus. Here, we report the design and synthesis of a new series of di- and tri- substituted benzimidazole derivatives including their kinetic and structural characterization as PTP1B inhibitors and hypoglycemic activity. Results show that compounds 43, 44, 45, and 46 are complete mixed type inhibitors with a Ki of 12.6 µM for the most potent (46). SAR type analysis indicates that a chloro substituent at position 6(5), a ß-naphthyloxy at position 5(6), and a p-benzoic acid attached to the linker 2-thioacetamido at position 2 of the benzimidazole nucleus, was the best combination for PTP1B inhibition and hypoglycemic activity. In addition, molecular dynamics studies suggest that these compounds could be potential selective inhibitors from other PTPs such as its closest homologous TCPTP, SHP-1, SHP-2 and CDC25B. Therefore, the compounds reported here are good hits that provide structural, kinetic, and biological information that can be used to develop novel and selective PTP1B inhibitors based on benzimidazole scaffold.
Assuntos
Benzimidazóis/farmacologia , Glicemia/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Hipoglicemiantes/farmacologia , Simulação de Dinâmica Molecular , Proteína Tirosina Fosfatase não Receptora Tipo 1/antagonistas & inibidores , Animais , Benzimidazóis/síntese química , Benzimidazóis/química , Relação Dose-Resposta a Droga , Desenho de Fármacos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Feminino , Teste de Tolerância a Glucose , Hipoglicemiantes/síntese química , Hipoglicemiantes/química , Estrutura Molecular , Proteína Tirosina Fosfatase não Receptora Tipo 1/metabolismo , Ratos , Ratos Wistar , Relação Estrutura-AtividadeRESUMO
Giardiasis is highly prevalent in the developing world, and treatment failures with the standard drugs are common. This work deals with the proposal of omeprazole as a novel antigiardial drug, focusing on a giardial glycolytic enzyme used to follow the cytotoxic effect at the molecular level. We used recombinant technology and enzyme inactivation to demonstrate the capacity of omeprazole to inactivate giardial triosephosphate isomerase, with no adverse effects on its human counterpart. To establish the specific target in the enzyme, we used single mutants of every cysteine residue in triosephosphate isomerase. The effect on cellular triosephosphate isomerase was evaluated by following the remnant enzyme activity on trophozoites treated with omeprazole. The interaction of omeprazole with giardial proteins was analyzed by fluorescence spectroscopy. The susceptibility to omeprazole of drug-susceptible and drug-resistant strains of Giardia lamblia was evaluated to demonstrate its potential as a novel antigiardial drug. Our results demonstrate that omeprazole inhibits giardial triosephosphate isomerase in a species-specific manner through interaction with cysteine at position 222. Omeprazole enters the cytoplasmic compartment of the trophozoites and inhibits cellular triosephosphate isomerase activity in a dose-dependent manner. Such inhibition takes place concomitantly with the cytotoxic effect caused by omeprazole on trophozoites. G. lamblia triosephosphate isomerase (GlTIM) is a cytoplasmic protein which can help analyses of how omeprazole works against the proteins of this parasite and in the effort to understand its mechanism of cytotoxicity. Our results demonstrate the mechanism of giardial triosephosphate isomerase inhibition by omeprazole and show that this drug is effective in vitro against drug-resistant and drug-susceptible strains of G. lamblia.
Assuntos
Antiprotozoários/farmacologia , Inibidores Enzimáticos/farmacologia , Giardia lamblia/efeitos dos fármacos , Omeprazol/farmacologia , Proteínas de Protozoários/antagonistas & inibidores , Triose-Fosfato Isomerase/antagonistas & inibidores , Trofozoítos/efeitos dos fármacos , Albendazol/farmacologia , Cultura Axênica , Cisteína/química , Cisteína/metabolismo , Relação Dose-Resposta a Droga , Resistência a Medicamentos , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Giardia lamblia/enzimologia , Giardia lamblia/crescimento & desenvolvimento , Giardia lamblia/isolamento & purificação , Humanos , Metronidazol/farmacologia , Mutação , Nitrocompostos , Testes de Sensibilidade Parasitária , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espectrometria de Fluorescência , Tiazóis/farmacologia , Triose-Fosfato Isomerase/genética , Triose-Fosfato Isomerase/metabolismo , Trofozoítos/enzimologia , Trofozoítos/crescimento & desenvolvimentoRESUMO
BACKGROUND: We have previously proposed triosephosphate isomerase of Giardia lamblia (GlTIM) as a target for rational drug design against giardiasis, one of the most common parasitic infections in humans. Since the enzyme exists in the parasite and the host, selective inhibition is a major challenge because essential regions that could be considered molecular targets are highly conserved. Previous biochemical evidence showed that chemical modification of the non-conserved non-catalytic cysteine 222 (C222) inactivates specifically GlTIM. The inactivation correlates with the physicochemical properties of the modifying agent: addition of a non-polar, small chemical group at C222 reduces the enzyme activity by one half, whereas negatively charged, large chemical groups cause full inactivation. RESULTS: In this work we used mutagenesis to extend our understanding of the functional and structural effects triggered by modification of C222. To this end, six GlTIM C222 mutants with side chains having diverse physicochemical characteristics were characterized. We found that the polarity, charge and volume of the side chain in the mutant amino acid differentially alter the activity, the affinity, the stability and the structure of the enzyme. The data show that mutagenesis of C222 mimics the effects of chemical modification. The crystallographic structure of C222D GlTIM shows the disruptive effects of introducing a negative charge at position 222: the mutation perturbs loop 7, a region of the enzyme whose interactions with the catalytic loop 6 are essential for TIM stability, ligand binding and catalysis. The amino acid sequence of TIM in phylogenetic diverse groups indicates that C222 and its surrounding residues are poorly conserved, supporting the proposal that this region is a good target for specific drug design. CONCLUSIONS: The results demonstrate that it is possible to inhibit species-specifically a ubiquitous, structurally highly conserved enzyme by modification of a non-conserved, non-catalytic residue through long-range perturbation of essential regions.
Assuntos
Giardia lamblia/enzimologia , Mutagênese Sítio-Dirigida , Triose-Fosfato Isomerase/química , Triose-Fosfato Isomerase/metabolismo , Biocatálise , Sequência Conservada , Cristalografia por Raios X , Estabilidade Enzimática , Glicolatos/metabolismo , Cinética , Modelos Moleculares , Mutação , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Análise Espectral , Triose-Fosfato Isomerase/genéticaRESUMO
Antigenic drift of avian influenza viruses (AIVs) has been observed in chickens after extended vaccination program, similar to those observed with human influenza viruses. To evaluate the evolutionary properties of endemic AIV under high vaccination pressure (around 2 billion doses used in the last 12 years), we performed a pilot phylogenic analysis of the hemagglutinin (HA) gene of AIVs isolated from 1994 to 2006. This study demonstrates that Mexican low pathogenicity (LP) H5N2-AIVs are constantly undergoing genetic drifts. Recent AIV isolates (2002-2006) show significant molecular drifts when compared with the H5N2 vaccine-strain or other field isolates (1994-2000). This study also demonstrates that molecular drifts in the HA gene lineages follow a yearly trend, suggesting gradually cumulative sequence mutations. These findings might explain the increasing incidence of LP H5N2 AIV isolated from commercial avian farms. These findings support recent concerns about the challenge of AIV antigenic drift and influenza epidemics.