RESUMO
Hepatitis C is affecting millions of people around the globe annually, which leads to death in very high numbers. After many years of research, hepatitis C virus (HCV) remains a serious threat to the human population and needs proper management. The in silico approach in the drug discovery process is an efficient method in identifying inhibitors for various diseases. In our study, the interaction between Epigallocatechin-3-gallate, a component of green tea, and envelope glycoprotein E2 of HCV is evaluated. Epigallocatechin-3-gallate is the most promising polyphenol approved through cell culture analysis that can inhibit the entry of HCV. Therefore, various in silico techniques have been employed to find out other potential inhibitors that can behave as EGCG. Thus, the homology modelling of E2 protein was performed. The potential lead molecules were predicted using ligand-based as well as structure-based virtual screening methods. The compounds obtained were then screened through PyRx. The drugs obtained were ranked based on their binding affinities. Furthermore, the docking of the topmost drugs was performed by AutoDock Vina, while its 2D interactions were plotted in LigPlot+. The lead compound mms02387687 (2-[[5-[(4-ethylphenoxy) methyl]-4-prop-2-enyl-1,2,4-triazol-3-yl] sulfanyl]-N-[3(trifluoromethyl) phenyl] acetamide) was ranked on top, and we believe it can serve as a drug against HCV in the future, owing to experimental validation.
Assuntos
Catequina/análogos & derivados , Hepacivirus/efeitos dos fármacos , Hepatite C/tratamento farmacológico , Proteínas do Envelope Viral/genética , Antivirais/química , Antivirais/farmacologia , Catequina/química , Catequina/farmacologia , Hepacivirus/genética , Hepacivirus/patogenicidade , Hepatite C/virologia , Humanos , Ligantes , Simulação de Acoplamento Molecular , Polifenóis/química , Polifenóis/farmacologia , Chá/química , Envelope Viral/química , Proteínas do Envelope Viral/antagonistas & inibidores , Internalização do Vírus/efeitos dos fármacosRESUMO
Zika virus (ZIKV) is an emerging mosquito-borne flavivirus, which has recently caused global epidemics with its association with congenital Zika syndrome such as severe microcephaly. The recombinant ZIKV envelope (Env) glycoprotein is useful for immunological applications such as serodiagnosis of ZIKV infection and for monitoring immune responses in preclinical and clinical ZIKV vaccine developments. In this chapter, we describe the optimization of production of Zika virus envelope glycoprotein in Human Embryonic Kidney (HEK 293T) cells by small-scale expression followed by large-scale protein production. Small-scale expression of HEK 293T cells allows screening of a large number of vectors simultaneously to select the vectors with best secretory profiles for scale-up in Expi293 mammalian system to maximize the protein yield followed by purification for research and clinical applications.
Assuntos
Proteômica/métodos , Proteínas Recombinantes de Fusão , Testes Sorológicos/métodos , Transfecção/métodos , Proteínas do Envelope Viral , Antígenos CD4/química , Antígenos CD4/genética , Antígenos CD4/metabolismo , Calibragem , Ensaio de Imunoadsorção Enzimática/métodos , Ensaio de Imunoadsorção Enzimática/normas , Expressão Gênica , Produtos do Gene env/genética , Produtos do Gene env/isolamento & purificação , Produtos do Gene env/metabolismo , Células HEK293 , Humanos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Via Secretória , Envelope Viral/química , Envelope Viral/metabolismo , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/isolamento & purificação , Proteínas do Envelope Viral/metabolismo , Zika virus/química , Zika virus/genética , Zika virus/metabolismoRESUMO
We explored the stability of the dengue virus envelope (E) protein dimer since it is widely assumed that the E protein dimer is stabilized by drug ligands or antibodies in an acidic environment, neutralizing the virus's ability to fuse with human cells. During this process, a large conformational change of the E protein dimer is required. We performed Molecular Dynamics simulations to mimic the conformational change and stability of the dimer in neutral and acidic conditions with the well-tempered metadynamics method. Furthermore, as a few neutralizing antibodies discovered from dengue patients were reported, we used the same simulation method to examine the influence of a selected antibody on the dimer stability in both neutral and acidic conditions. We also investigated the antibody's influence on a point-mutated E protein that had been reported to interrupt the protein-antibody interaction and result in more than 95% loss of the antibody's binding ability. Our simulation results are highly consistent with the experimental conclusion that binding of the antibody to the E protein dimer neutralizes the virus, especially in a low pH condition, while the mutation of W101A or N153A significantly reduces the antibody's ability in stabilizing the E protein dimer. We demonstrate that well-tempered metadynamics can be used to accurately explore the antibody's interaction on large protein complexes such as the E protein dimer, and the computational approach in this work is promising in future antibody development.