Inhibition of Human Cytomegalovirus Replication by Benzimidazole ...

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Oct. 2004, p. 3918–3927 0066-4804/04/$08.00⫹0 DOI: 10.1128/AAC.48.10.3918–3927.2004 Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Vol. 48, No. 10

Inhibition of Human Cytomegalovirus Replication by Benzimidazole Nucleosides Involves Three Distinct Mechanisms David L. Evers,1,2 Gloria Komazin,1,2 Roger G. Ptak,1,2† Dongjin Shin,1 Brian T. Emmer, Leroy B. Townsend,2 and John C. Drach1,2* Department of Biologic and Materials Sciences, School of Dentistry,1 and Interdepartmental Graduate Program in Medicinal Chemistry, College of Pharmacy,2 University of Michigan, Ann Arbor, Michigan Received 3 December 2003/Returned for modification 8 January 2004/Accepted 19 April 2004

The benzimidazole nucleosides 2-bromo-5,6-dichloro-1-(␤-D-ribofuranosyl)benzimidazole (BDCRB) and 2-isopropylamino-5,6-dichloro-1-(␤-L-ribofuranosyl)benzimidazole (1263W94, or maribavir) are potent and selective inhibitors of human cytomegalovirus (HCMV) replication. These inhibitors act by two different mechanisms: BDCRB blocks the processing and maturation of viral DNA, whereas maribavir prevents viral DNA synthesis and capsid nuclear egress. In order to determine by which of these two mechanisms other benzimidazole nucleosides acted, we performed time-of-addition studies and other experiments with selected new analogs. We found that the erythrofuranosyl analog and the ␣-lyxofuranosyl analog acted late in the viral replication cycle, similar to BDCRB. In marked contrast, the ␣-5ⴕ-deoxylyxofuranosyl analog of 2,5,6-trichloro1-(␤-D-ribofuranosyl)benzimidazole (compound UMJD1311) acted early in the replication cycle, too early to be consistent with either mechanism. Similar to other reports on early acting inhibitors of herpesviruses, compound 1311 was multiplicity of infection dependent, an observation that could not be reproduced with UV-inactivated virus. HCMV isolates resistant to BDCRB and maribavir were sensitive to compound 1311, as were viruses resistant to ganciclovir, cidofovir, and foscarnet. The preincubation of host cells with compound 1311 and removal prior to the addition of HCMV did not produce an antiviral cellular response. We conclude that this newly discovered early mode of action occurs at a stage of viral replication after entry to cells but prior to viral DNA synthesis, thereby strongly suggesting that the trisubstituted benzimidazole nucleoside series possesses three distinct biochemical modes of action for inhibition of HCMV replication. gence of cross-resistant strains has been described in clinical settings (15, 17). To alleviate the clinical problems associated with the cross-resistance demonstrated for drugs targeting HCMV viral DNA polymerase, it would be useful to have new drugs with different molecular targets. There is also an unmet need for less toxic and more orally bioavailable therapeutics. In 1995 it was reported that 2,5,6-trichloro-1-(␤-D-ribofuranosyl)benzimidazole (TCRB) and the 2-bromo analog (BDCRB) are potent and selective inhibitors of HCMV replication (Fig. 1) (43). These (S. S. Good, B. S. Owens, L. B. Townsend, and J. C. Drach, Abstr. 7th Int. Conf. Antivir. Res., abstr. 128, 1994) compounds have a novel mechanism of action that does not involve the inhibition of DNA synthesis but does prevent the cleavage of high-molecular-weight viral DNA concatemers to monomeric genomic lengths (47). Resistance to these compounds has been mapped to the UL56 (Q204R) and UL89 (D344E) putative viral gene products (22, 47). These are two of at least seven viral gene products (UL104, UL93, UL89, UL77, UL56, UL52, and UL51) identified as required for viral DNA cleavage and packaging by homology with bacteriophage and herpes simplex virus genes (9). The HCMV UL56 and UL89 proteins form the two subunits of the HCMV putative terminase (36). The large subunit encoded by UL56 is involved in DNA binding and capsid association (7, 36), whereas the small subunit encoded by UL89 is required for cleavage of genomic DNA (36). The ATPase activity of the large subunit UL56 is partially inhibited by BDCRB (37), and high concentrations of BDCRB partially inhibit the UL89-associated nuclease activity (36). Although TCRB and BDCRB are excellent inhibitors of

Human cytomegalovirus (HCMV) causes significant morbidity and mortality in immunocompromised populations. It is a common opportunistic disease in AIDS and bone marrow transplant patients and is often a factor in their deaths (1). HCMV infection has been implicated in increased risk of organ transplant rejection (4), restenosis of coronary arteries following angioplasty (50), and exacerbation of inflammatory bowel diseases (5). It is also a leading cause of birth defects such as deafness and mental retardation (1). Current therapies for HCMV infection include the nucleoside analogs ganciclovir (GCV) (12), its ester prodrug valganciclovir (13), and cidofovir (CDV) (19), plus the pyrophosphate analog foscarnet (PFA) (10) and the antisense phosphorothioate oligonucleotide fomivirsen (29). These drugs suffer from disadvantages which include poor oral bioavailability for all but valganciclovir and certain toxicities. At therapeutic doses GCV is toxic to bone marrow progenitor cells (13), whereas CDV and PFA are toxic to the kidneys and fomivirsen is toxic to the retina (2, 14, 19). Following phosphorylation of GCV to its monophosphate by the viral protein kinase encoded by the UL97 gene, GCV is phosphorylated to its triphosphate, which acts as an inhibitor of the UL54 HCMV DNA polymerase (42). CDV diphosphate and PFA share the same molecular target as GCV triphosphate (17). Drug-resistant strains of HCMV have been reported for all three drugs, and the emer* Corresponding author. Mailing address: School of Dentistry, 1011 N. University Ave., University of Michigan, Ann Arbor, MI 48109-1078. Phone: (734) 763-5579. Fax: (734) 764-4497. E-mail: [email protected]. † Present address: Southern Research Institute, Frederick, MD 21701. 3918

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FIG. 1. Structures of compounds used in this study. Compounds were synthesized, purchased, or obtained as gifts as described in Materials and Methods.

HCMV in vitro, they are less effective in vivo due to rapid metabolic cleavage of the sugar from the heterocycle (Good et al., Abstr. 7th Int. Conf. Antivir. Res., abstr. 128). A synthetic effort to improve upon this limitation produced several other promising compounds (44) including maribavir (Fig. 1) (G. W. Koszalka, S. D. Chamberlain, R. J. Harvey, L. W. Frick, S. S. Good, M. G. Davis, A. Smith, J. C. Drach, L. B. Townsend, and K. K. Biron, Abstr. 9th Int. Conf. Antivir. Res., abstr. 43, 1996). Maribavir is an L-ribosyl benzimidazole which also is a potent and selective inhibitor of HCMV replication (6). However, its mechanism of action is different from that of BDCRB and

involves the inhibition of viral DNA synthesis (6) and viral egress (24, 49). Mutations in the HCMV UL97 protein kinase (6) and in UL27 (21; S. Chou, A. E. Senters, R. H. Waldemer, M. G. Davis, and K. K. Biron, Abstr. 9th Int. Cytomegalovirus Workshop, 1st Int. Betaherpesvirus Workshop, abstr. H.03, 2003) have been shown to produce resistance to this drug. It has advanced to phase II clinical trials for the treatment of HCMV infections (25). The effort at synthesis centered on the benzimidazole nucleoside pharmacophore has resulted in many potent and selective antiviral compounds (6, 20, 27, 43, 44, 51). Since BDCRB

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is an inhibitor of viral DNA cleavage and processing, whereas maribavir acts by different mechanisms, we explored the structure-activity relationships of this series more extensively with respect to mechanisms of action against HCMV. We report herein that there is at least one new benzimidazole nucleoside which appears to act by a third mechanism distinct from the mechanisms by which BDCRB and maribavir act. MATERIALS AND METHODS Compounds. Benzimidazole nucleosides UMJD853 (27), UMJD1049 (18), UMJD1311 (27), TCRB, and BDCRB (43) and pyrrolo[2,3-d]pyrimidines UMJD1028 (34) and UMJD1369 (45) were synthesized and characterized as described previously. Maribavir was synthesized at GlaxoSmithKline and was provided through the courtesy of K. K. Biron. GCV was provided by Hoffman La Roche (Palo Alto, Calif.). CDV was kindly provided by M. J. M. Hitchcock of Gilead Sciences (Foster City, Calif.), and PFA was purchased from Sigma (St. Louis, Mo.). Stock solutions of all compounds were prepared at concentrations of 10 mg/ml in dimethyl sulfoxide and stored at ⫺20°C. Compounds were added to cultures such that the resulting concentrations of dimethyl sulfoxide never exceeded 0.05% by volume. Cell culture procedures. The routine growth of normal human diploid cells was performed in minimal essential medium with Earle’s salts [MEM(E)] with 10% fetal bovine serum (FBS). The two cell lines used were human foreskin fibroblasts (HFF) derived in our laboratory and MRC-5 cells, a human embryonic lung cell line obtained from the American Type Culture Collection (ATCC CCL 171). Cells were routinely passaged at 1:2 dilutions according to conventional procedures by using 0.05% trypsin with 0.02% EDTA in HEPES-buffered saline (38, 46). Virus strains and virological procedures. The Towne strain, plaque-purified isolate Po, of HCMV was kindly provided by M. F. Stinski, University of Iowa. HCMV strains D10, r56, and C4 were selected for resistance to TCRB from wild-type Towne as described previously (22). The AD169 strain of HCMV was obtained from ATCC and plaque purified in the laboratory of K. K. Biron, GlaxoSmithKline. The following strains were also provided by K. K. Biron: strain 2916r (6), derived from wild-type AD169 and resistant to maribavir; HCMV strains 4760 Rec Pol A (3) and 1117 3-1-2, which were derivatives of AD169; and HCMV clinical isolates 17517 and 48041. Stocks of HCMV were prepared by infecting HFF at a multiplicity of infection (MOI) of 0.01 PFU per cell according to a procedure described previously (46). Virus titers were determined by using monolayer cultures of HFF, also as described previously (31). HCMV plaque reduction assay. HFF were planted at 85,000 cells per well in 24-well cluster plates and were infected 24 h later with HCMV at 100 PFU/well in MEM(E) with 10% FBS. Following an initial 1-h adsorption, medium containing selected drug dilutions and 0.5% methylcellulose was added. All drug concentrations were tested in at least duplicate by using eight 1:3 dilutions from a starting concentration of 100 ␮M. After incubation at 37°C for 8 to 10 days in an atmosphere of 5% CO2, cells were fixed and stained with crystal violet, and plaques were enumerated by light microscopy. Drug effects were calculated as a percentage of reduction in number of plaques in the presence of each drug concentration compared to the number observed in the absence of drug. HCMV yield reduction assay. HFF were planted at 12,500 cells/well in 96-well plates and incubated overnight; medium was removed, and the cultures were inoculated with HCMV at various MOIs as described above. After virus adsorption, 300 ␮l of fresh medium containing a test compound was added to each well in quadruplicate. Eight 1:3 dilutions from a starting concentration of 100 ␮M were used for each compound. The addition of medium alone served as virus (positive) controls. Plates were incubated at 37°C for 7 days and subjected to one cycle of freezing and thawing; 100-␮l aliquots from each of the 12 wells of a given row were transferred to the first row of a fresh 96-well monolayer culture of HFF. Contents were mixed and serially diluted 1:3 across the remaining seven columns of the secondary plate. Cultures were incubated, plaques were enumerated, and titers were calculated (31). Data analysis. Dose-response relationships were used to quantify drug effects by linearly regressing the percent inhibition of parameters derived in the preceding assays (except for yield experiments) against log drug concentrations. For yield experiments, the log of viral titer was plotted against the log drug concentration. The 50% inhibitory concentrations (IC50s) and 90% inhibitory concentrations (IC90s; yield experiments) were calculated from the linear portions of the regression lines. Statistical results were obtained using the Student’s t test function of Microsoft Excel.

ANTIMICROB. AGENTS CHEMOTHER. HCMV time-of-addition study. As described above, HFF at 85,000 cells per well in 24-well cluster plates were infected with HCMV (Towne strain) at a concentration of 100 PFU/well. At infection and selected times postinfection, media were replaced with either fresh media or media containing selected virus inhibitory but nontoxic drug concentrations plus 0.5% methylcellulose and 5% FBS. After incubation at 37°C for 8 to 10 days in an atmosphere of 5% CO2, cells were fixed and stained with crystal violet, and plaques were enumerated by light microscopy. Alternatively, to emphasize the differences between inhibitors of viral DNA synthesis and packaging, virus was added to cells in MEM(E), and virus-infected cells were incubated in MEM(E) with 4% FBS at 34°C for 8 to 10 days. Drug effects were calculated as a percentage of reduction in the number of plaques in the presence of each drug concentration compared to the number observed in the absence of drug; percentages are presented as the means ⫾ standard deviations (SD) of duplicate results for each drug at each time point. Drug interaction assays and analysis. Drug combination assays were performed by using an HCMV enzyme-linked immunosorbent assay (ELISA) procedure previously described (33) for HCMV and a dye technique for cytotoxicity (32). The three-dimensional method developed by Prichard and Shipman (MacSynergy II) (30) was used to analyze drug-drug interactions. Briefly, data derived from three to six replicate plates were used to construct dose-response surfaces. Theoretical additive interactions were calculated from the dose-response curves for each drug used alone. This calculated surface was subtracted from the experimentally determined dose-response surface to reveal regions of nonadditive activity. Data are interpreted as follows. If the resulting plane appeared as a horizontal plane at 0% inhibition, the interactions between the two drugs are additive. Depressions in the plane indicate antagonism, whereas peaks above the plane indicate synergistic interactions between the two drugs. Confidence intervals (95%) around each of the points that defined the dose-response surface were calculated from the replicate data to provide limits for the experimental doseresponse surface. If the upper confidence limits of the experimental data were less than the calculated additive surface, antagonism would be considered significant at that confidence level. Conversely, if the lower confidence limits of the experimental data were greater than the calculated additive surface, the synergy would be considered significant. Finally, if the calculated additive surface were contained within the confidence limits, the interaction would be considered to be additive. Volumes of drug-drug interaction were quantified by integrating the area above or below the theoretical plane of additivity. UV inactivation of HCMV. A stock suspension of infectious HCMV (Towne strain) was prepared as above. Aliquots of stock virus were transferred to uncovered six-well plates (6 ml/well) on ice at a distance of 1 to 1.5 in. beneath the midpoint of the two bulbs of a Spectroline shortwave UV lamp (provides 1,100 ␮W/cm2 at 10 in.) (XX-15F; Spectronics Corp.) for a duration of 45 min (41). Under these conditions, the titer of infectious HCMV was reduced four to five orders of magnitude. Preincubation and drug washout experiments. Cells were incubated with fixed noncytotoxic but virus-inhibitory concentrations (5 to 10 ␮M) of compounds from the time of seeding until just before infection with HCMV (24 h). Drug was removed from the wells of 24-well plates by washing three times [1.5 ml of MEM(E) plus 10% FBS for 2 min], cells were infected, and the subsequent overlay contained no drug. Standard plaque inhibition assay conditions were employed at the same concentration of drugs. Both conditions were evaluated for the inhibition of plaque formation compared to virus (positive) controls.

RESULTS Time-of-addition studies. Our initial effort to distinguish between benzimidazole nucleosides which act by the inhibition of viral DNA cleavage and processing from those that inhibit viral DNA synthesis was to examine the time in the viral replication cycle at which these compounds act. Because HCMV replicates in a sequential cascade of immediate-early, early, and then late gene products (28), at some time point it is too late for any given drug to inhibit viral replication. In this assay, inhibitors of viral DNA cleavage and processing lose activity after inhibitors of viral DNA synthesis. It has previously been established that BDCRB, TCRB and the 5⬘-deoxy analog of TCRB act late in the viral replication cycle via inhibition of viral DNA cleavage and processing (23, 47). Results from a time-of-addition study (Fig. 2) reflected this mechanism, as

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FIG. 2. HCMV time-of-addition study under conditions that emphasize the differences between viral DNA synthesis and DNA processing. Benzimidazole nucleosides 853, 1311, maribavir, and BDCRB and a GCV control were added to cells infected with HCMV at time zero and at the indicated time points. To emphasize the differences between late-acting inhibitors, virus-infected cells were incubated with a lower amount of FBS at 34°C instead of 37°C, which is ordinarily used as in the experiment shown in Fig. 3. The inhibition of viral replication was measured by plaque reduction. Data are presented as the means ⫾ SDs of duplicate experiments. The concentrations of compounds used in the experiments were below cytotoxic levels and were as follows: 30 ␮M BDCRB, 10 ␮M 1311, 20 ␮M 853, 20 ␮M maribavir, and 30 ␮M GCV.

BDCRB lost activity at time points late in the replication cycle, after activity of the DNA synthesis inhibitors GCV and maribavir was lost. The closely related benzimidazole nucleoside UMJD853, which differs from TCRB only in that the 4⬘-hydroxymethyl group is “down” relative to the plane of the sugar ring (see Fig. 1), also lost activity late in the replication cycle. In contrast, we were surprised to observe that compound UMJD1311 lost activity early in the replication cycle, at times too early to be consistent with either known mechanism of action. The data shown in Fig. 2 are from a time-of-addition study optimized to separate the effects of inhibitors of DNA synthesis from inhibitors of DNA processing by using a lower temperature and serum concentration to extend the HCMV replication cycle to approximately 120 h. To more carefully determine the stage of viral replication upon which compound 1311 acts, we repeated the time-of-addition study under more standard conditions (Fig. 3). This experiment demonstrated that compound 1311 lost activity when added between 24 and 48 h postinfection in a manner similar to the previously described early acting pyrrolo[2,3-d]pyrimidine UMJD1028 (20). Again, the activities of GCV and BDCRB were lost later in the replication cycle. All drug concentrations used in these experiments were below the concentrations that produced cytotoxicity in uninfected cells. Because both of the foregoing experiments were performed in a plaque reduction format, another experiment was performed in a yield reduction format by using an input MOI of

0.5 PFU/ml. In this experiment, 10 ␮M 1311 was compared to both of the very closely related benzimidazole nucleosides 853 (100 ␮M) and 1049 (10 ␮M) (Fig. 1); GCV and TCRB (each 100 ␮M) served as later-acting control compounds. Results of this experiment were nearly identical to the two other experiments; 1311 lost activity between 32 and 56 h, GCV lost activity at 64 to 80 h, and TCRB, 853, and 1049 lost activity after 80 h. Because of the unexpected nature of the preceding results with UMJD1311, it was compared to GCV in a more detailed time-of-addition study in which multiple concentrations of each compound were used to establish dose-response curves at selected times by using a yield reduction assay. As expected, IC90s for GCV were the same at all times examined up to 48 h postinfection (Fig. 4). In contrast, potent IC90s for 1311 were consistently observed when the compound was added from 24 h before infection through 2 h postinfection (Fig. 4). The potency of compound 1311 was approximately 10-fold less when it was added at 24 h postinfection and was virtually nonexistent when the compound was added at 48 h. We conclude that 1311 does not affect viral entry and acts very early in the HCMV replication cycle. MOI dependence. Following the determination that compound 1311 acted at an early stage in the viral replication cycle, we examined the possibility that benzimidazole 1311 exhibited biological properties similar to those of the nonnucleoside pyrrolo[2,3-d]pyrimidine series studied previously in our laboratory. These early acting pyrrolo[2,3-d]pyrimidines are known

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ANTIMICROB. AGENTS CHEMOTHER.

FIG. 3. HCMV time-of-addition study for benzimidazole nucleosides 1311 and BDCRB with GCV and pyrrolo[2,3-d]pyrimidine 1028 control compounds. Drugs were added to cells infected with HCMV at time zero and at the indicated time points. Inhibition of viral replication was measured by plaque reduction assays under standard conditions. Data are presented as the means ⫾ SDs of triplicate experiments. The concentration of compound 1311 was 15 ␮M; the concentration of each control compound was 75 ␮M.

to be MOI dependent, meaning that their antiviral potencies decreased as the ratio of input virus to cells increased (20). We examined the antiviral potencies of compound 1311 and the closely related benzimidazole nucleosides 853 and 1049 (Fig. 1) by yield reduction assay at four MOIs (Table 1). In contrast to benzimidazoles 853, 1049, BDCRB, and GCV, compound 1311 was MOI dependent, losing three orders of magnitude in antiviral potency with an increase of four orders of magnitude of input virus. Interestingly, maribavir also showed some MOI dependence but considerably less than 1311. This supports the observation that MOI dependence does not negate the clinical potential and further development of an antiviral compound (8). These results also confirmed that compound 1311 inhibits HCMV differently than does BDCRB. Cross-resistance to BDCRB and maribavir. To further explore the mode of action of compound 1311, we determined the susceptibility of BDCRB- and maribavir-resistant viruses to 1311. Viruses D10, r56, and C4 were derived from the Towne strain and are resistant to BDCRB (22, 47). D10 bears a mutation in HCMV UL89 (D344E), r56 has a mutation in HCMV UL56 (Q204R), and C4 bears both mutations and is more resistant to BDCRB than either D10 or r56. The BDCRBresistant viruses were susceptible to compound 1311 and to GCV, as measured by a plaque reduction assay (Table 2). Interestingly, these viruses were resistant to the structurally similar compounds 853 and 1049. We speculate that the lower level of resistance of 853 compared to BDCRB and 1049 stemmed from the fact that it has a poorer in vitro selectivity index, reaching cytotoxicity at concentrations above 50 ␮M (27).

The extent of cross-resistance to compound 1311 by HCMV resistant to maribavir was determined by yield reduction assay. Compared to the parent AD169 strain, the maribavir-resistant HCMV strain 2916r (6) was 300-fold less sensitive to maribavir (Table 2) but remained sensitive to control compounds GCV and BDCRB. The susceptibilities of AD169 and 2916r to compound 1311 were the same within experimental error (Table 2). Thus, we found no cross-resistance between compound 1311 and the parent compounds BDCRB and maribavir. We recognize that evidence presented in Table 2 is by itself not definitive for a third mechanism. Nonetheless, together with the preceding and following results, the common and consistent conclusion is that compound 1311 acts by a mechanism different from BDCRB and maribavir. Cross-resistance to GCV, CDV, and PFA. GCV, CDV, and PFA are the most commonly used drugs for the treatment of HCMV infections (16). Therefore, we examined the susceptibility of GCV-, CDV-, and PFA-resistant viruses to the early acting benzimidazole nucleoside 1311. Table 3 shows that isolate 17517 was susceptible to GCV, while 48041 was resistant to GCV due to a mutation (L595S) in the UL97 protein kinase (K. K. Biron, personal communication). BDCRB and compound 1311 were equally active against both strains. This indicated that these benzimidazoles are active not only against clinical isolates of HCMV but also against GCV-resistant HCMV. The ultimate molecular target of nucleoside analogs currently used to treat HCMV infections is the UL54 viral DNA polymerase (15, 17). Strains 4760 and 1117 are viruses that contain mutations in UL54 shown to confer resistance to PFA

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FIG. 4. Yield reduction potencies for compound 1311 and GCV when compounds were added at different times. Compounds were added to cells at eight different concentrations (from 100 to 0.05 ␮M) at the indicated times, and virus was added at time zero. Dose-response experiments were performed at each time point and were used to determine IC90s. Data are presented as the means ⫾ SDs of triplicate experiments. When added at 48 h post infection, the IC90 of compound 1311 was ⬎100 ␮M (*).

and GCV plus CDV, respectively (3). We found that both strains remained susceptible to BDCRB and the early acting benzimidazole nucleoside 1311 (Table 3). The lack of crossresistance to these drugs confirms the other experiments described above, suggesting that the mode of action of the early acting benzimidazole nucleosides is different from that of GCV, CDV, and PFA, all of which are late-acting viral DNA synthesis inhibitors. Studies with drug combinations. Since compound 1311 acted early in the viral replication cycle while GCV acted late, we hypothesized that the use of these two compounds together would increase the effect over each drug acting alone. The effects of compound 1311 and GCV in combination on HCMV replication were measured by an HCMV ELISA (33). Figure 5 presents the data obtained from a three-dimensional analysis (30) of drug-drug interactions. Since the effects of the two drugs in combination were found to be greater than that of additivity (volume of interaction, 100 ␮M), compound 1311 and GCV interact in a synergistic manner. This is in contrast to the parent compounds BDCRB and maribavir, which have been shown to interact with GCV in an additive manner (16). UV-inactivated virus. Time-of-addition studies appeared to indicate that the mode of antiviral action for early acting pyrrolo[2,3-d]pyrimidines and benzimidazoles required the de novo expression of viral proteins. In an effort to determine the

cause(s) of this MOI dependence, we examined the possibility that UV-inactivated virus could be sufficient to reproduce the MOI effect observed for intact virus particles. Because limited UV irradiation degrades viral DNA without significant disruption of viral proteins (39, 40), we examined its effect on the activity of selected compounds. Following the establishment of conditions for the UV inactivation of HCMV, yield reduction assays were performed at

TABLE 1. MOI dependence of benzimidazole nucleosides and GCV IC90 (␮M) in yield reduction assay at the following MOIs (PFU/cell)a:

Compound 0.001

853 1049 1311 GCV Maribavir BDCRB

b

ND ND 0.77 ⫾ 0.40 4.0 ⫾ 1.9 ND 0.41 ⫾ 0.08

0.01

0.1

1.0

5.9 ⫾ 0.6 0.41 ⫾ 0.14 1.7 ⫾ 0.6 3.2 ⫾ 0.6 0.11 ⫾ 0.03 0.45 ⫾ 0.05

9.2 ⫾ 0.9 0.46 ⫾ 0.19 6.6 ⫾ 1.0 2.4 ⫾ 0.7 0.49 ⫾ 0.12 0.31 ⫾ 0.02

5.8 ⫾ 1.2 0.50 ⫾ 0.14 52 ⫾ 5 4.4 ⫾ 3.8 1.2 ⫾ 0.14 0.48 ⫾ 0.10

a Inhibition of infectious HCMV (Towne strain) titer production was measured at eight drug concentrations ranging from 100 to 0.05 ␮M. Data are presented as the means ⫾ SDs of IC90s determined in quadruplicate. b ND, not determined.

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ANTIMICROB. AGENTS CHEMOTHER. TABLE 2. Drug susceptibilities of HCMV strains resistant to BDCRB and maribavir

Towne

853 1049 1311 GCV BDCRB Maribavir

IC90 (␮M) in yield reduction against the following virusesb:

IC50 (␮M) in plaque reduction against the following virusesa:

Compound

24 ⫾ 2 0.61 ⫾ 0.10 0.66 ⫾ 0.42 13.3 ⫾ 1.5 1.0 ⫾ 0.1 ND

D10

r56 c

56 ⫾ 1* 4.6 ⫾ 0.3* 0.54 ⫾ 0.16 12.7 ⫾ 1.2 11.8 ⫾ 0.8* ND

C4

51 ⫾ 3* 5.3 ⫾ 0.4* 0.81 ⫾ 0.08 13.3 ⫾ 0.6 8.8 ⫾ 0.7* ND

AD169 d

56 ⫾ 2* 25 ⫾ 5* 0.95 ⫾ 0.18 12.0 ⫾ 5.0 37.7 ⫾ 2.5* ND

ND ND 0.82 ⫾ 0.10 1.2 ⫾ 0.4 0.36 ⫾ 0.12 0.19 ⫾ 0.09

2916

ND ND 0.70 ⫾ 0.18 1.5 ⫾ 0.3 0.38 ⫾ 0.05 57 ⫾ 3*

a Inhibition of HCMV plaque formation was measured by using at least six drug concentrations ranging from 100 to 0.05 ␮M. Data are presented as the means ⫾ SDs of IC50s determined in at least triplicate. HCMV strains D10, r56, and C4 were derived from Towne (wild-type strain) (described in references 24 and 45). D10 bears a mutation in UL89 (D344E), r56 contains a mutation in UL56 (Q204R), and C4 has both mutations. b Inhibition of HCMV titer production was measured at eight drug concentrations ranging from 100 to 0.05 ␮M. Data are presented as the means ⫾ SDs of IC90s determined in at least quadruplicate. HCMV strain 2916r was derived from AD169 (wild-type) (described in reference 6). Strain 2916r contains a mutation in UL97 (I397R). c *, P ⬍ 0.005 as determined by the Student’s t test function of Microsoft Excel. d ND, not determined.

an MOI of 0.005 PFU of live virus/cell with up to 50 times the amount of UV-inactivated virus. Table 4 shows that the antiviral potencies of control compounds BDCRB, maribavir, and GCV, as well as test compounds 1311 and 1028, were either unaffected by or failed to show a dose-response relationship with the amount of input UV-inactivated virus. These results support the hypothesis that the MOI dependence of compound 1311 requires viral replication or viral particles with an intact genome, rather than simply resulting from the cellular sequelae of virion-associated proteins. Preincubation studies. Based upon literature precedent (8) and our own inability to select for drug-resistant HCMV, we hypothesized that early acting compounds might specifically inhibit a cellular, rather than a viral, target. In this case, an antiviral effect (such as interferon) could be induced in cells prior to HCMV infection. For example, the antiviral effects of 5,6-dichloro-1-(␤-D-ribofuranosyl)benzimidazole against human immunodeficiency virus have been traced to this compound’s inhibition of a cellular kinase (11). Thus, the ability of selected pyrrolo[2,3-d]pyrimidines and compound 1311 to elicit an antiviral response following their removal from the media was examined. CDV was used as a positive control due to its long intracellular pharmacokinetic half-life (19). The data presented in Fig. 6 suggest that benzimidazole nucleosides BDCRB, maribavir, and 1311 did not continue to elicit a cellular antiviral response because little antiviral activity persisted following their removal from cells. In contrast, pyrrolo[2,3-

d]pyrimidines 1028 and 1369 continued to display 50 to 60% of their inhibitory potencies under the same removal conditions. DISCUSSION We have presented three lines of evidence strongly suggesting that the benzimidazole nucleosides have a third mechanism of action against HCMV. First, in contrast to parent compounds, 1311 acts early in the viral replication cycle. Second, unlike BDCRB, 1311 is MOI dependent. Third, viruses resistant to BDCRB and maribavir remain sensitive to compound 1311. Complementing the time-of-addition studies that place the mode of action of these inhibitors at the immediate-early or early stage of viral replication is the lack of cross-resistance to GCV-, CDV-, and PFA-resistant viruses. Our efforts to determine the mode of action of new benzimidazole nucleosides–which led to the conclusions stated above–also provided insight into structure-activity relationships among other analogs. For example, the benzimidazole nucleosides 853, 1049, 5⬘-deoxy-TCRB, and TCRB have the same 2,5,6-trichlorobenzimidazole heterocycle but differ in their respective carbohydrates at positions 4 and 5 (Fig. 1). Nonetheless, all these compounds appear to act as DNA processing inhibitors (reference 23 and the present study). Thus, the putative HCMV benzimidazole nucleoside binding site responsible for the inhibition of DNA processing must tolerate considerable change to, or even elimination of, the 5⬘ position

TABLE 3. Activity of benzimidazole nucleoside 1311 against HCMV strains resistant to GCV, CDV, and PFA Compound

1311 GCV BDCRB PFA CDV

IC50 (␮M) against the following virusesa: 17517

48041

AD169

4760 Rec Pol A

1117 3-1-2

0.57 ⫾ 0.23 3.2 ⫾ 2.6 0.46 ⫾ 0.24 NDc ND

0.82 ⫾ 0.35 27 ⫾ 8*b 0.64 ⫾ 0.05 ND ND

0.82 ⫾ 0.10 11.8 ⫾ 3.9 0.58 ⫾ 0.05 50 ⫾ 13 0.44 ⫾ 0.04

0.87 ⫾ 0.07 14.3 ⫾ 1.5 0.50 ⫾ 0.11 190 ⫾ 10* 0.40 ⫾ 0.07

0.96 ⫾ 0.07 48.3 ⫾ 5.5* 0.53 ⫾ 0.04 52 ⫾ 11 2.27 ⫾ 0.67*

a Inhibition of HCMV plaque formation was measured by using at least six drug concentrations ranging from 100 to 0.05 ␮M. Data are presented as the means ⫾ SDs of IC50s determined in at least triplicate. HCMV strains 17517 (GCV sensitive) and 48041 (GCV resistant) are a matched pair of clinical isolates. Strain 48041 is resistant to GCV due to a mutation (L595S) in UL97 (K. K. Biron, personal communication). HCMV strains 4760 Rec Pol A (PFA resistant) and 1117 3-1-2 (CDV resistant) were derived from AD169 (wild type) and manifest their resistance to PFA or GCV and CDV due to mutations in the UL54 viral DNA polymerase gene (3). b *, P ⬍ 0.005 as determined by the Student’s t test function of Microsoft Excel. c ND, not determined.

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FIG. 5. Interaction against HCMV between compounds 1311 and GCV. The extent of viral inhibition from combinations of compound 1311 and GCV was assessed by ELISA. Data are presented as a three-dimensional surface area of inhibition above the level expected based on a model of additive interaction (29). The results shown are those computed to be statistically significant at a 95% confidence interval.

of TCRB. However, it does not tolerate all changes. Compared to the DNA processing inhibitor 5⬘-deoxy-TCRB, where the 5⬘-methyl group is “up” relative to the plane of the carbohydrate ring (24), the down methyl group in compound 1311 gives a compound that does not inhibit DNA processing but acts much earlier in the HCMV replication cycle. It has been said that “benzimidazoles will be a really BIG fishing hole” (44). We believe that our present studies demonstrate that the benzimidazole nucleosides represent a robust pharmacophore for the design of HCMV inhibitors. We are unaware of any other pharmacophore that represents three distinct modes of antiviral action. Studies with a series of aryl thiourea inhibitors of herpes simplex virus type 1 found a mechanism of action related to that of TCRB which involved inhibition of both viral DNA cleavage and processing, but the molecular target involved only a single gene (48). In drug discovery, a model of the interactions of a series of compounds with a single protein target is often considered in the optimization and development of lead compounds. We suggest that when developing pharmacophore models from data derived from a whole-cell assay, it is wise to consider the possibility that there may be multiple molecular targets with different binding modes. Although we were unable to determine the molecular basis for the activity of compound 1311, several possibilities were

eliminated. HCMV protein IE1p72 was considered because it is the major immediate-early HCMV transactivator protein (27). We found, however, that wild-type virus propagated on cells expressing IE1p72 in trans was no less susceptible to compound 1311 than wild-type virus grown in normal cells. This indicates that the major immediate-early transactivator of HCMV and its sequelae are unlikely to be the ultimate molecular targets of compound 1311. Additionally, we have shown similar data to

TABLE 4. Effect of UV-inactivated HCMV on the antiviral potency of selected compounds Compound

1028 1311 1369 Maribavir GCV BDCRB

IC90 (␮M) in yield reduction assay in the presence of UV-inactivated virus (PFU/cell)a None

0.0025

0.025

0.25

0.27 ⫾ 0.24 0.34 ⫾ 0.06 0.010 ⫾ 0.001 0.12 ⫾ 0.02 2.0 ⫾ 0.4 0.11 ⫾ 0.03

0.25 ⫾ 0.12 0.47 ⫾ 0.05 0.013 ⫾ 0.001 0.08 ⫾ 0.02 1.5 ⫾ 0.4 0.11 ⫾ 0.01

0.64 ⫾ 0.63 0.34 ⫾ 0.02 0.014 ⫾ 0.003 0.11 ⫾ 0.02 2.1 ⫾ 0.2 0.10 ⫾ 0.04

0.32 ⫾ 0.24 0.30 ⫾ 0.15 0.019 ⫾ 0.008 0.11 ⫾ 0.05 3.0 ⫾ 0.1 0.11 ⫾ 0.02

a Inhibition of viral titer production in HFF infected with HCMV Towne strain (0.005 PFU/cell) in the presence of the indicated amounts of UV-inactivated virus. Yields were measured at eight drug concentrations from 120 to 0.02 ␮M. IC90s are reported as the means ⫾ SDs of three experiments.

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ANTIMICROB. AGENTS CHEMOTHER.

FIG. 6. The effect of preincubation and removal of compounds prior to infection compared to adding drug at 1 h postinfection. Shaded bars quantify the percent inhibition compared to the control without drug for a 24-h preincubation with compound, which was then washed out prior to infection with virus. Open bars quantify the percent inhibition compared to the control without drug for the plaque reduction assay under standard conditions; the same concentrations of drugs were added at 1 h postinfection.

rule out the same hypothesis for the antiviral mode of action of the pyrrolo[2,3-d]pyrimidine nucleoside analogs. Preincubation of cells with antiviral compounds and their subsequent removal have provided the only differences we observed in the antiviral biology of early acting benzimidazoles and pyrrolo[2,3-d]pyrimidines. The addition of test compounds 24 h prior to infection and washing out before HCMV infection did not result in activity versus HCMV for the benzimidazole nucleosides, but it did for the pyrrolo[2,3-d]pyrimidines and CDV. Presumably, the activity for CDV was due to its unusually long intracellular pharmacokinetic half-life (19). Although we cannot rule out pharmacokinetic effects, our data support the possibility that the pyrrolo[2,3-d]pyrimidines exert their antiviral effects via inhibition of a cellular target, or induction of an antiviral state, whereas early acting benzimidazoles exert their antiviral effects via the inhibition of a viral target. There are a number of parallels between the biological activity of early acting benzimidazole nucleosides and previously reported compounds. Pyrrolo[2,3-d]pyrimidine nucleoside analogs (20), nonnucleoside benzothiophenes (8), tetrahydroindolizines (40), and thiazolo[4,5-d]pyrimidine nucleoside analogs (26) have all been reported to be MOI dependent early acting inhibitors of HCMV. As with studies of these compound series, we have been unsuccessful in our attempts to select drug-resistant viruses to early acting benzimidazoles (data not presented). Some investigators have speculated that the inability to isolate drug-resistant virus is indicative of a mode of antiviral action involving a cellular target (8, 35), but we have no data to support this hypothesis for the early acting benzimidazole nucleosides.

In summary, we have presented evidence which strongly suggests that there is a third mechanism of action whereby benzimidazole nucleosides inhibit HCMV. This robust pharmacophore has produced compounds that act early in the HCMV replication cycle (1311), at the stage of viral DNA synthesis (maribavir), or at viral DNA maturation and processing (BDCRB). ACKNOWLEDGMENTS The authors thank Souad A. Barnat for performing certain preliminary experiments. These studies were supported by research grants U19-AI31718 and P01-AI46390 from the National Institute of Allergy and Infectious Diseases and by research funds from the University of Michigan. D.L.E., who was supported in part by NIH training grant GM07767, also thanks the American Chemical Society Division of Medicinal Chemistry and Bristol-Myers Squibb for the generous support of a predoctoral fellowship. REFERENCES 1. Alford, C. A., and W. J. Britt. 1993. Cytomegalovirus, p. 227–255. In B. Roizman, R. J. Whitley, and C. Lopez (ed.), The human herpesviruses. Raven Press, New York, N.Y. 2. Amin, H. I., E. Ai, H. R. McDonald, and R. N. Johnson. 2000. Retinal toxic effects associated with intravitreal fomivirsen. Arch. Ophthalmol. 118:426– 427. 3. Baldanti, F., M. R. Underwood, S. C. Stanat, K. K. Biron, S. Chou, A. Sarasini, E. Silini, and G. Gerna. 1996. Single amino acid changes in the DNA polymerase confer foscarnet resistance and slow-growth phenotype, while mutations in UL97-encoded phosphotransferase confer ganciclovir resistance in three double-resistant human cytomegalovirus strains recovered from patients with AIDS. J. Virol. 70:1390–1395. 4. Balfour, H. H., Jr. 1979. Cytomegalovirus: the troll of transplantation. Arch. Int. Med. 139:280. 5. Berk, T., S. J. Gordon, H. Y. Choi, and H. S. Cooper. 1985. Cytomegalovirus

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