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Resistance to the 2'-F-2'-C-methylguanosine monophosphate nucleotide hepatitis C virus (HCV) inhibitors PSI-352938 and PSI-353661 was associated with a combination of amino acid changes (changes of S to G at position 15 [S15G], C223H, and V321I) within the genotype 2a nonstructural protein 5B (NS5B), an RNA-dependent RNA polymerase. To understand the role of these residues in viral replication, we examined the effects of single and multiple point mutations on replication fitness and inhibition by a series of nucleotide analog inhibitors. An acidic residue at position 15 reduced replicon fitness, consistent with its proximity to the RNA template. A change of the residue at position 223 to an acidic or large residue reduced replicon fitness, consistent with its proposed proximity to the incoming nucleoside triphosphate (NTP). A change of the residue at position 321 to a charged residue was not tolerated, consistent with its position within a hydrophobic cavity. This triple resistance mutation was specific to both genotype 2a virus and 2'-F-2'-C-methylguanosine inhibitors. A crystal structure of the NS5B S15G/C223H/V321I mutant of the JFH-1 isolate exhibited rearrangement to a conformation potentially consistent with short primer-template RNA binding, which could suggest a mechanism of resistance accomplished through a change in the NS5B conformation, which was better tolerated by genotype 2a virus than by viruses of other genotypes., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

PSI-352938 is a novel cyclic phosphate prodrug of β-D-2'-deoxy-2'-α-fluoro-2'-β-C-methylguanosine-5'-monophosphate with potent anti-HCV activity. In order to inhibit the NS5B RNA-dependent RNA polymerase, PSI-352938 must be metabolized to the active triphosphate form, PSI-352666. During in vitro incubations with PSI-352938, significantly larger amounts of PSI-352666 were formed in primary hepatocytes than in clone A hepatitis C virus (HCV) replicon cells. Metabolism and biochemical assays were performed to define the molecular mechanism of PSI-352938 activation. The first step, removal of the isopropyl group on the 3',5'-cyclic phosphate moiety, was found to be cytochrome P450 (CYP) 3A4 dependent, with other CYP isoforms unable to catalyze the reaction. The second step, opening of the cyclic phosphate ring, was catalyzed by phosphodiesterases (PDEs) 2A1, 5A, 9A, and 11A4, all known to be expressed in the liver. The role of these enzymes in the activation of PSI-352938 was confirmed in primary human hepatocytes, where prodrug activation was reduced by inhibitors of CYP3A4 and PDEs. The third step, removal of the O(6)-ethyl group on the nucleobase, was shown to be catalyzed by adenosine deaminase-like protein 1. The resulting monophosphate was consecutively phosphorylated to the diphosphate and to the triphosphate PSI-352666 by guanylate kinase 1 and nucleoside diphosphate kinase, respectively. In addition, formation of nucleoside metabolites was observed in primary hepatocytes, and ecto-5'-nucleotidase was able to dephosphorylate the monophosphate metabolites. Since CYP3A4 is highly expressed in the liver, the CYP3A4-dependent metabolism of PSI-352938 makes it an effective liver-targeted prodrug, in part accounting for the potent antiviral activity observed clinically.

PSI-7977, a prodrug of 2'-F-2'-C-methyluridine monophosphate, is the purified diastereoisomer of PSI-7851 and is currently being investigated in phase 3 clinical trials for the treatment of hepatitis C. In this study, we profiled the activity of PSI-7977 and its ability to select for resistance using a number of different replicon cells. Results showed that PSI-7977 was active against genotype (GT) 1a, 1b, and 2a (strain JFH-1) replicons and chimeric replicons containing GT 2a (strain J6), 2b, and 3a NS5B polymerase. Cross-resistance studies using GT 1b replicons confirmed that the S282T change conferred resistance to PSI-7977. Subsequently, we evaluated the ability of PSI-7977 to select for resistance using GT 1a, 1b, and 2a (JFH-1) replicon cells. S282T was the common mutation selected among all three genotypes, but while it conferred resistance to PSI-7977 in GT 1a and 1b, JFH-1 GT 2a S282T showed only a very modest shift in 50% effective concentration (EC(50)) for PSI-7977. Sequence analysis of the JFH-1 NS5B region indicated that additional amino acid changes were selected both prior to and after the emergence of S282T. These include T179A, M289L, I293L, M434T, and H479P. Residues 179, 289, and 293 are located within the finger and palm domains, while 434 and 479 are located on the surface of the thumb domain. Data from the JFH-1 replicon variants showed that amino acid changes within the finger and palm domains together with S282T were required to confer resistance to PSI-7977, while the mutations on the thumb domain serve to enhance the replication capacity of the S282T replicons.

PSI-352938 is a novel cyclic phosphate prodrug of β-D-2'-deoxy-2'-α-fluoro-2'-β-C-methylguanosine 5'-monophosphate that has potent activity against hepatitis C virus (HCV) in vitro. The studies described here characterize the in vitro anti-HCV activity of PSI-352938, alone and in combination with other inhibitors of HCV, and the cross-resistance profile of PSI-352938. The effective concentration required to achieve 50% inhibition for PSI-352938, determined using genotype 1a-, 1b-, and 2a-derived replicons stably expressed in the Lunet cell line, were 0.20, 0.13, and 0.14 μM, respectively. The active 5'-triphosphate metabolite, PSI-352666, inhibited recombinant NS5B polymerase from genotypes 1 to 4 with comparable 50% inhibitory concentrations. In contrast, PSI-352938 did not inhibit the replication of hepatitis B virus or human immunodeficiency virus in vitro. PSI-352666 did not significantly affect the activity of human DNA and RNA polymerases. PSI-352938 and its cyclic phosphate metabolites did not affect the cyclic GMP-mediated activation of protein kinase G. Clearance studies using replicon cells demonstrated that PSI-352938 cleared cells of HCV replicon RNA and prevented replicon rebound. An additive to synergistic effect was observed when PSI-352938 was combined with other classes of HCV inhibitors, including alpha interferon, ribavirin, NS3/4A inhibitors, an NS5A inhibitor, and nucleoside/nucleotide and nonnucleoside inhibitors. Cross-resistance studies showed that PSI-352938 remained fully active against replicons containing the S282T or the S96T/N142T amino acid alteration. Replicons that contain mutations conferring resistance to various classes of nonnucleoside inhibitors also remained sensitive to inhibition by PSI-352938. PSI-352938 is currently being evaluated in a phase I clinical study in genotype 1-infected individuals.

The hepatitis C virus (HCV) NS5B RNA polymerase facilitates the RNA synthesis step during the HCV replication cycle. Nucleoside analogs targeting the NS5B provide an attractive approach to treating HCV infections because of their high barrier to resistance and pan-genotype activity. PSI-7851, a pronucleotide of beta-D-2'-deoxy-2'-fluoro-2'-C-methyluridine-5'-monophosphate, is a highly active nucleotide analog inhibitor of HCV for which a phase 1b multiple ascending dose study of genotype 1-infected individuals was recently completed (M. Rodriguez-Torres, E. Lawitz, S. Flach, J. M. Denning, E. Albanis, W. T. Symonds, and M. M. Berry, Abstr. 60th Annu. Meet. Am. Assoc. Study Liver Dis., abstr. LB17, 2009). The studies described here characterize the in vitro antiviral activity and cytotoxicity profile of PSI-7851. The 50% effective concentration for PSI-7851 against the genotype 1b replicon was determined to be 0.075+/-0.050 microM (mean+/-standard deviation). PSI-7851 was similarly effective against replicons derived from genotypes 1a, 1b, and 2a and the genotype 1a and 2a infectious virus systems. The active triphosphate, PSI-7409, inhibited recombinant NS5B polymerases from genotypes 1 to 4 with comparable 50% inhibitory concentrations. PSI-7851 is a specific HCV inhibitor, as it lacks antiviral activity against other closely related and unrelated viruses. PSI-7409 also lacked any significant activity against cellular DNA and RNA polymerases. No cytotoxicity, mitochondrial toxicity, or bone marrow toxicity was associated with PSI-7851 at the highest concentration tested (100 microM). Cross-resistance studies using replicon mutants conferring resistance to modified nucleoside analogs showed that PSI-7851 was less active against the S282T replicon mutant, whereas cells expressing a replicon containing the S96T/N142T mutation remained fully susceptible to PSI-7851. Clearance studies using replicon cells demonstrated that PSI-7851 was able to clear cells of HCV replicon RNA and prevent viral rebound.

beta-D-2'-Deoxy-2'-fluoro-2'-C-methylcytidine (PSI-6130) is a potent inhibitor of hepatitis C virus (HCV) RNA replication in an HCV replicon assay. The 5'-triphosphate of PSI-6130 is a competitive inhibitor of the HCV RNA-dependent RNA polymerase (RdRp) and acts as a nonobligate chain terminator. Recently, it has been shown that the metabolism of PSI-6130 also results in the formation of the 5'-triphosphate of the uridine congener, beta-D-2'-deoxy-2'-fluoro-2'-C-methyluridine (PSI-6206; RO2433). Here we show that the formation of the 5'-triphosphate of RO2433 (RO2433-TP) requires the deamination of PSI-6130 monophosphate and that RO2433 monophosphate is subsequently phosphorylated to the corresponding di- and triphosphates by cellular UMP-CMP kinase and nucleoside diphosphate kinase, respectively. RO2433-TP is a potent inhibitor of the HCV RdRp; however, both enzymatic and cell-based assays show that PSI-6130 triphosphate is a more potent inhibitor of the HCV RdRp than RO2433-TP.

Beta-D-2'-deoxy-2'-fluoro-2'-C-methylcytidine (PSI-6130) is a potent specific inhibitor of hepatitis C virus (HCV) RNA synthesis in Huh-7 replicon cells. To inhibit the HCV NS5B RNA polymerase, PSI-6130 must be phosphorylated to the 5'-triphosphate form. The phosphorylation of PSI-6130 and inhibition of HCV NS5B were investigated. The phosphorylation of PSI-6130 by recombinant human 2'-deoxycytidine kinase (dCK) and uridine-cytidine kinase 1 (UCK-1) was measured by using a coupled spectrophotometric reaction. PSI-6130 was shown to be a substrate for purified dCK, with a Km of 81 microM and a kcat of 0.007 s-1, but was not a substrate for UCK-1. PSI-6130 monophosphate (PSI-6130-MP) was efficiently phosphorylated to the diphosphate and subsequently to the triphosphate by recombinant human UMP-CMP kinase and nucleoside diphosphate kinase, respectively. The inhibition of wild-type and mutated (S282T) HCV NS5B RNA polymerases was studied. The steady-state inhibition constant (Ki) for PSI-6130 triphosphate (PSI-6130-TP) with the wild-type enzyme was 4.3 microM. Similar results were obtained with 2'-C-methyladenosine triphosphate (Ki=1.5 microM) and 2'-C-methylcytidine triphosphate (Ki=1.6 microM). NS5B with the S282T mutation, which is known to confer resistance to 2'-C-methyladenosine, was inhibited by PSI-6130-TP as efficiently as the wild type. Incorporation of PSI-6130-MP into RNA catalyzed by purified NS5B RNA polymerase resulted in chain termination.

Racivir [RCV; (+/-)-beta-2',3'-dideoxy-5-fluoro-3'-thiacytidine], a 50:50 racemic mixture of the two beta nucleoside enantiomers, is currently in development for the treatment of human immunodeficiency virus type 1 (HIV-1) infections. RCV was administered once a day orally for 14 days at doses of 200, 400, or 600 mg in combination with stavudine and efavirenz to HIV-1-infected treatment-naïve male volunteers in a phase Ib/IIa study. Six volunteers at each dose were monitored for a total of 35 days for tolerance, pharmacokinetics, and plasma HIV RNA levels. RCV in combination with stavudine and efavirenz was well tolerated at all doses tested. Pharmacokinetic parameters were dose proportional, and the maximum concentration of drug in serum at all doses exceeded the 90% effective concentration for wild-type HIV-1. Viral loads dropped as expected in all dosage groups, with mean reductions from 1.13 to 1.42 log10 by day 4 and 2.02 to 2.43 log10 by day 14. HIV RNA levels remained suppressed for more than 2 weeks in the absence of any additional therapy, with mean viral loads ranging from 2.1 to 2.6 log10 below baseline through day 28. By day 35, HIV RNA levels began to increase but still remained >1 log10 below baseline levels.

2'-Deoxy-2'-fluorocytidine (FdC) is a potent inhibitor of the hepatitis C virus RNA replicon in culture, and FdC-5'-triphosphate is an effective inhibitor of the NS5B polymerase. Dynamic profiling of cell growth in an antiviral assay showed that FdC caused cytostasis due to an S-phase arrest. These observations demonstrate that FdC treatment is affecting both a viral target and a cellular target.

A base-modified nucleoside analogue, beta-D-N(4)-hydroxycytidine (NHC), was found to have antipestivirus and antihepacivirus activities. This compound inhibited the production of cytopathic bovine viral diarrhea virus (BVDV) RNA in a dose-dependant manner with a 90% effective concentration (EC(90)) of 5.4 microM, an observation that was confirmed by virus yield assays (EC(90) = 2 microM). When tested for hepatitis C virus (HCV) replicon RNA reduction in Huh7 cells, NHC had an EC(90) of 5 microM on day 4. The HCV RNA reduction was incubation time and nucleoside concentration dependent. The in vitro antiviral effect of NHC was additive with recombinant alpha interferon-2a and could be prevented by the addition of exogenous cytidine and uridine but not of other natural ribo- or 2'-deoxynucleosides. When HCV RNA replicon cells were cultured in the presence of increasing concentrations of NHC (up to 40 micro M) for up to 45 cell passages, no resistant replicon was selected. Similarly, resistant BVDV could not be selected after 20 passages. NHC was phosphorylated to the triphosphate form in Huh7 cells, but in cell-free HCV NS5B assays, synthetic NHC-triphosphate (NHC-TP) did not inhibit the polymerization reaction. Instead, NHC-TP appeared to serve as a weak alternative substrate for the viral polymerase, thereby changing the mobility of the product in polyacrylamide electrophoresis gels. We speculate that incorporated nucleoside analogues with the capacity of changing the thermodynamics of regulatory secondary structures (with or without introducing mutations) may represent an important class of new antiviral agents for the treatment of RNA virus infections, especially HCV.

The antiviral efficacies and cytotoxicities of 2',3'- and 4'-substituted 2',3'-didehydro-2',3'-dideoxycytidine analogs were evaluated. All compounds were tested (i) against a wild-type human immunodeficiency virus type 1 (HIV-1) isolate (strain xxBRU) and lamivudine-resistant HIV-1 isolates, (ii) for their abilities to inhibit hepatitis B virus (HBV) production in the inducible HepAD38 cell line, and (iii) for their abilities to inhibit bovine viral diarrhea virus (BVDV) production in acutely infected Madin-Darby bovine kidney cells. Some compounds demonstrated potent antiviral activities against the wild-type HIV-1 strain (range of 90% effective concentrations [EC(90)s], 0.14 to 5.2 micro M), but marked increases in EC(90)s were noted when the compounds were tested against the lamivudine-resistant HIV-1 strain (range of EC(90)s, 53 to >100 micro M). The beta-L-enantiomers of both classes of compounds were more potent than the corresponding beta-D-enantiomers. None of the compounds showed antiviral activity in the assay that determined their abilities to inhibit BVDV, while two compounds inhibited HBV production in HepAD38 cells (EC(90), 0.25 micro M). The compounds were essentially noncytotoxic in human peripheral blood mononuclear cells and HepG2 cells. No effect on mitochondrial DNA levels was observed after a 7-day incubation with the nucleoside analogs at 10 micro M. These studies demonstrate that (i) modification of the sugar ring of cytosine nucleoside analogs with a 4'-thia instead of an oxygen results in compounds with the ability to potently inhibit wild-type HIV-1 but with reduced potency against lamivudine-resistant virus and (ii) the antiviral activity of beta-D-2',3'-didehydro-2',3'-dideoxy-5-fluorocytidine against wild-type HIV-1 (EC(90), 0.08 micro M) and lamivudine-resistant HIV-1 (EC(90) = 0.15 micro M) is markedly reduced by introduction of a 3'-fluorine in the sugar (EC(90)s of compound 2a, 37.5 and 494 micro M, respectively).

Highly active antiretroviral therapy (HAART) is the standard treatment for infection with the human immunodeficiency virus (HIV). HAART regimens consist of protease inhibitors or nonnucleoside reverse transcriptase inhibitors combined with two or more nucleoside reverse transcriptase inhibitors (NRTIs). DPC 817, 2',3'-didehydro-2',3'-dideoxy-5-fluorocytidine (PSI 5582 D-D4FC) is a potent inhibitor of HIV type 1 replication in vitro. Importantly, DPC 817 retains activity against isolates harboring mutations in the reverse transcriptase gene that confer resistance to lamivudine (3TC) and zidovudine (AZT), which are frequent components of initial HAART regimens. DPC 817 combines this favorable resistance profile with rapid uptake and conversion to the active metabolite DPC 817-triphosphate, which has an intracellular half-life of 13 to 17 h. Pharmacokinetics in the rhesus monkey suggest low clearance of parent DPC 817 and a plasma half-life longer than that of either AZT or 3TC. Together, these properties suggest that DPC 817 may be useful as a component of HAART regimens in individuals with resistance to older NRTI agents.

AT-61, a member of a novel class of phenylpropenamide derivatives, was found to be a highly selective and potent inhibitor of human hepatitis B virus (HBV) replication in four different human hepatoblastoma cell lines which support the replication of HBV (i.e., HepAD38, HepAD79, 2.2.15, and transiently transfected HepG2 cells). This compound was equally effective at inhibiting both the formation of intracellular immature core particles and the release of extracellular virions, with 50% effective concentrations ranging from 0.6 to 5.7 microM. AT-61 (27 microM) was able to reduce the amount of HBV covalently closed circular DNA found in the nuclei of HepAD38 cells by >99%. AT-61 at concentrations of >27 microM had little effect on the amount of viral RNA found within the cytoplasms of induced HepAD38 cells but reduced the number of immature virions which contained pregenomic RNA by >99%. The potency of AT-61 was not affected by one of the mutations responsible for (-)-beta-L-2', 3'-dideoxy-3' thiacytidine (3TC) resistance in HBV, and AT-61 acted synergistic with 3TC to inhibit HBV replication. AT-61 (81 microM) was not cytotoxic or antiproliferative to several cell lines and had no antiviral effect on woodchuck or duck HBV, human immunodeficiency virus type 1, herpes simplex virus type 1, vesicular stomatitis virus, or Newcastle disease virus. Therefore, we concluded that the antiviral activity of AT-61 is specific for HBV replication and most likely occurs at one of the steps between the synthesis of viral RNA and the packaging of pregenomic RNA into immature core particles.

We report the development and isolation of a cell line, termed HepAD38, that replicates human hepatitis B virus (HBV) under conditions that can be regulated with tetracycline. In the presence of the antibiotic, this cell line is free of virus due to the repression of pregenomic (pg) RNA synthesis. Upon removal of tetracycline from the culture medium, the cells express viral pg RNA, accumulate subviral particles in the cytoplasm that contain DNA intermediates characteristic of viral replication, and secrete virus-like particles into the supernatant. Since the HepAD38 cell line can produce high levels of HBV DNA, it should be useful for analyses of the viral replication cycle that depend upon viral DNA synthesis in a synchronized fashion. In addition, this cell line has been formatted into a high-throughput, cell-based assay that permits the large-scale screening of diverse compound libraries for new classes of inhibitors of HBV replication.

XM323 represents a novel class of potent inhibitors of human immunodeficiency virus (HIV) protease. In vitro studies have shown that inhibition of this enzyme translates into potent inhibition of replication of HIV type 1 (HIV-1) and HIV-2. The inhibition of virus replication was assessed with three assays designed to measure the production of infectious virus, viral RNA, or p24 antigen. The production of mature infectious virions was measured with a yield reduction assay. By this assay, several strains and isolates of HIV-1 and HIV-2 were shown to be susceptible to XM323 in two lymphoid cell lines (MT-2 and H9) and in normal peripheral blood mononuclear cells, with a concentration required for 90% inhibition (IC90) of 0.12 +/- 0.04 microM (mean +/- standard deviation). The production of HIV-1(RF) RNA was measured with an RNA hybridization-capture assay. With this assay, XM323 was shown to be a potent inhibitor of HIV-1(RF) replication, with an IC90 of 0.063 +/- 0.032 microM. A third measure of virus replication, the production of p24 viral antigen, an essential protein component of the virion, was determined with the AIDS Clinical Trial Group-Department of Defense peripheral blood mononuclear cell consensus assay. This assay was used for expanded testing of XM323 against 28 clinical isolates and laboratory strains of HIV-1. XM323 was shown to be equally effective against zidovudine-susceptible and zidovudine-resistant isolates of HIV-1, with an overall IC90 of 0.16 +/- 0.06 microM.

The antiherpesvirus agent (E)-5-(2-bromovinyl)-2'-deoxyuridine caused marked alterations in the synthesis and processing of several herpes simplex virus type 1 (HSV-1)-infected-cell polypeptides. Analogous to other thymidine analogs, there was a dose-dependent decrease in several beta and gamma polypeptides and an accumulation of HSV-1 thymidine kinase. In contrast to the action of other thymidine analogs, there were alterations in alpha polypeptides, including an increase in the synthesis and phosphorylation of infected-cell polypeptide 4b and a decrease in the synthesis of infected-cell polypeptide 27. The phosphorylation of several other HSV-1 phosphoproteins was mildly inhibited. (E)-5-(2-Bromovinyl)-2'-deoxyuridine inhibited the glycosylation of the major HSV-1 glycoproteins, and this activity appeared to be independent of the incorporation of the drug into the viral DNA. Thus, the alterations in HSV-1 polypeptide expression appear to be due to the presence of the drug in a low-molecular-weight form as well as its presence in the viral DNA. This suggests that this analog or a phosphorylated derivative might act as an inhibitor of an enzyme(s) responsible for posttranslational modification of polypeptides.

WIN 51711 (5-[7-[4-(4,5-dihydro-2-oxazolyl)phenoxy]heptyl]-3-methylisoxazole), a new antipicornavirus drug, is a potent inhibitor of human entero- and rhinoviruses at concentrations not inhibitory to HeLa cell growth. In plaque reduction assays, WIN 51711 reduced plaque formation by 9 enteroviruses and 33 rhinoviruses, with MICs of 0.004 to 0.17 and 0.004 to 6.2 micrograms/ml, respectively. Addition of WIN 51711 to infected cells at concentrations of 0.02 to 5.0 micrograms/ml reduced the yield of picornaviruses by 90%. Other RNA viruses (nonpicornaviruses) and DNA viruses were unaffected by the compound.

WIN 51711, a potent new antipicornavirus drug, has been shown to inhibit an early event in the replication cycle of human poliovirus type 2 and human rhinovirus type 2. WIN 51711 was not virucidal and had no measurable effect on the adsorption of [3H]uridine-labeled virions to cells. When virion penetration of the plasma membrane was determined through loss of sensitivity to neutralizing antisera, WIN 51711 had no effect on poliovirus penetration, but inhibited rhinovirus penetration by 40%. In the presence of WIN 51711, exposure of neutral red-encapsidated virus-infected cells to light at 3 h postinfection resulted in a 3-log reduction in the number of infectious centers, indicating that WIN 51711 maintained the viral RNA in the encapsidated state after penetrating the cell membrane. The inhibition of uncoating by WIN 51711 in the neutral red assay was found to be concentration dependent, with a concentration of 0.03 micrograms/ml resulting in a 90% inhibition of uncoating. Sucrose gradient sedimentation of lysates from whole cells infected with [3H]uridine-labeled poliovirus showed that poliovirions remained intact in the presence of WIN 51711, but were uncoated in the absence of drug. WIN 51711 also prevented thermal inactivation of poliovirus infectivity, indicating a direct stabilizing effect of this compound on virion capsid conformation.

WIN 54954 (5-[5-[2,6-dichloro-4-(4,5-dihydro-2-oxazolyl)phenoxy]pentyl]-3- methylisoxazole) is a new member of the class of broad-spectrum antipicornavirus compounds known to bind in a hydrophobic pocket within virion capsid protein VP1. In plaque reduction assays, WIN 54954 reduced plaque formation of 50 of 52 rhinovirus serotypes (MICs ranged from 0.007 to 2.2 micrograms/ml). A concentration of 0.28 microgram/ml was effective in inhibiting 80% of the 52 serotypes tested (EC80). WIN 54954 was also effective in inhibiting 15 commonly isolated enteroviruses, with an EC80 of 0.06 microgram/ml. Furthermore, WIN 54954 was effective in reducing the yield of two selected enteroviruses in cell culture by 90% at concentrations approximately equal to their MICs. The therapeutic efficacy of intragastrically administered WIN 54954 was assessed in suckling mice infected with coxsackievirus A-9 or echovirus type 9 (Barty) 2.5 days prior to initiation of therapy. Single daily doses of 2 and 100 mg/kg protected 50% of the mice from developing paralysis (PD50) following infection with coxsackievirus A-9 and echovirus-9, respectively. At the PD50 doses for these two viruses, levels of WIN 54954 in serum were maintained above the in vitro MICs for a significant portion of the dosing interval. The dose-dependent reduction in viral titers observed in coxsackievirus A-9-infected mice correlated well with the therapeutic dose response. The potency and spectrum of WIN 54954 make it a potentially useful compound for the treatment of human enterovirus and rhinovirus infections.