RESUMO
Dengue virus (DENV) is the predominant mosquito-borne viral pathogen that infects humans with an estimated 50 to 100 million infections per year worldwide. Over the past 50 years, the incidence of dengue disease has increased dramatically and the virus is now endemic in more than 100 countries. Moreover, multiple serotypes of DENV are now found in the same geographic region, increasing the likelihood of more severe forms of disease. Despite extensive research, there are still no approved vaccines or therapeutics commercially available to treat DENV infection. Here we report the results of a high-throughput screen of a chemical compound library using a whole-virus assay that identified a novel small-molecule inhibitor of DENV, ST-610, that potently and selectively inhibits all four serotypes of DENV replication in vitro. Sequence analysis of drug-resistant virus isolates has identified a single point mutation, A263T, in the NS3 helicase domain that confers resistance to this compound. ST-610 inhibits DENV NS3 helicase RNA unwinding activity in a molecular-beacon-based helicase assay but does not inhibit nucleoside triphosphatase activity based on a malachite green ATPase assay. ST-610 is nonmutagenic, is well tolerated (nontoxic) in mice, and has shown efficacy in a sublethal murine model of DENV infection with the ability to significantly reduce viremia and viral load compared to vehicle controls.
Assuntos
Benzoxazóis/uso terapêutico , Vírus da Dengue/efeitos dos fármacos , Vírus da Dengue/enzimologia , Dengue/tratamento farmacológico , RNA Helicases/antagonistas & inibidores , Proteínas não Estruturais Virais/antagonistas & inibidores , Replicação Viral/efeitos dos fármacos , Animais , Linhagem Celular , Vírus da Dengue/genética , Vírus da Dengue/patogenicidade , Cães , Farmacorresistência Viral/genética , Células HeLa , Humanos , Camundongos , RNA Helicases/genética , Células Vero , Proteínas não Estruturais Virais/genéticaRESUMO
Dengue viruses (DENV) infect 50 to 100 million people worldwide per year, of which 500,000 develop severe life-threatening disease. This mosquito-borne illness is endemic in most tropical and subtropical countries and has spread significantly over the last decade. While there are several promising vaccine candidates in clinical trials, there are currently no approved vaccines or therapeutics available for treatment of dengue infection. Here, we describe a novel small-molecule compound, ST-148, that is a potent inhibitor of all four serotypes of DENV in vitro. ST-148 significantly reduced viremia and viral load in vital organs and tended to lower cytokine levels in the plasma in a nonlethal model of DENV infection in AG129 mice. Compound resistance mapped to the DENV capsid (C) gene, and a direct interaction of ST-148 with C protein is suggested by alterations of the intrinsic fluorescence of the protein in the presence of compound. Thus, ST-148 appears to interact with the DENV C protein and inhibits a distinct step(s) of the viral replication cycle.
Assuntos
Antivirais/farmacologia , Proteínas do Capsídeo/antagonistas & inibidores , Proteínas do Capsídeo/genética , Vírus da Dengue/efeitos dos fármacos , Dengue/tratamento farmacológico , Compostos Heterocíclicos com 3 Anéis/farmacologia , Tiadiazóis/farmacologia , Viremia/tratamento farmacológico , Sequência de Aminoácidos , Animais , Antivirais/síntese química , Proteínas do Capsídeo/química , Linhagem Celular , Chlorocebus aethiops , Cricetinae , Dengue/virologia , Vírus da Dengue/classificação , Vírus da Dengue/genética , Modelos Animais de Doenças , Cães , Escherichia coli/genética , Compostos Heterocíclicos com 3 Anéis/síntese química , Humanos , Concentração Inibidora 50 , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Tiadiazóis/síntese química , Carga Viral/efeitos dos fármacos , Ensaio de Placa Viral , Viremia/virologia , Replicação Viral/efeitos dos fármacosRESUMO
Cantagalo virus (CTGV) is the etiologic agent of a pustular disease in dairy cows and dairy workers in Brazil with important economical and occupational impacts. Nevertheless, no antiviral therapy is currently available. ST-246 is a potent inhibitor of orthopoxvirus egress from cells and has proved its efficacy in cell culture and in animal models. In this work, we evaluated the effect of ST-246 on CTGV replication. Plaque reduction assays indicated that CTGV is 6-38 times more susceptible to the drug than VACV-WR and cowpox virus, respectively, with an EC50 of 0.0086µM and a selective index of >11,600. The analysis of ß-gal activity expressed by recombinant viruses in the presence of ST-246 confirmed these results. In addition, ST-246 had a greater effect on the reduction of CTGV spread in comet tail assays and on the production of extracellular virus relative to VACV-WR. Infection of mice with CTGV by tail scarification generated primary lesions at the site of scarification that appeared less severe than those induced by VACV-WR. Animals infected with CTGV and treated with ST-246 at 100mg/kg for 5days did not develop primary lesions and virus yields were inhibited by nearly 98%. In contrast, primary lesions induced by VACV-WR were not affected by ST-246. The analysis of F13 (p37) protein from CTGV revealed a unique substitution in residue 217 (D217N) not found in other orthopoxviruses. Construction of recombinant VACV-WR containing the D217N polymorphism did not lead to an increase in the susceptibility to ST-246. Therefore, it is still unknown why CTGV is more susceptible to the antiviral effects of ST-246 compared to VACV-WR. Nonetheless, our data demonstrates that ST-246 is a potent inhibitor of CTGV replication that should be further evaluated as a promising anti-CTGV therapy.
