RESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19). The NSP15 endoribonuclease enzyme, known as NendoU, is highly conserved and plays a critical role in the ability of the virus to evade the immune system. NendoU is a promising target for the development of new antiviral drugs. However, the complexity of the enzyme's structure and kinetics, along with the broad range of recognition sequences and lack of structural complexes, hampers the development of inhibitors. Here, we performed enzymatic characterization of NendoU in its monomeric and hexameric form, showing that hexamers are allosteric enzymes with a positive cooperative index, and with no influence of manganese on enzymatic activity. Through combining cryo-electron microscopy at different pHs, X-ray crystallography and biochemical and structural analysis, we showed that NendoU can shift between open and closed forms, which probably correspond to active and inactive states, respectively. We also explored the possibility of NendoU assembling into larger supramolecular structures and proposed a mechanism for allosteric regulation. In addition, we conducted a large fragment screening campaign against NendoU and identified several new allosteric sites that could be targeted for the development of new inhibitors. Overall, our findings provide insights into the complex structure and function of NendoU and offer new opportunities for the development of inhibitors.
Asunto(s)
SARS-CoV-2 , Humanos , Regulación Alostérica , Secuencia de Aminoácidos , COVID-19 , Microscopía por Crioelectrón , Endorribonucleasas/metabolismo , SARS-CoV-2/metabolismo , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/químicaRESUMEN
The Zika virus (ZIKV), discovered in Africa in 1947, swiftly spread across continents, causing significant concern due to its recent association with microcephaly in newborns and Guillain-Barré syndrome in adults. Despite a decrease in prevalence, the potential for a resurgence remains, necessitating urgent therapeutic interventions. Like other flaviviruses, ZIKV presents promising drug targets within its replication machinery, notably the NS3 helicase (NS3Hel) protein, which plays critical roles in viral replication. However, a lack of structural information impedes the development of specific inhibitors targeting NS3Hel. Here we applied high-throughput crystallographic fragment screening on ZIKV NS3Hel, which yielded structures that reveal 3D binding poses of 46 fragments at multiple sites of the protein, including 11 unique fragments in the RNA-cleft site. These fragment structures provide templates for direct design of hit compounds and should thus assist the development of novel direct-acting antivirals against ZIKV and related flaviviruses, thus opening a promising avenue for combating future outbreaks.
RESUMEN
The 2015/16 Zika virus (ZIKV) epidemic led to almost 1 million confirmed cases in 84 countries and was associated to the development of congenital microcephaly and Guillain-Barré syndrome. More recently, a ZIKV African lineage was identified in Brazil raising concerns about a future outbreak. The long-term consequences of viral infection emphasizes the need for the development of effective anti-ZIKV drugs. In this study, we developed and characterized a ZIKV replicon cell line for the screening of viral replication inhibitors. The replicon system was developed by engineering the IRES-Neo cassette into the 3' UTR terminus of the ZIKV Rluc DNA construct. After in vitro transcription, replicon RNA was used to transfect BHK-21 cells, that were selected with G418, thus generating the BHK-21-RepZIKV_IRES-Neo cell line. Through this replicon-based cell system, we identified two molecules with potent anti-ZIKV activities, an imidazonaphthyridine and a riminophenazine, both from the MMV/DNDi Pandemic Response Box library of 400 drug-like compounds. The imidazonaphthyridine, known as RO8191, showed remarkable selectivity against ZIKV, while the riminophenazine, the antibiotic Clofazimine, could act as a non-nucleoside analog inhibitor of viral RNA-dependent RNA polymerase (RdRp), as evidenced both in vitro and in silico. The data showed herein supports the use of replicon-based assays in high-throughput screening format as a biosafe and reliable tool for antiviral drug discovery.
Asunto(s)
Infección por el Virus Zika , Virus Zika , Antivirales/farmacología , Antivirales/uso terapéutico , Clofazimina/farmacología , Clofazimina/uso terapéutico , Ensayos Analíticos de Alto Rendimiento , Humanos , Replicón , Replicación Viral , Virus Zika/fisiologíaRESUMEN
BACKGROUND: The Yellow Fever virus (YFV) is transmitted by mosquitos and causes an infection with symptoms including fever, headaches and nausea. In 20-50% of the cases, the disease may evolve to a visceral stage, reaching high mortality rates. YFV NS2B-NS3 protease has been identified as an important drug target. METHODS: Herein, we describe the crystal structure of the NS2B-NS3 protease from the 2017 YFV Brazilian circulating strain using X-ray crystallography. Furthermore, we used a combination of biochemical and biophysical assays to characterize the enzyme and investigate the impact of the polymorphisms observed in different YFV circulating strains. RESULTS: Surprisingly, the crystal structure of YFV protease seems to adopt the closed conformation without the presence of a binding partner. Although D88E and K121R mutants exhibited a lower affinity for the substrate, both revealed to be more processive, resulting in a similar catalytic efficiency in relation to the WT protease. Still, both mutants showed an accentuated decrease in stability when compared with the WT. CONCLUSIONS: The crystal structure of YFV NS2B-NS3 in closed conformation might be an important tool for the development of new drugs, as well as understanding the activation mechanism of viral proteases. Biochemical analyses indicate that the NS2B-NS3 protease of the circulating strain of YFV is more stable than previous strains. GENERAL SIGNIFICANCE: The YFV NS2B-NS3 protease is the first flaviviral structure described in its closed conformation when in a free form, implying that external factors might induce the activation of the enzyme.