rupintrivir and Enterovirus-Infections

We describe the development, optimisation, and validation of an automated, cell-based and high-throughput screening assay using existing luminescence-based ATPlite reagents for identifying antiviral compounds that inhibit enterovirus replication. Antiviral efficacy was determined by measuring the ATP levels in cells that were protected from the viral cytopathic effect (CPE) by the antiviral compounds pleconaril and rupintrivir. CPE-based assay conditions were optimised at a cell density of 5000 cells/well and a viral infection dose of 100 CCID

Topics: Animals; Antiviral Agents; Chlorocebus aethiops; Cytopathogenic Effect, Viral; Enterovirus; Enterovirus Infections; HeLa Cells; Humans; Isoxazoles; Luminescent Measurements; Microbial Sensitivity Tests; Oxadiazoles; Oxazoles; Phenylalanine; Pyrrolidinones; Valine; Vero Cells

Cyanohydrin derivatives as enterovirus 71 (EV71) 3C protease (3C(pro)) inhibitors have been synthesized and assayed for their biochemical and antiviral activities. Compared with the reported inhibitors, cyanohydrins (1S,2S,2'S,5S)-16 and (1R,2S,2'S,5S)-16 exhibited significantly improved activity and attractive selectivity profiles against other proteases, which were a result of the specific interactions between the cyanohydrin moiety and the catalytic site of 3C(pro). Cyanohydrin as an anchoring group with high selectivity and excellent inhibitory activity represents a useful choice for cysteine protease inhibitors.

Topics: 3C Viral Proteases; Antiviral Agents; Crystallography, X-Ray; Cysteine Endopeptidases; Enterovirus A, Human; Enterovirus Infections; Humans; Molecular Docking Simulation; Nitriles; Protein Binding; Viral Proteins

Antivirals against enterovirus 71 (EV71) are urgently needed. We demonstrate that the novel enteroviral protease inhibitor (PI) SG85 and capsid binder (CB) vapendavir efficiently inhibit the in vitro replication of 21 EV71 strains/isolates that are representative of the different genogroups A, B, and C. The PI rupintrivir, the CB pirodavir, and the host-targeting compound enviroxime, which were included as reference compounds, also inhibited the replication of all isolates. Remarkably, the CB compound pleconaril was devoid of any anti-EV71 activity. An in silico docking study revealed that pleconaril-unlike vapendavir and pirodavir-lacks essential binding interactions with the viral capsid. Vapendavir and SG85 (or analogues) should be further explored for the treatment of EV71 infections. The data presented here may serve as a reference when developing yet-novel inhibitors.

Topics: Antiviral Agents; Benzimidazoles; Capsid; Capsid Proteins; Drug Resistance, Viral; Enterovirus A, Human; Enterovirus Infections; Isoxazoles; Molecular Docking Simulation; Oximes; Phenylalanine; Piperidines; Protease Inhibitors; Protein Binding; Pyridazines; Pyrrolidinones; Sulfonamides; Valine; Virus Replication

Enterovirus-71 (EV71) infections can cause life-threatening diseases with neurological symptoms. Currently, no direct targeting antivirals are available to combat severe EV71 infection. Rupintrivir (AG7088) is a compound originally designed for Rhinovirus 3C protease. Previous computational analyses by us and crystallography studies by others suggested that rupintrivir is also a high affinity inhibitor to EV71 3C. Thus, we aimed to further evaluate its anti-EV71 activity in vivo at clinically acceptable doses. It was observed that administration of rupintrivir in suckling mice largely protected them from limb paralysis and dramatically improved survival (38.5% DMSO vs. 90.9% at 0.1mg/kg, p=0.006). Histological, immunohistochemical and quantitative RT-PCR analyses confirmed that rupintrivir profoundly alleviated virus induced necrotizing myositis, suppressed viral RNA and blocked EV71 VP1 expression in various tissues. In conclusion, we established that rupintrivir can strongly contain the spread of EV71 infection in vivo at a clinically acceptable dose (as low as 0.1mg/kg). As its safety has been fully tested in previous clinical trials, rupintrivir is suitable for immediate evaluation of potential benefits in EV71-infected individuals with life-threatening neurological symptoms.

Topics: Animals; Antiviral Agents; Drug Evaluation, Preclinical; Enterovirus A, Human; Enterovirus Infections; Female; Humans; Infant; Isoxazoles; Male; Mice; Phenylalanine; Pyrrolidinones; Valine; Virus Replication

Enterovirus 71 (EV71) can cause severe diseases and even lead to death in children. There is no vaccine or specific antiviral therapy to prevent or cure EV71 infection. Although interferon (IFN)-α has been used in the treatment of several viral infections, we found that IFN-α alone was ineffective in restricting EV71 replication in Vero cells.. Through a bioinformatics analysis, several cellular proteins in the IFN response pathway were identified as susceptible substrates that might be degraded by the EV71-encoded 3C protease (3C(pro)).. Indeed, IRF9 was shown to be vulnerable to 3C(pro) cleavage, as revealed by enzyme-based and cell-based assays. Thus, the IFN-mediated antiviral mechanism compromised by the viral 3C(pro) in EV71-infected cells may be accountable, at least partially, for that IFN-α cannot inhibit EV71 replication. Because rupintrivir (AG7088) is known to be an effective EV71 inhibitor, we investigated the effects of the combination of rupintrivir and IFN-α on EV71 replication and found that they strongly synergized with each other in inhibiting EV71 replication.. Because rupintrivir was shown to be generally tolerable in earlier clinical investigations, it is worth evaluating whether a combination of rupintrivir and IFN-α could be an effective treatment for EV71.

Topics: Algorithms; Animals; Antiviral Agents; Blotting, Western; Chlorocebus aethiops; DNA Primers; Down-Regulation; Drug Synergism; Drug Therapy, Combination; Enterovirus A, Human; Enterovirus Infections; Humans; Interferon-alpha; Interferon-Stimulated Gene Factor 3, gamma Subunit; Isoxazoles; Phenylalanine; Polymerase Chain Reaction; Pyrrolidinones; Recombinant Proteins; Rhabdomyosarcoma; Tumor Cells, Cultured; Valine; Vero Cells; Virus Replication

To evaluate the suitability of rupintrivir against Enterovirus 71 (EV71) induced severe clinical symptoms using computational methods.. The structure of EV71 3C protease was predicted by homology modeling. The binding free energies between rupintrivir and EV71 3C and human rhinovirus 3C protease were computed by molecular dynamics and molecular mechanics Poisson-Boltzmann/surface area and molecular mechanics generalized-born/surface area methods. EV71 3C fragments obtained from clinical samples collected during May to July 2008 in Shanghai were amplified by reverse-transcription and polymerase chain reaction and sequenced.. We observed that rupintrivir had favorable binding affinity with EV71 3C protease (-10.76 kcal/mol). The variability of the 3C protein sequence in isolates of various outbreaks, including those obtained in our hospital from May to July 2008, were also analyzed to validate the conservation of the drug binding pocket.. Rupintrivir, whose safety profiles had been proved, is an attractive candidate and can be quickly utilized for treating severe EV71 infection.

Topics: 3C Viral Proteases; Amino Acid Sequence; Antiviral Agents; Cysteine Endopeptidases; Enterovirus A, Human; Enterovirus Infections; Humans; Isoxazoles; Molecular Sequence Data; Phenylalanine; Protease Inhibitors; Pyrrolidinones; Retrospective Studies; Treatment Outcome; Valine; Viral Proteins

A real-time assay system that allows monitoring of intracellular human enterovirus (HEV) protease activity was established using the principle of fluorescence resonance energy transfer (FRET). It was accomplished by engineering cells to constitutively express a genetically encoded FRET probe. The FRET-based probe was designed to contain an enterovirus 71 3C protease (3C(pro)) cleavage motif flanked by the FRET pair composed of green fluorescent protein 2 and red fluorescent protein 2 (DsRed2). Efficient FRET from the stable line was detected in a real-time manner by fluorescence microscopy, and the disruption of FRET was readily monitored upon HEV infection. The level of the repressed FRET was proportional to the input virus titer and the infection duration as measured by the fluorometric method. The FRET biosensor cell line was also responsive to other related HEV serotypes, but not to the phylogenetically distant herpes simplex virus, which was confirmed by Western blot analysis. The FRET biosensor was then utilized to develop a format for the determination of antiviral susceptibility, as the reduced FRET appeared to reflect viral replication. Evaluations of the FRET biosensor system with representative HEV serotypes demonstrated that their susceptibilities to a 3C(pro) inhibitor, rupintrivir, were all accurately determined. In summary, this novel FRET-based system is a means for rapid detection, quantification, and drug susceptibility testing for HEVs, with potential for the development of a high-throughput screening assay.

Topics: 3C Viral Proteases; Antiviral Agents; Biosensing Techniques; Blotting, Western; Cell Fusion; Cell Line; Cysteine Endopeptidases; Enterovirus A, Human; Enterovirus Infections; Fluorescence Resonance Energy Transfer; Fluorometry; Green Fluorescent Proteins; HeLa Cells; Herpesvirus 1, Human; Humans; Image Processing, Computer-Assisted; Isoxazoles; Microbial Sensitivity Tests; Phenylalanine; Plasmids; Pyrrolidinones; Recombinant Proteins; Transfection; Valine; Viral Plaque Assay; Viral Proteins