Selección de blanco terapéutico en la catepsina B para el desarrollo de fármacos contra el cáncer de mama / Therapeutic targeting of Cathepsin B for the development of drugs for breast cancer

RESUMEN Introducción: el cáncer de mama se ha incrementado en un 50 % en las dos últimas décadas. La catepsina B es una proteasa que participa en el proceso de tumurogénesis. Uno de los problemas actuales es la aparición de resistencias a fármacos. La búsqueda de nuevas alternativas terapéuticas puede reducir su morbimortalidad. Objetivo: caracterizar in silico estructural y funcionalmente la región conservada en la catepsina B como blanco terapéutico potencial en el tratamiento del cáncer de mama Métodos: con el uso de la herramienta ENTREZ del NCBI se obtuvieron 2 485 secuencias de la catepsina B. Las secuencias son sometidas a un alineamiento múltiple empleando Clustall Omega 1.2.4. Se realiza la caracterización estructural y funcional de la proteasa en estudio a partir de las bases de datos InterPro, Prosite, Uniprot y UniprotKB. Con el empleo del visualizador Jalview se seleccionó la mayor zona conservada de las especies de catepsina B. Resultados: la proteasa participa en la regulación de la actividad catalítica, proteólisis, regulación negativa de la muerte celular, procesos catabólicos del colágeno y posee actividad hidrolasa. El análisis múltiple permitió la visualización de las características aminoacídicas del sitio activo de la catepsina B y la selección de la región proteica más conservada. Conclusiones: la zona conservada de la catepsina B constituye un blanco potencial en el desarrollo de inhibidores como fármacos contra el cáncer de mama. Los análisis in silico reducen costo de las investigaciones actuales y contribuyen a la bioseguridad farmacológica.

ABSTRACT Introduction: breast cancer has increased by 50% in the last two decades. Cathepsin B is a protease involved in the process of tumorigenesis. One of the current problems is the emergence of drug resistance. The search for new therapeutic alternatives can reduce its morbidity and mortality. Objective: in-silico structural and functional characterization of the conserved region in Cathepsin B as a potential therapeutic target in the treatment of breast cancer. Methods: using the NCBI ENTREZ tool 2,485 Cathepsin B sequences were obtained. The sequences were subjected to multiple alignments using Clustall Omega 1.2.4. Structural and functional characterization of the protease under study was performed using the InterPro, Prosite, Uniprot and UniprotKB databases. Using the Jalview viewer, the largest conserved area of Cathepsin B species was chosen. Results: the protease is involved in the regulation of catalytic activity, proteolysis, negative regulation of cell death, collagen catabolic processes and possesses hydrolase activity. The multiple analyses allowed the visualization of the aminoacid characteristics of the active site of Cathepsin B and the selection of the most conserved protein region. Conclusions: the conserved region of Cathepsin B constitutes a potential target in the development of inhibitors as drugs against breast cancer. In-silico analysis reduces the cost of current research and contributes to pharmacological biosafety.

Avaliação da resistência de formas mutantes da enzima L-asparaginase a proteases séricas humanas / Evaluation of L-asparaginase mutant forms resistance to human serum proteases

O tratamento para a Leucemia Linfoblástica Aguda LLA utiliza, entre outros fármacos, a enzima L-asparaginase (ASNase) proveniente da bactéria Escherichia coli. Reações imunológicas estão entre os problemas do tratamento com ASNase, e a formação de anticorpos contra essa proteína pode impedir o sucesso no tratamento. Duas cisteíno proteases lisossomais estão relacionadas com a degradação de ASNase nos seres humanos, a Catepsina B (CTSB) e Asparagina Endopeptidase (AEP). Em estudos prévios do nosso grupo obteve-se mutantes de ASNase resistentes a degradação por CTSB e/ou AEP in vitro. Nesse trabalho avaliamos essas mutantes quanto a sua citotoxicidade em linhagens celulares de leucemia e conduzimos estudos in vivo, aplicando as proteoformas de ASNases em camundongos Balb C para avaliar a atividade asparaginase sérica das enzimas ao longo do tempo, bem como obter informações sobre a formação de anticorpos contra essas proteoformas. Nos ensaios de citotoxicidade, duas das proteoformas testadas tiveram efeito citotóxico semelhante a forma selvagem, enquanto uma outra proteoforma tem a citotoxicidade sensivelmente reduzida. Já nos ensaios in vivo, uma proteoforma demonstrou meia vida sérica maior da atividade asparaginásica, e duas proteoformas causaram reduzida formação de anticorpos. Juntos, esses resultados colaboram para a obtenção de uma nova geração de ASNases com melhor biodisponibilidade, e efeitos adversos reduzidos, gerando a possibilidade de menores doses e frequência de aplicações

The Treatment for Acute Lymphoblastic Leukemia (ALL) includes the biopharmaceutical L-asparaginase (ASNase) from Escherichia coli. Immunological reactions are among the problems of treatment using ASNase, and the antibodies formation protein may prevent success in treatment. Lysosomal cysteine proteases are related to ASNase degradation, Cathepsin B (CTSB) and Asparagine Endopeptidase (AEP). In previous studies, ASNase mutants resistant to CTSB and / or AEP degradation in vitro were obtained. In this work, mutants were evaluated in cytotoxicity in ALL cell lines and, in vivo studies, applying doses of the wild and mutant ASNases in Balb C mice to evaluate serum asparaginase activity of the enzymes over time, as well as to obtain information on the formation of antibodies against these proteoforms. Regarding to the cytotoxicity, two proteoforms among the tested had similar cytotoxicity than the wild-type. While another proteoform had the cytotoxicity severely reduced. One proteoform have demonstrated greater serum half-life of asparaginase activity, while two other mutants caused reduced antibody formation. Together, these results collaborate to obtain a new generation of ASNases with increased bioavailability and reduced side effects, generating the possibility of lower doses and frequency of applications

Analysis of heparanase isoforms and cathepsin B in the plasma of patients with gastrointestinal carcinomas: analytical cross-sectional study / Análises das isoformas de heparanase e da catepsina B em plasma de pacientes com carcinomas gastrointestinais: estudo transversal analítico

CONTEXT AND OBJECTIVE: Heparanase-1 degrades heparan sulfate and has been correlated with tumor progression. Although the isoform heparanase-2 has no catalytic activity, it seems to be important for modulating heparanase-1 activity. Cathepsin B is a proteinase involved in tumor metastasis. The aim of this study was to analyze heparanase isoform expression and cathepsin B activity in plasma samples from patients with gastrointestinal carcinomas, compared with healthy individuals (control group). DESIGN AND SETTING: This was an analytical cross-sectional study. Peripheral blood samples were collected at a Brazilian public hospital, from 21 patients with histopathological diagnoses of gastrointestinal carcinomas and from 43 healthy individuals. The analyses were performed in two Brazilian medical schools. METHODS: Heparanase isoforms were identified and quantified in plasma samples by means of Western blot. The enzymatic activities of heparanase-1 and cathepsin B were also measured. RESULTS: The results demonstrated that the expression of both heparanase isoforms was significantly greater in plasma samples from gastrointestinal carcinoma patients, compared with the control group. Logistic regression analysis showed that increased heparanase-1 and heparanase-2 expression was exclusively dependent on the ...

CONTEXTO E OBJETIVO: A heparanase-1 degrada heparam sulfato e está relacionada à progressão de tumor. Apesar de a isoforma heparanase-2 não possuir atividade catalítica, parece ser importante para modular a atividade da heparanase-1. A catepsina B é uma proteinase envolvida na metástase de tumores. O objetivo deste estudo foi analisar a expressão das isoformas de heparanase e atividade da catepsina B em amostras de plasma de pacientes com carcinomas gastrointestinais, comparando-se com indivíduos saudáveis (grupo controle). TIPO DE ESTUDO E LOCAL: Este é um estudo transversal analítico. Foram coletadas amostras de sangue periférico, em hospital público brasileiro, de 21 pacientes com diagnóstico histopatológico de carcinoma gastrointestinal e 43 indivíduos saudáveis. As análises foram realizadas em duas faculdades de medicina brasileiras. MÉTODOS: As isoformas da heparanase foram identificadas e quantificadas em amostras de plasma por Western blot. As atividades enzimáticas de heparanase-1 e catepsina B foram também mensuradas. RESULTADOS: Os resultados demonstraram que as expressões das isoformas de heparanase foram significativamente maiores nas amostras de plasma de pacientes com carcinoma gastrointestinal em comparação com ...