Article Figures & Data
Figures
- FIG. 1.
Structures of benzimidazole ribonucleosides. The asterisk indicates the position of the tritium label in [3H]BDCRB. The moieties at positions R1 and R2 were as follows: in DRB, H and OH, respectively; in TCRB, Cl and OH, respectively; in BDCRB, Br and OH, respectively; in 5′-TCRB, Cl and H, respectively; and in 5′-BDCRB, Br and H, respectively.
- FIG. 2.
Reverse-phase HPLC of extracts from uninfected CEM cells incubated with [3H]BDCRB. The cells were incubated with 10 μCi of (9.1 μM) [3H]BDCRB per ml for 48 h, after which nucleosides and nucleotides were isolated by trichloroacetic acid precipitation and Freon extraction. Reverse-phase separations were performed on a μBondapak C18 column (3.9 by 300 mm), as detailed in the text. The retention times of naturally occurring nucleoside monophosphates (NMP’s) and BDCRB-5′-MP were determined by addition of standards to the extract and are indicated by horizontal bars.
- FIG. 3.
Cesium sulfate isopycnic gradient separation of cellular RNA and DNA. CEM cells were incubated with [3H]BDCRB (•) at 1 μM (1.1 μCi/ml) for 48 h, and the amount of labeled acid-precipitable material was determined. Parallel incubations were performed under identical conditions with 1 μCi of [3H]Urd per ml (○) as a marker for incorporation into RNA and DNA (following metabolism to dTTP). (Inset) Nature of gradient determined by refractive index (R.I.).
- FIG. 4.
Anion-exchange HPLC analysis of an extract from HCMV-infected HFF cells incubated with [3H]BDCRB. Following infection at an MOI of 0.15, the cells were incubated with 10 μCi of (9.1 μM) [3H]BDCRB per ml for 5 days. Nucleosides and nucleotides were extracted and separated by anion-exchange HPLC on an Ultrasil-AX 10-μm column (4.6 by 250 mm), as detailed in the text. The dotted line is the response from the UV detector; the line defined by solid circles is the counts per minute from labeled metabolites. Horizontal bars indicate the ranges in which naturally occurring nucleoside monophosphates (NMP’s) and nucleoside diphosphates (NDP’s) eluted. The retention time of BDCRB-5′-MP was determined by the addition of a nonradioactive standard to the infected cell extract. That of BDCRB-5′-diphosphate (BDCRB-5′-DP) was determined in a separate run.
- FIG. 5.
Benzimidazole nucleosides inhibited HCMV replication but not viral DNA synthesis. Selected concentrations of TCRB (▪) and 5′-dTCRB (▵) were incubated in duplicate wells with HFF cells infected with HCMV at 3 PFU/cell. Viral DNA was detected by DNA-DNA hybridization, and yield was quantified by serial dilution of supernatants across 96-well culture plates seeded with HFF cells.
- FIG. 6.
Inhibition of cleavage of concatemeric DNA to monomeric genomic units by benzimidazole nucleoside analogs. Selected concentrations of TCRB (▪), BDCRB (◊), 5′-dTCRB (▵), and 1263W94 (•) were incubated in duplicate wells with HFF cells infected with HCMV at 3 PFU/cell. After 3 days, the cells were suspended in agarose and digested to release DNA. The DNA was separated by contour-clamped homogeneous electric field separation, transferred to a nylon membrane, and probed for HCMV-specific sequences. The amount of genomic-length monomeric DNA is expressed as a percentage of the total amount of viral DNA in the well. In the absence of test compounds, monomeric DNA was 25% ± 5% of the total DNA in three determinations.
Tables
- TABLE 1.
Activities of benzimidazole nucleosides against HCMV isolates sensitive and resistant to TCRB and BDCRB
Compound Mean ± SD IC50 or IC90 (μM)a for virus isolateb: Towne (wild type) B11 (UL89) D10 (UL89) C4 (UL56 + UL89) TCRB 2.8 ± 1.1 16 ± 4.9 17 ± 7.6 56 ± 31 BDCRB 1.2 ± 0.78 12 ± 6.7 7.3 ± 5.8 28 ± 7.4 5′-dTCRB 0.58 ± 0.46c 2.9 ± 0.5c 3.1 ± 0.14c 13d 5′-dBDCRB 0.15 ± 0.001c 5′-dBDCRBe 0.06 ± 0.01 3.0 ± 0.8 GCV 1.5 ± 0.6 3.1 ± 0.2c 4.6 ± 2.1 5.5 ± 3.2 ↵ a Results are from plaque reduction assays performed in duplicate with the indicated isolates of HCMV selected for resistance to TCRB, as described in the text. The 50% inhibitory concentration (IC50s) were calculated from three to six separate experiments with at least four drug concentrations each, unless marked otherwise.
↵ b The genotype of each isolate is given in parentheses. The UL number specifies the gene with the mutation that confers resistance to TCRB; the UL89 mutation is D344E, and the UL56 mutation is Q204R (25). B11 and D10 are different isolates with the same mutation that results in resistance to TCRB, which we have described previously (25).
↵ c The experiment was performed twice, in duplicate, by using at least four drug concentrations.
↵ d The experiment was performed once, in duplicate, by using five drug concentrations.
↵ e The results are presented as 90% inhibitory concentrations (IC90s) from two yield reduction experiments performed in duplicate or triplicate wells by using at least five drug concentrations.
- TABLE 2.
Mode-of-action comparisons among benzimidazole nucleosides
Test compound IC90a (μM) by: IC50 (μM) for DNA synthesisd Maturation assayb Yield assayc BDCRB 0.08 0.29 ± 0.15 >25e 5′-dBDCRB <0.04e 0.018 ± 0.003 >5 5′-dTCRB 0.03 0.36 ± 0.26 >25 1263W94 27 0.55 ± 0.06 0.3 ↵ a IC90, 90% inhibitory concentration.
↵ b The maturation assay was a single-cycle yield reduction experiment in which 20 μM BDCRB was removed from HCMV-infected cultures and replaced at 96 h postinfection by the test compound.
↵ c Data are from two or three single-cycle yield reduction experiments in which the test compound was present for the entire experiment.
↵ d The assay was a single-cycle yield experiment in which the amount of DNA was measured by a hybridization assay. IC50, 50% inhibitory concentration.
↵ e The 50 or 90% inhibitory concentration was not reached at the noted highest or lowest concentration tested.
