RESUMO
A high-throughput molecular docking approach was successfully applied for the selection of potential inhibitors of the Influenza RNA-polymerase which act by targeting the PA-PB1 protein-protein interaction. Commercially available compounds were purchased and biologically evaluated in vitro using an ELISA-based assay. As a result, some compounds possessing a 3-cyano-4,6-diphenyl-pyridine nucleus emerged as effective inhibitors with the best ones showing IC50 values in the micromolar range.
Assuntos
Inibidores Enzimáticos/farmacologia , Vírus da Influenza A/enzimologia , Piridinas/farmacologia , RNA Polimerase Dependente de RNA/antagonistas & inibidores , Proteínas Virais/antagonistas & inibidores , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/química , Ensaios de Triagem em Larga Escala , Modelos Moleculares , Estrutura Molecular , Ligação Proteica/efeitos dos fármacos , Piridinas/química , RNA Polimerase Dependente de RNA/metabolismo , Relação Estrutura-Atividade , Proteínas Virais/metabolismoRESUMO
The influenza RNA polymerase complex, which consists of the three subunits PA, PB1, and PB2, is a promising target for the development of new antiviral drugs. A large library of benzofurazan compounds was synthesized and assayed against influenza virus A/WSN/33 (H1N1). Most of the new derivatives were found to act by inhibiting the viral RNA polymerase complex through disruption of the complex formed between subunits PA and PB1. Docking studies were also performed to elucidate the binding mode of benzofurazans within the PB1 binding site in PA and to identify amino acids involved in their mechanism of action. The predicted binding pose is fully consistent with the biological data and lays the foundation for the rational development of more effective PA-PB1 inhibitors.
Assuntos
Antivirais/síntese química , Benzoxazóis/química , RNA Polimerases Dirigidas por DNA/antagonistas & inibidores , Inibidores Enzimáticos/síntese química , Vírus da Influenza A Subtipo H1N1/enzimologia , Proteínas Virais/antagonistas & inibidores , Antivirais/farmacologia , Benzoxazóis/farmacologia , Sítios de Ligação , RNA Polimerases Dirigidas por DNA/metabolismo , Inibidores Enzimáticos/farmacologia , Células HEK293 , Humanos , Simulação de Dinâmica Molecular , Ligação Proteica , Mapas de Interação de Proteínas/efeitos dos fármacos , Estrutura Terciária de Proteína , Subunidades Proteicas/antagonistas & inibidores , Subunidades Proteicas/metabolismo , Proteínas Virais/metabolismo , Replicação Viral/efeitos dos fármacosRESUMO
The identification of a novel hit compound inhibitor of the protein-protein interaction between the influenza RNA-polymerase PA and PB1 subunits has been accomplished by means of high-throughput screening. A small family of structurally related molecules has been synthesized and biologically evaluated with most of the compounds showing micromolar potency of inhibition against viral replication.
Assuntos
Antivirais/toxicidade , Benzoxazóis/química , RNA Polimerases Dirigidas por DNA/metabolismo , Inibidores Enzimáticos/síntese química , Vírus da Influenza A/efeitos dos fármacos , Animais , Antivirais/síntese química , Antivirais/química , Benzoxazóis/síntese química , Benzoxazóis/toxicidade , RNA Polimerases Dirigidas por DNA/química , Cães , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/toxicidade , Vírus da Influenza A/enzimologia , Células Madin Darby de Rim Canino , Domínios e Motivos de Interação entre Proteínas/efeitos dos fármacos , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Relação Estrutura-AtividadeRESUMO
The interaction between the catalytic subunit Pol and the processivity subunit UL42 of herpes simplex virus DNA polymerase has been characterized structurally and mutationally and is a potential target for novel antiviral drugs. We developed and validated an assay for small molecules that could disrupt the interaction of UL42 and a Pol-derived peptide and used it to screen approximately 16,000 compounds. Of 37 "hits" identified, four inhibited UL42-stimulated long-chain DNA synthesis by Pol in vitro, of which two exhibited little inhibition of polymerase activity by Pol alone. One of these specifically inhibited the physical interaction of Pol and UL42 and also inhibited viral replication at concentrations below those that caused cytotoxic effects. Thus, a small molecule can inhibit this protein-protein interaction, which provides a starting point for the discovery of new antiviral drugs.
