Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2012 Mar;86(5):2632-40.
doi: 10.1128/JVI.05416-11. Epub 2011 Dec 28.

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

Affiliations
Free PMC article

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

Ken Dower et al. J Virol. .
Free PMC article

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 drug-resistant 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.

Figures

Fig 1
Fig 1
(A) Screen summary. Confluent A549 cells were infected with a fluorescence reporter vaccinia virus at a high MOI. A diversity-oriented synthesis library was screened for compounds that reduced reporter expression at 12 h postinfection. (B) Structure and synthesis of CMLDBU6128, identified from this screen. (C) Late viral reporter expression from high-MOI infections of A549 and HeLa cells (12 h postinfection) and cell cytotoxicity determination (24 h) over a dose range of CMLDBU6128. (D) Sequential low-MOI–high-MOI infection. A549 cells were infected with Venus-expressing reporter virus at a low MOI, and infections proceeded for 48 h (primary infection). Medium exchange was followed by high-MOI infection with an mCherry-expressing reporter virus for 24 h (secondary infection). Images were taken using a 2.5× objective.
Fig 2
Fig 2
(A) Single-cycle growth yields on A549 cells. (B and C) Multicycle growth yields on A549 and Vero cells, respectively. (D) Phase-contrast microscopy of cell monolayers of A549 and Vero cells at the endpoint of multistep growth for panels B and C (10× objective). All growth curves were created with vaccinia virus strain Western Reserve. (E) Time course of vaccinia virus growth in the presence of DMSO (filled circles) or CMLDBU6128 (open circles). The log value is listed above each point. Compound concentrations used were as follows: 20 μM CMLDBU6128, 5 μM ST-246, and 1 μg/ml of AraC.
Fig 3
Fig 3
(A) A549 cells were infected with early Venus (EV; A), intermediate Venus (IV; B), and late Venus (LV; C) reporter viruses at a high MOI in the presence of DMSO or CMLDBU6128. Viral reporter fluorescence was measured hourly for 12 h. (D) Promoter-dependent Venus mRNA accumulation over a time course of infection with EV, IV, or LV virus. (E) Microscopy of A549 cells after high-MOI infection with TrpV reporter virus, which contains early Venus, intermediate mCherry, and late TagBFP in a single virus. Uninfected and DMSO- or CMLDBU6128-treated cells at 12 h postinfection are shown.
Fig 4
Fig 4
(A) Venus DNA copy number per A549 cell equivalent (c.e.) after high-MOI infection with LV reporter virus. Venus copy numbers were calculated from a standard curve of Venus-containing plasmid. (B) High-magnification microscopy after infection of A549 cells with Venus-A4L virus. DMSO- or CMLDBU6128-treated cells are shown at 1, 8, and 24 h postinfection. Cells were fixed and stained with DAPI before visualization of viral DNA factories (DAPI) and Venus-A4L (yellow fluorescent protein [YFP]). Images were taken using a 63× objective. Arrowheads point to representative perinuclear viral factories.
Fig 5
Fig 5
(A) [35S]methionine labeling of cells to visualize active translation. Densitometry values are provided normalized to 12-h mock-infected cells (−). A representative slice of the Coomassie blue-stained gel is also shown. (B) Time course analysis of Venus expression in A549 cells subjected to various treatments. The legend indicates the treatments given at 0 h and 10 h. Reporter fluorescence was measured hourly for 12 h after the initial 10-h treatment (until 22 h). “LV” indicates high-MOI infection with the late Venus reporter virus. For example, LV+DMSO/CMLDBU6128 indicates an initial treatment of high-MOI LV reporter virus in the presence of DMSO, followed 10 h later by medium removal and replacement with CMLDBU6128-containing medium.
Fig 6
Fig 6
(A) Fluorescent foci from low-MOI infection of A549 cell monolayers with parental LV virus or two selection pools (DR1 and DR2) in the absence or presence of CMLDBU6128. Images were taken using a 2.5× objective. (B) Single-cycle growth yields for A549 cells infected with four plaque-purified viruses: two from pool DR1 (DR1a and DR1b) and two from pool DR2 (DR2b and DR2c). (C) Coding changes identified by whole-genome sequencing. DR1a and DR1b had an identical J6R V576G mutation, and DR2b and DR2c had an identical J6R A954V mutation. (D) Marker rescue experiments using fragments containing the DR1 (J6R V576G) mutation or DR2 (J6R A954V) mutation. The graph shows fold increases in viral progeny following 2 days of incubation with CMLDBU6128 relative to mock rescue using the corresponding fragment from the wild-type virus.
Fig 7
Fig 7
Single-cycle growth yields for A549 cells infected with monkeypox virus Zaire 1979 (MPOX), wild-type cowpox virus (CPX), or vaccinia virus (VACV) IHDJ in the absence or presence of CMLDBU6128.
Fig 8
Fig 8
(A) Chemical structures of the (R)-enantiomer (R)-CMLDBU6128 (left) and of nevirapine (right). (B) Computationally generated overlay of (R)-CMLDBU6128 and nevirapine. The overlay was generated using the OpenEye Scientific Software shape similarity comparison program ROCS.

Similar articles

See all similar articles

Cited by 6 articles

See all "Cited by" articles

Publication types

MeSH terms

LinkOut - more resources

Feedback