Identification of a Pyridopyrimidinone Inhibitor of Orthopoxviruses from a Diversity-Oriented Synthesis Library

Abstract

Orthopoxviruses include the prototypical vaccinia virus, the emerging infectious agent monkeypox virus, and the potential biothreat variola virus (the causative agent of smallpox). There is currently no FDA-approved drug for humans infected with orthopoxviruses. We screened a diversity-oriented synthesis library for new scaffolds with activity against vaccinia virus. This screen identified a nonnucleoside analog that blocked postreplicative intermediate and late gene expression. Viral genome replication was unaffected, and inhibition could be elicited late in infection and persisted upon drug removal. Sequencing of drugresistant viruses revealed mutations predicted to be on the periphery of the highly conserved viral RNA polymerase large subunit. Consistent with this, the compound had broad-spectrum activity against orthopoxviruses in vitro. These findings indicate that novel chemical synthesis approaches are a potential source for new infectious disease therapeutics and identify a potentially promising candidate for development to treat orthopoxvirus-infected individuals.

Activation of Stress Response Pathways Promotes Formation of Antiviral Granules and Restricts Virus Replication

“…Fluorescent virus reporter assays using LREV (late mCherry-and early VENUS-expressing) virus were performed as previously described (29,30). 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) cytotoxicity assays were performed as previously described (31,32).…”

“…6E) were added at 1 hpi, fixed at 7 hpi, and processed for immunofluorescence as described above. Concentrations of 1 g/ml 1-␤-D-arabinofuranosylcytosine (AraC) (catalog number C6645; Sigma), 50 M isatin ␤-thiosemicarbazone (IBT) (catalog number NC9075202; Fisher Scientific), 50 g/ml rifampin (catalog number R3501; Sigma), and 5 M ST-246 (a kind gift of Siga Labs, Corvallis, OR) were used as previously described (30,34,35). Type 1 interferon (IFN) was added 24 h prior to infection at 200 U/ml (human beta 1a interferon, catalog number 11415-1; PBL Interferon Source).…”

Vaccinia Reporter Viruses for Quantifying Viral Function at All Stages of Gene Expression

“…Infection and observation via fluorescence microscope (see Figure 2B) allows for a cell-by-cell examination of viral stage progression, which could be useful in a mixed-cell culture setting, such as infection of a tissue, or in viral or bacterial co-infection conditions. Additionally, while these viruses were created for the purpose of antiviral screening 8 , we expect them to be of equal usefulness in basic research investigating the life cycle of vaccinia virus. For example, these viruses can easily be modified using traditional techniques to either lack or overexpress viral proteins; the kinetics of virus gene expression and spread can then be monitored in real time for all stages of viral transcription.…”

“…Due to the complexity of poxvirus replication and the lack of existing tools available to assay real-time changes in viral gene expression, we have developed a suite of single and multi stage reporter viruses 7,8 . As described in previous publications, these viruses express one, two, or three spectrally distinct fluorescent proteins from native vaccinia promoters during early, intermediate, or late stages in infection.…”

“…Dilute control compounds into infection media. Recommended control compounds include: 3.6 μM (1 μg/ml) 1-β-DArabinofuranosylcytosine (AraC), 50 μM (11.7 μg/ml) isatin β -thiosemicarbazone (IBT), 60 μM (50 μg/ml) Rifampicin, or 5 μM (1.9 μg/ ml) ST-246 8,15,16 . See Representative Results for expected effects.…”