RESUMO
BACKGROUND: Peptide drugs are advantageous because they are subject to rational design and exhibit highly diverse structures and broad biological activities. The NS2B-NS3 protein is a particularly promising flavivirus therapeutic target, with extensive research on the development of inhibitors as therapeutic candidates, and was used as a model in this work to determine the mechanism by which GA-Hecate inhibits ZIKV replication. OBJECTIVE: The present study aimed to evaluate the potential of GA-Hecate, a new antiviral developed by our group, against the Brazilian Zika virus and to evaluate the mechanism of action of this compound on the flavivirus NS2B-NS3 protein. METHODS: Solid-phase peptide Synthesis, High-Performance Liquid Chromatography, and Mass Spectrometry were used to obtain, purify, and characterize the synthesized compound. Real-time and enzymatic assays were used to determine the antiviral potential of GA-Hecate against ZIKV. RESULTS: The RT-qPCR results showed that GA-Hecate decreased the number of ZIKV RNA copies in the virucidal, pre-treatment, and post-entry assays, with 5- to 6-fold fewer RNA copies at the higher nontoxic concentration in Vero cells (HNTC: 10 µM) than in the control cells. Enzymatic and kinetic assays indicated that GA-Hecate acts as a competitive ZIKV NS2B-NS3 protease inhibitor with an IC50 of 32 nM and has activity against the yellow fever virus protease. CONCLUSION: The results highlight the antiviral potential of the GA-Hecate bioconjugate and open the door for the development of new antivirals.
Assuntos
Antivirais , Proteínas não Estruturais Virais , Replicação Viral , Zika virus , Zika virus/efeitos dos fármacos , Antivirais/farmacologia , Antivirais/química , Proteínas não Estruturais Virais/metabolismo , Proteínas não Estruturais Virais/antagonistas & inibidores , Animais , Chlorocebus aethiops , Células Vero , Replicação Viral/efeitos dos fármacos , Serina Endopeptidases/metabolismo , Peptídeos/farmacologia , Peptídeos/química , RNA Helicases/metabolismo , RNA Helicases/antagonistas & inibidores , Infecção por Zika virus/tratamento farmacológico , Infecção por Zika virus/virologia , Humanos , Inibidores de Proteases/farmacologia , Inibidores de Proteases/química , Proteases Virais , Nucleosídeo-Trifosfatase , RNA Helicases DEAD-boxRESUMO
Since late 2016, a yellow fever virus (YFV) variant carrying a set of nine amino acid variations has circulated in South America. Three of them were mapped on the methyltransferase (MTase) domain of viral NS5 protein. To assess whether these changes affected viral infectivity, we synthesized YFV carrying the MTase of circulating lineage as well as its isoform with the residues of the previous strains (NS5 K101R, NS5 V138I, and NS5 G173S). We observed a slight difference in viral growth properties and plaque phenotype between the two synthetic YFVs. However, the MTase polymorphisms associated with the Brazilian strain of YFV (2016-2019) confer more susceptibility to the IFN-I. In addition, in vitro MTase assay revealed that the interaction between the YFV MTase and the methyl donor molecule (SAM) is altered in the Brazilian MTase variant. Altogether, the results reported here describe that the MTase carrying the molecular signature of the Brazilian YFV circulating since 2016 might display a slight decrease in its catalytic activity but virtually no effect on viral fitness in the parameters comprised in this study. The most marked influence of these residues stands in the immune escape against the antiviral response mediated by IFN-I.
Assuntos
Interferon Tipo I , Vírus da Febre Amarela , Vírus da Febre Amarela/fisiologia , Interferon Tipo I/genética , Aminoácidos , Evasão da Resposta Imune , Brasil , Metiltransferases/metabolismo , Proteínas não Estruturais Virais/genéticaRESUMO
The Chikungunya virus (CHIKV) causes Chikungunya fever, a disease characterized by symptoms such as arthralgia/polyarthralgia. Currently, there are no antivirals approved against CHIKV, emphasizing the need to develop novel therapies. The imidazonaphthyridine compound (RO8191), an interferon-α (IFN-α) agonist, was reported as a potent inhibitor of HCV. Here RO8191 was investigated for its potential to inhibit CHIKV replication in vitro. RO8191 inhibited CHIKV infection in BHK-21 and Vero-E6 cells with a selectivity index (SI) of 12.3 and 37.3, respectively. Additionally, RO8191 was capable to protect cells against CHIKV infection, inhibit entry by virucidal activity, and strongly impair post-entry steps of viral replication. An effect of RO8191 on CHIKV replication was demonstrated in BHK-21 through type-1 IFN production mechanism and in Vero-E6 cells which has a defective type-1 IFN production, also suggesting a type-1 IFN independent mode of action. Molecular docking calculations demonstrated interactions of RO8191 with the CHIKV E proteins, corroborated by the ATR-FTIR assay, and with non-structural proteins, supported by the CHIKV-subgenomic replicon cells assay.
Assuntos
Febre de Chikungunya , Vírus Chikungunya , Interferon Tipo I , Animais , Chlorocebus aethiops , Humanos , Febre de Chikungunya/tratamento farmacológico , Antivirais/farmacologia , Antivirais/uso terapêutico , Simulação de Acoplamento Molecular , Replicação Viral , Células Vero , Interferon Tipo I/farmacologiaRESUMO
With almost half of the world population living at risk, tropical infectious diseases cause millions of deaths every year in developing countries. Considering the lack of economic prospects for investment in this field, approaches aiming the rational design of compounds, such as structure-based drug discovery (SBDD), fragment screening, target-based drug discovery, and drug repurposing are of special interest. Herein, we focused in the advances on the field of SBDD targeting arboviruses such as dengue, yellow fever, zika and chikungunya enzymes of the RNA replication complex (RC) and enzymes involved in a variety of pathways essential to ensure parasitic survival in the host, for malaria, Chagas e leishmaniasis diseases. We also highlighted successful examples such as promising new inhibitors and molecules already in preclinical/clinical phase tests, major gaps in the field and perspectives for the future of drug design for tropical diseases.