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Clinical and Diagnostic Laboratory Immunology, November 2001, p. 1279-1281, Vol. 8, No. 6
Albany Medical College, Albany, New
York,1 and GlaxoSmithKline, Research
Triangle Park, North Carolina2
Received 25 May 2001/Returned for modification 5 June 2001/Accepted 20 August 2001
The average 50% inhibitory concentration (IC50) values
for AD169 were 0.22 ± 0.09 µM 1263W94 and 5.36 ± 0.12 µM ganciclovir. For 35 human cytomegalovirus (HCMV) clinical isolates
the average IC50 was 0.42 ± 0.09 µM 1263W94, and
for 26 ganciclovir-susceptible HCMV clinical isolates the average
IC50 was 3.78 ± 1.62 µM ganciclovir. Nine HCMV
clinical isolates that were resistant to ganciclovir were completely
susceptible to 1263W94.
Human cytomegalovirus (HCMV) causes
considerable morbidity and mortality in the immunocompromised host
(18, 19). Organ transplant recipients suffer from
retinitis, gastrointestinal disease, hepatitis, and pneumonia caused by
HCMV infections, whereas AIDS patients suffer from HCMV-induced
retinitis and other complications (1). The current Food
and Drug Administration-approved chemotherapies for HCMV infections
consist of ganciclovir, foscarnet, cidofovir, and fomivirsen (5,
16, 17, 20). These antiviral drugs are effective against
infections caused by HCMV; however, they are not ideal because of their
toxicity and poor bioavailability. Furthermore, long-term treatment
with these drugs often leads to the selection of drug-resistant mutants
(6, 8, 9). Due to the problems associated with the
currently used antiviral compounds for HCMV infection, there is an
active search for more useful compounds to combat infections with HCMV.
The benzimidazole ribonucleosides represent a new class of antiviral
compounds that inhibit HCMV replication by blocking the processing of
progeny viral DNA (4, 10, 22). In an attempt to make a
more stable derivative of benzimidazole riboside
2-bromo-5,6-dichloro-1- In this report, we show that 1263W94 inhibits the replication of the
AD169 laboratory strain of HCMV and 35 HCMV clinical isolates at drug
concentrations that are approximately 10-fold less than those required
by ganciclovir. Nine of the 35 HCMV clinical isolates are resistant to
ganciclovir, and several are also resistant to foscarnet and cidofovir
(2, 3, 7, 8, 9, 11). All of these drug-resistant HCMV
clinical isolates are susceptible to 1263W94. These results show that
1263W94 inhibits the replication of both ganciclovir-susceptible and
single- and multiple-drug-resistant HCMV clinical isolates, confirming
reports that 1263W94 has a mode of action different from that of
ganciclovir, foscarnet, and cidofovir (4). These results
also suggest that this drug is potentially useful for treating patients
infected with HCMV clinical isolates that are resistant to the
currently used antiviral drugs.
Determination of IC50 values of 1263W94 and ganciclovir
for HCMV laboratory strains and clinical isolates by FACS
analysis.
Confluent human foreskin fibroblast cell
monolayers (Clontech, San Diego, Calif.) were infected at low
multiplicity of infection with the AD169 laboratory strain of HCMV, the
1263W94-resistant derivative of AD169, or 35 HCMV clinical isolates in
the presence of various concentrations of 1263W94 or ganciclovir. The
cells were harvested, permeabilized with methanol, and treated with fluorescein isothiocyanate-labeled monoclonal antibodies (MAbs) to
the HCMV immediate-early (IE) or late antigens (direct
fluorescent-antibody reagent 5090 or MAb 1G5.2; Chemicon
International, Inc., Temecula, Calif.), and their drug susceptibilities
were determined by the flow cytometry drug susceptibility (FACS) assay
as described previously (12-14). Nine of the 35 HCMV
clinical isolates were resistant to ganciclovir. The average 50%
inhibitory concentration (IC50) values for the AD169
laboratory strain were 0.22 ± 0.09 µM 1263W94 and 5.36 ± 0.12 µM ganciclovir on the basis of analysis of cells expressing the
IE antigen and 0.31 ± 0.22 µM 1263W94 and 3.44 ± 1.01 µM ganciclovir on the basis of analysis of cells expressing the late
antigen. The IC50 values for 2916rA were 57.08 ± 5.01 µM 1263W94 and 2.34 ± 0.92 µM ganciclovir using the IE antigen and
>20 µM 1263W94 and 2.11 ± 0.98 µM ganciclovir using the late
antigen. The IC50 values for the 35 clinical isolates are
summarized in Table 1. The
IC50 values ranged from 0.11 to 1.22 µM 1263W94, with an
average IC50 value of 0.42 ± 0.22 µM 1263W94, using
the IE antigen. The IC50 values ranged from 0.15 to 1.0 µM 1263W94, with an average IC50 value of 0.40 ± 0.17 µM 1263W94, using the late antigen. The IC50 values
for 26 ganciclovir-susceptible HCMV clinical isolates ranged from 1.22 to 7.59 µM, with an average IC50 value of 3.78 ± 1.62 µM ganciclovir using the IE antigen and ranged from 2.63 to 7.79 µM, with an average IC50 value of 3.76 ± 1.13 µM
ganciclovir, using the late antigen. Average IC50 values
for drug-susceptible HCMV clinical isolates for either drug obtained by
FACS analysis of HCMV-infected cells expressing either the IE or late
antigens were statistically identical. The average IC50
values of 1263W94 for these HCMV clinical isolates were similar to the
average IC50 values for the AD169 laboratory strain.
Compound 1263W94 was 24 times more potent than ganciclovir against the
AD169 laboratory strain of HCMV and approximately 10 times more
effective than ganciclovir for inhibiting the replication of
ganciclovir-susceptible HCMV clinical isolates in human foreskin
fibroblast cell monolayers. The data in Table 1 show that nine of the
HCMV clinical isolates are ganciclovir resistant (IC50
values of greater than 9 µM ganciclovir). Since all 35 HCMV clinical
isolates were susceptible to 1263W96, the 9 ganciclovir-resistant HCMV
clinical isolates were susceptible to 1263W94. These results show
that 1263W94 inhibits the replication of ganciclovir-resistant HCMV
clinical isolates and suggest that 1263W94 may be useful for the
treatment of patients with ganciclovir-resistant HCMV disease.
1071-412X/01/$04.00+0 DOI: 10.1128/CDLI.8.6.1279-1281.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Inhibition of Ganciclovir-Susceptible and
-Resistant Human Cytomegalovirus Clinical Isolates by the
Benzimidazole L-Riboside 1263W94
ABSTRACT
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-D-ribofuranosyl benzimidazole
(BDCRB), the L form of the compound, was synthesized (4). The L-riboside benzimidazole analogue of
BDCRB, 1263W94, has potent activity against HCMV laboratory strains and
clinical isolates as well as Epstein-Barr virus (4, 23).
Preliminary studies suggest that 1263W94 inhibits HCMV replication by
blocking viral DNA synthesis, but not by an effect on the viral DNA
polymerase or the phosphotransferase encoded by the UL97 gene
(4).
TABLE 1.
IC50 values of 1263W94 and ganciclovir
for HCMV clinical isolates by FACS
Comparison of IC50 values of 1263W94 for HCMV clinical
isolates determined by FACS assay, PRA, and the DNA hybridization
assay.
To determine if the FACS assay yields IC50
values for 1263W94 similar to those obtained with more-traditional
methods such as the plaque reduction assay (PRA) (21) and
the DNA hybridization assay (8, 9), the IC50
values for selected numbers of ganciclovir-susceptible and
ganciclovir-resistant HCMV clinical isolates were determined by all
three methods. The data are presented in Table
2. The average IC50 values of
1263W94 for 11 HCMV clinical isolates obtained with the FACS assay, the
PRA, and the DNA hybridization assay were 0.38 ± 0.13, 0.35 ± 0.17, and 0.08 ± 0.04 µM, respectively. The average
ganciclovir IC50 values for nine ganciclovir-susceptible HCMV clinical isolates obtained for the FACS assay, PRA, and the DNA
hybridization assay were 3.54 ± 1.74, 3.46 ± 2.27, and 0.58 ± 0.34 µM, respectively. V917401-r is resistant to ganciclovir, and
MR11979-r is resistant to ganciclovir, foscarnet, and cidofovir (14). For these isolates the IC50 values of
ganciclovir were higher, but not those of 1263W94. The IC50
values of 1263W94 from the PRA are very similar to those obtained with
the FACS assay for all of the HCMV clinical isolates. However, there is
more variability between the PRA and the FACS assay for ganciclovir. The DNA hybridization assays gave substantially lower IC50
values than either the FACS assay or the PRA for both compounds. This comparison between the FACS assay and the PRA confirms the utility of
the FACS assay for determining IC50 values and extends its use to antiviral compounds that inhibit HCMV replication by a mechanism
different from that of ganciclovir. An analysis of bias and precision
of the IC50 values obtained by the FACS assay and the PRA
for the ganciclovir-susceptible clinical isolates showed a less than
twofold difference.
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ACKNOWLEDGMENTS |
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MR11979, V917401, and ganciclovir were provided by the Virology Quality Assurance Program, Division of AIDS, NIAID. 1263W94 was provided by GlaxoSmithKline.
This work was supported in part by grants AI45350 and AI45257 from the National Institutes of Health and a grant from GlaxoSmithKline.
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FOOTNOTES |
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* Corresponding author. Mailing address: Center for Immunology and Microbial Disease, Albany Medical College, Mail Code 151, 47 New Scotland Ave., Albany, NY 12208. Phone: (518) 262-5174. Fax: (518) 262-5748. E-mail: mcsharj{at}mail.amc.edu.
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Clinical and Diagnostic Laboratory Immunology, November 2001, p. 1279-1281, Vol. 8, No. 6
1071-412X/01/$04.00+0 DOI: 10.1128/CDLI.8.6.1279-1281.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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