RESUMO
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.
RESUMO
Chikungunya virus (CHIKV) is the causative agent of Chikungunya fever, an acute febrile and arthritogenic illness with no effective treatments available. The development of effective therapeutic strategies could be significantly accelerated with detailed knowledge of the molecular components behind CHIKV replication. However, drug discovery is hindered by our incomplete understanding of their main components. The RNA-dependent RNA-polymerase (nsP4-CHIKV) is considered the key enzyme of the CHIKV replication complex and a suitable target for antiviral therapy. Herein, the nsP4-CHIKV was extensively characterized through experimental and computational biophysical methods. In the search for new molecules against CHIKV, a compound designated LabMol-309 was identified as a strong ligand of the nsp4-CHIKV and mapped to bind to its active site. The antiviral activity of LabMol-309 was evaluated in cellular-based assays using a CHIKV replicon system and a reporter virus. In conclusion, this study highlights the biophysical features of nsP4-CHIKV and identifies a new compound as a promising antiviral agent against CHIKV infection.
Assuntos
Febre de Chikungunya , Vírus Chikungunya , Antivirais/uso terapêutico , Vírus Chikungunya/genética , Humanos , Ligantes , RNA/metabolismo , RNA Polimerase Dependente de RNA , Replicação ViralRESUMO
Zika virus (ZIKV) is a dangerous human pathogen and no antiviral drugs have been approved to date. The chalcones are a group of small molecules that are found in a number of different plants, including Angelica keiskei Koidzumi, also known as ashitaba. To examine chalcone anti-ZIKV activity, three chalcones, 4-hydroxyderricin (4HD), xanthoangelol (XA), and xanthoangelol-E (XA-E), were purified from a methanol-ethyl acetate extract from A. keiskei. Molecular and ensemble docking predicted that these chalcones would establish multiple interactions with residues in the catalytic and allosteric sites of ZIKV NS2B-NS3 protease, and in the allosteric site of the NS5 RNA-dependent RNA-polymerase (RdRp). Machine learning models also predicted 4HD, XA and XA-E as potential anti-ZIKV inhibitors. Enzymatic and kinetic assays confirmed chalcone inhibition of the ZIKV NS2B-NS3 protease allosteric site with IC50s from 18 to 50 µM. Activity assays also revealed that XA, but not 4HD or XA-E, inhibited the allosteric site of the RdRp, with an IC50 of 6.9 µM. Finally, we tested these chalcones for their anti-viral activity in vitro with Vero cells. 4HD and XA-E displayed anti-ZIKV activity with EC50 values of 6.6 and 22.0 µM, respectively, while XA displayed relatively weak anti-ZIKV activity with whole cells. With their simple structures and relative ease of modification, the chalcones represent attractive candidates for hit-to-lead optimization in the search of new anti-ZIKV therapeutics.
Assuntos
Angelica , Chalcona , Chalconas , Infecção por Zika virus , Zika virus , Angelica/química , Animais , Chalcona/farmacologia , Chalconas/química , Chalconas/farmacologia , Chlorocebus aethiops , Humanos , RNA , RNA Polimerase Dependente de RNA , Células Vero , Replicação ViralRESUMO
Antiviral drug discovery requires the development of reliable biochemical and cellular assays that can be performed in high-throughput screening (HTS) formats. The flavivirus non-structural (NS) proteins are thought to co-translationally assemble on the endoplasmic reticulum (ER) membranes, forming the replication complex (RC). The NS3 and NS5 are the most studied enzymes of the RC and constitute the main targets for drug development due to their crucial roles in viral genome replication. NS3 protease domain, which requires NS2B as its cofactor, is responsible for the cleavage of the immature viral polyprotein into the mature NS proteins, whereas NS5 RdRp domain is responsible for the RNA replication. Herein, we describe in detail the protocols used in replicon-based screenings and enzymatic assays to test large compound libraries for inhibitors of the Zika virus (ZIKV) replication. Replicons are self-replicating subgenomic systems expressed in mammalian cells, in which the viral structural genes are replaced by a reporter gene. The inhibitory effects of compounds on viral RNA replication can be easily evaluated by measuring the reduction in the reporter protein activity. The replicon-based screenings were performed using a BHK-21 ZIKV replicon cell line expressing Renilla luciferase as a reporter gene. To characterize the specific targets of identified compounds, we established in-vitro fluorescence-based assays for recombinantly expressed NS3 protease and NS5 RdRp. The proteolytic activity of the viral protease was measured by using the fluorogenic peptide substrate Bz-nKRR-AMC, while the NS5 RdRp elongation activity was directly detected by the increase of the fluorescent signal of SYBR Green I during RNA elongation, using the synthetic biotinylated self-priming template 3'UTR-U30 (5'-biotin-U30-ACUGGAGAUCGAUCUCCAGU-3').
Assuntos
Infecção por Zika virus , Zika virus , Animais , Antivirais/metabolismo , Ensaios de Triagem em Larga Escala , Mamíferos , Replicação Viral , Zika virus/genética , Infecção por Zika virus/tratamento farmacológicoRESUMO
Dioxygenases are nonheme iron enzymes that biodegrade recalcitrant compounds, such as catechol and derivatives, released into the environment by modern industry. Intradiol dioxygenases have attracted much attention due to the interest in their use for bioremediation, which has demanded efforts towards understanding their action mechanism and also how to control it. The role of unexpected amphipatic molecules, observed in crystal structures of intradiol dioxygenases, during catalysis has been poorly explored. We report results obtained with the intradiol enzyme chlorocatechol 1,2-dioxygenase (1,2-CCD) from Pseudomonas putida subjected to delipidation. The delipidated enzyme is more stable and shows more cooperative thermal denaturation. The kinetics changes from Michaelis-Menten to a cooperative scheme, indicating that conformational changes propagate between monomers in the absence of amphipatic molecules. Furthermore, these molecules inhibit catalysis, yielding lower v(max) values. To the best of our knowledge, this is the first report concerning the effects of amphipatic molecules on 1,2-CCD function.