Targeting flavivirus RNA dependent RNA polymerase through a pyridobenzothiazole inhibitor

AbstractRNA dependent RNA polymerases (RdRp) are essential enzymes for flavivirus replication. Starting from an in silico docking analysis we identified a pyridobenzothiazole compound, HeE1-2Tyr, able to inhibit West Nile and Dengue RdRps activity in vitro, which proved effective against different flaviviruses in cell culture. Crystallographic data show that HeE1-2Tyr binds between the fingers domain and the priming loop of Dengue virus RdRp (Site 1). Conversely, enzyme kinetics, binding studies and mutational analyses suggest that, during the catalytic cycle and assembly of the RdRp-RNA complex, HeE1-2Tyr might be hosted in a distinct binding site (Site 2). RdRp mutational studies, driven by in silico docking analysis, allowed us to locate the inhibition Site 2 in the thumb domain. Taken together, our results provide innovative concepts for optimization of a new class of anti-flavivirus compounds

Discovery of novel dengue virus NS5 methyltransferase non-nucleoside inhibitors by fragment-based drug design

International audienc

A single mutation in the chikungunya virus RNA polymerase causes resistance to Favipiravir (T-705)

T-705, also known as Favipiravir, is a small-molecule inhibitor that is currently in clinical development for the treatment of influenza virus infections. This molecule also inhibits the replication of a broad spectrum of other RNA viruses. In the host cell, T-705 is converted into its active metabolite, the ribonucleoside analogue T-705RTP. However, the precise molecular mechanism of action against influenza virus or any other virus remains to be elucidated. We here demonstrate that T-705 inhibits the replication of laboratory strains and clinical isolates of chikungunya virus (CHIKV) [EC50 = 2-25 µM]. When given orally to CHIKV-infected AG129 mice in a pre- or post-exposure scenario, T-705 reduced virus-induced mortality by 85% and 65%, respectively. T-705 resistant CHIKV variants all acquired a K291R mutation in motif F1 of the RNA-dependent RNA polymerase. Reverse-engineering of this mutation in an infectious clone of CHIKV corroborated the link between the mutant genotype and the compound-resistant phenotype. Interestingly, the lysine in motif F1 is also highly conserved in positive-stranded RNA viruses in general, again highlighting the importance of this amino acid. Although our observations do not exclude additional mechanisms of action against other viruses, this study lifts an important tip of the veil on the precise molecular mechanism of the antiviral activity of T-705 against (alpha)viruses and may help to design other potent broad-spectrum antivirals. Furthermore, this is the first report of resistance of any of the viruses that are susceptible to T-705.status: publishe

Novel 2-phenyl-5-[(E)-2-(thiophen-2-yl)ethenyl]-1,3,4-oxadiazole and 3-phenyl-5-[(E)-2-(thiophen-2-yl)ethenyl]-1,2,4-oxadiazole derivatives as dengue virus inhibitors targeting NS5 polymerase

International audienceno abstrac

Japanese encephalitis in a French traveler to Nepal

International audienceJapanese encephalitis is frequent in Asia, with a severe prognosis, but rare in travelers. Culex mosquitoes transmit Japanese encephalitis virus. Risk factors are destination, duration of stay, summer and fall seasons, outdoor activities, and type of accommodation. We report the case of a French traveler to Nepal with neutralization-based serological confirmed Japanese encephalitis. He presented classical clinical (viral syndrome before an encephalitis status with behavioral disorder, global hypotonia, mutism, movement disorders, seizure , and coma), radiological (lesions of thalami, cortico-spinal tracts, and brainstem) and biological features (lymphocytic meningitis). Nowadays, the presence of Japanese encephalitis virus in Nepal, including mountain areas, is established but Japanese encephalitis remains rare in travelers returning from this area and neurologist physicians need to become familiar with this. We recommend vaccination for travelers spending a long period of time in Nepal and having at-risk outdoor activities

Functionalized 2,1-benzothiazine 2,2-dioxides as new inhibitors of Dengue NS5 RNA-dependent RNA polymerase

Over recent years, many RNA viruses have been “re-discovered”, including life-threatening flaviviruses, such as Dengue, Zika, and several encephalitis viruses. Since no specific inhibitors are currently available to treat these infections, there is a pressing need for new therapeutics. Among the flaviviral proteins, NS5 RNA-dependent RNA polymerase (RdRp) represents a validated target being essential for viral replication and it has no human analog. To date, few NS5 RdRp inhibitor chemotypes have been reported and no inhibitors are currently in clinical development. In this context, after an in vitro screening against Dengue 3 NS5 RdRp of our in-house HCV NS5B inhibitors focused library, we found that 2,1-benzothiazine 2,2-dioxides are promising non-nucleoside inhibitors of flaviviral RdRp with compounds 8 and 10 showing IC50 of 0.6 and 0.9 μM, respectively. Preliminary structure-activity relationships indicated a key role for the C-4 benzoyl group and the importance of a properly functionalized C-6 phenoxy moiety to modulate potency. Compound 8 acts as non-competitive inhibitor and its proposed pose in the so-called N pocket of the RdRp thumb domain allowed to explain the key contribution of the benzoyl and the phenoxy moieties for the ligand binding

Approved drugs screening against the nsP1 capping enzyme of Venezuelan equine encephalitis virus using an immuno-based assay

International audienceAlphaviruses such as the Venezuelan equine encephalitis virus (VEEV) are important human emerging pathogens transmitted by mosquitoes. They possess a unique viral mRNA capping mechanism catalyzed by the viral non-structural protein nsP1, which is essential for virus replication. The alphaviruses capping starts by the methylation of a GTP molecule by the N7-guanine methyltransferase (MTase) activity; nsP1 then forms a covalent link with m7GMP releasing pyrophosphate (GT reaction) and the m7GMP is next transferred onto the 5'-diphosphate end of the viral mRNA to form a cap-0 structure. The cap-0 structure decreases the detection of foreign viral RNAs, prevents RNA degradation by cellular exonucleases, and promotes viral RNA translation into proteins. Additionally, reverse-genetic studies have demonstrated that viruses mutated in nsP1 catalytic residues are both impaired towards replication and attenuated. The nsP1 protein is thus considered an attractive antiviral target for drug discovery. We have previously demonstrated that the guanylylation of VEEV nsP1 can be monitored by Western blot analysis using an antibody recognizing the cap structure. In this study, we developed a high throughput ELISA screening assay to monitor the GT reaction through m7GMP-nsP1 adduct quantitation. This assay was validated using known nsP1 inhibitors before screening 1220 approved compounds. 18 compounds inhibiting the nsP1 guanylylation were identified, and their IC50 determined. Compounds from two series were further characterized and shown to inhibit the nsP1 MTase activity. Conversely, these compounds barely inhibited a cellular MTase demonstrating their specificity towards nsP1. Analogues search and SAR were also initiated to identify the active pharmacophore features. Altogether the results show that this HT enzyme-based assay is a convenient way to select potent and specific hit compounds targeting the viral mRNA capping of Alphaviruses