The thymidine analog 3'-azido-3'-deoxythymidine (BW A509U, azidothymidine) can inhibit human immunodeficiency virus (HIV) replication effectively in the 50-500 nM range [Mitsuya, H., Weinhold, K. J., Furman, P. A., St. Clair, M. H., Nusinoff-Lehrman, S., Gallo, R. C., Bolognesi, D., Barry, D. W. & Broder, S. (1985) Proc. Naul. Acad. Sci. USA 82,[7096][7097][7098][7099][7100]. In contrast, inhibition of the growth of uninfected human fibroblasts and lymphocytes has been observed only at concentrations above 1 mM. The nature of this selectivity was investigated. Azidothymidine anabolism to the 5'-mono-, -di-, and -triphosphate derivatives was similar in uninfected and HIV-infected cells. The level of azidothymidine monophosphate was high, whereas the levels of the di-and triphosphate were low (c5 MuM and <2 MuM, respectively).Cytosolic thymidine kinase (EC 2.7.1.21) was responsible for phosphorylation of azidothymidine to its monophosphate. Purified thymidine kinase catalyzed the phosphorylations of thymidine and azidothymidine with apparent K. values of 2.9MuM and 3.0 ,uM. The maximal rate of phosphorylation with azidothymidine was equal to 60% of the rate with thymidine. Phosphorylation of azidothymidine monophosphate to the diphosphate also appeared to be catalyzed by a host-cell enzyme, thymidylate kinase (EC 2.7.4.9). The apparent Km value for azidothymidine monophosphate was 2-fold greater than the value for dTMP (8.6 ,uM vs. 4.1 MM), but the maximal phosphorylation rate was only 0.3% of the dTMP rate. These kinetic constants were consistent with the anabolism results and indicated that azidothymidine monophosphate is an alternative-substrate inhibitor of thymidylate kinase. This conclusion was reflected in the observation that cells incubated with azidothymidine had reduced intracellular levels of dTTP. ICso (concentration of inhibitor that inhibits enzyme activity 50%) values were determined for azidothymidine triphosphate with HYIV reverse transcriptase and with immortalized human lymphocyte (H9 cell) DNA polymerase a. Azidothymidine triphosphate competed about 100-fold better for the HIV reverse transcriptase than for the cellular DNA polymerase a. The results reported here suggest that azidothymidine is nonselectively phosphorylated but that the triphosphate derivative efficiently and selectively binds to the HIV reverse transcriptase. Incorporation of azidothymidylate into a growing DNA strand should terminate DNA elongation and thus inhibit DNA synthesis.

The acquired immune deficiency syndrome (AIDS) is thought to result from infection of T cells by a pathogenic human retrovirus, human T-lymphotropic virus type III (HTLV-III) or lymphadenopathy-associated virus (LAV). In this report, we describe the antiviral effects of a thymidine analogue,3'-azido-3'-deoxythymidine (BW A509U), which, as a triphosphate, inhibits the reverse transcriptase of HTLV-III/LAV. This agent blocks the expression of the p24 gag protein of HTLV-III/LAV in H9 cells following exposure to virus. The drug also inhibits the cytopathic effect of HTLV-IIIB (a virus derived from a pool of American patients) and HTLV-III/RF-II (an isolate obtained from a Haitian patient that differs by about 20% in the amino acid sequence of the envelope gene from several isolates of HTLV-III/LAV, including HTLV-IIIB, analyzed so far). 3'-Azido-3'-deoxythymidine also completely blocks viral replication as assessed by reverse transcriptase production in normal human peripheral blood mononuclear cells exposed to HTLV-IIIB. Finally, at concentrations of 3'-azido-3'-deoxythymidine that block the in vitro infectivity and cytopathic effect of HTLV-IIIB, the in vitro immune functions of normal T cells remain basically intact.

Hepatitis C virus (HCV) is a global health problem requiring novel approaches for effective treatment of this disease. The HCV NS5B polymerase has been demonstrated to be a viable target for the development of HCV therapies. β-d-2'-Deoxy-2'-α-fluoro-2'-β-C-methyl nucleosides are selective inhibitors of the HCV NS5B polymerase and have demonstrated potent activity in the clinic. Phosphoramidate prodrugs of the 5'-phosphate derivative of the β-d-2'-deoxy-2'-α-fluoro-2'-β-C-methyluridine nucleoside were prepared and showed significant potency in the HCV subgenomic replicon assay (<1 μM) and produced high levels of triphosphate 6 in primary hepatocytes and in the livers of rats, dogs, and monkeys when administered in vivo. The single diastereomer 51 of diastereomeric mixture 14 was crystallized, and an X-ray structure was determined establishing the phosphoramidate stereochemistry as Sp, thus correlating for the first time the stereochemistry of a phosphoramidate prodrug with biological activity. 51 (PSI-7977) was selected as a clinical development candidate.

A phosphoramidate prodrug of 2-deoxy-2-␣-fluoro-␤-Cmethyluridine-5-monophosphate, PSI-7851, demonstrates potent anti-hepatitis C virus (HCV) activity both in vitro and in vivo. PSI-7851 is a mixture of two diastereoisomers, PSI-7976 and PSI-7977, with PSI-7977 being the more active inhibitor of HCV RNA replication in the HCV replicon assay. To inhibit the HCV NS5B RNA-dependent RNA polymerase, PSI-7851 must be metabolized to the active triphosphate form. The first step, hydrolysis of the carboxyl ester by human cathepsin A (CatA) and/or carboxylesterase 1 (CES1), is a stereospecific reaction. Western blot analysis showed that CatA and CES1 are both expressed in primary human hepatocytes. However, expression of CES1 is undetectable in clone A replicon cells. Studies with inhibitors of CatA and/or CES1 indicated that CatA is primarily responsible for hydrolysis of the carboxyl ester in clone A cells, although in primary human hepatocytes, both CatA and CES1 contribute to the hydrolysis. Hydrolysis of the ester is followed by a putative nucleophilic attack on the phosphorus by the carboxyl group resulting in the spontaneous elimination of phenol and the production of an alaninyl phosphate metabolite, PSI-352707, which is common to both isomers. The removal of the amino acid moiety of PSI-352707 is catalyzed by histidine triad nucleotide-binding protein 1 (Hint1) to give the 5-monophosphate form, PSI-7411. siRNA-mediated Hint1 knockdown studies further indicate that Hint1 is, at least in part, responsible for converting PSI-352707 to PSI-7411. PSI-7411 is then consecutively phosphorylated to the diphosphate, PSI-7410, and to the active triphosphate metabolite, PSI-7409, by UMP-CMP kinase and nucleoside diphosphate kinase, respectively.Nucleoside analogs have long been the backbone therapy for the treatment of viral diseases such as HIV, HBV, and HSV infections (1-5). Recent studies have suggested that nucleoside analogs may be useful for treating hepatitis C virus (HCV) 3 infection (4, 6 -8). The most advanced anti-HCV nucleoside, RG7128, is a diisobutyrate nucleoside prodrug of ␤-D-2Ј-deoxy-2Ј-␣-fluoro-2Ј-␤-C-methylcytidine (PSI-6130) and is currently in phase IIb clinical studies. PSI-6130 demonstrated potent activity in the subgenomic HCV replicon assay (9); the incubation of radiolabeled PSI-6130 with either replicon cells or primary human hepatocytes resulted in the formation of the 5Ј-mono-, di-, and triphosphate metabolites of . The triphosphate metabolite (PSI-6130-TP) was shown to be a potent inhibitor of HCV NS5B RNA-directed RNA polymerase (RdRp) (11). However, incubation of replicon cells with the uridine analog, PSI-6206, resulted in no inhibition of HCV RNA production due to the inability of PSI-6206 to be phosphorylated by cellular nucleoside kinases to its monophosphate, 12). Biochemical studies showed that PSI-7411 was consecutively phosphorylated to its diphosphate, PSI-7410, by UMP-CMP kinase and its triphosphate, PSI-7409, by nucleoside diphosphate kinase (12). Inhibition studies using the replic...

A guanine derivative with an acyclic side chain, 2-hydroxyethoxymethyl, at position 9 has potent antiviral activity [dose for 50% inhibition (ED50) = 0.1 ,uM] against herpes simplex virus type 1. This acyclic nucleoside analog, termed acycloguanosine, is converted to a monophosphate by a virusspecified pyrimidine deoxynucleoside (thymidine) kinase and is subsequently converted to acycloguanosine di-and triphosphates. In the uninfected host cell (Vero) these phosphorylations of acycloguanosine occur to a very limited extent. Acycloguanosine triphosphate inhibits herpes simplex virus DNA polymerase (DNA nucleotidyltransferase) 10-30 times more effectively than cellular (HeLa S3) DNA polymerase. These factors contribute to the drug's selectivity; inhibition of growth of the host cell requires a 3000-fold greater concentration of drug than does the inhibition of viral multiplication. There is, moreover, the strong possibility of chain termination of the viral DNA by incorporation of acycloguanosine.The identity of the kinase that phosphorylates acycloguanosine was determined after separation of the cellular and virus-specified thymidine kinase activities by affinity chromatography, by reversal studies with thymidine, and by the lack of monophosphate formation in a temperature-sensitive, thymidine kinase-deficient mutant of the KOS strain of herpes simplex virus type 1 (tsAl).

3'-Dideoxy-S-fluoro-3'-thiacytidine (FTC) has been shown to be a potent and selective compound against human immunodeficiency virus type 1 in acutely infected primary human lymphocytes. FTC is also active against human immunodeficiency virus type 2, simian immunodeficiency virus, and feline immunodeficiency virus in various cell culture systems, including human monocytes. The antiviral activity can be prevented by 2'-deoxycytidine, but not by other natural nucleosides, suggesting that FTC must be phosphorylated to be active and 2'-deoxycytidine kinase is responsible for the phosphorylation. By using chiral columns or enzymatic techniques, the two enantiomers ofFTC were separated. The (-)-(o-enantiomer of FTC was about 20-fold more potent than the (+)-,i-enantiomer against human immunodeficiency virus type 1 in peripheral blood mononuclear cells and was also effective in thymidine kinase-deficient CEM cells. Racemic FEC and its enantiomers were nontoxic to human lymphocytes and other cell lines at concentrations of up to 100 ,uM. Studies with human bone marrow cells indicated that racemic FTC and its (-)-enantiomer had a median inhibitory concentration of >30 FiM. The (+)-enantiomer was significantly more toxic than the (-)-enantiomer to myeloid progenitor cells. The susceptibilities to FTC of pretherapy isolates in comparison with those of posttherapy 3'-azido-3'-deoxythymidine-resistant viruses in human lymphocytes were not substantially different. Similar results were obtained with well-defined 2',3'-dideoxyinosine-and nevirapine-resistant viruses.(-)-FTC-5'-triphosphate competitively inhibited human immunodeficiency virus type 1 reverse transcriptase, with an inhibition constant of 2.9 ,IM, when a poly(I) . oligo(dC)1-24 template primer was used. A two-to threefold decreased affinity was noted for the (+)-enantiomer. By using sequencing analysis, racemic FTC-5'-triphosphate was shown to be a potent DNA chain terminator when human immunodeficiency virus reverse transcriptase was used. These results suggest that further development of the (-)-1-enantiomer of FTC is warranted as an antiviral agent for infections caused by human immunodeficiency viruses.

Hepadnaviruses replicate by reverse transcription, which takes place in the cytoplasm of the infected hepatocyte. Viral RNAs, including the pregenome, are transcribed from a covalently closed circular (ccc) viral DNA that is found in the nucleus. Inhibitors of the viral reverse transcriptase can block new DNA synthesis but have no direct effect on the up to 50 or more copies of cccDNA that maintain the infected state. Thus, during antiviral therapy, the rates of loss of cccDNA, infected hepatocytes (1 or more molecules of cccDNA), and replicating DNAs may be quite different. In the present study, we asked how these losses compared when woodchucks chronically infected with woodchuck hepatitis virus were treated with L-FMAU [1-(2-fluoro-5-methyl-␤-L-arabinofuranosyl) uracil], an inhibitor of viral DNA synthesis. Viremia was suppressed for at least 8 months, after which drug-resistant virus began replicating to high titers. In addition, replicating viral DNAs were virtually absent from the liver after 6 weeks of treatment. In contrast, cccDNA declined more slowly, consistent with a half-life of ϳ33 to 50 days. The loss of cccDNA was comparable to that expected from the estimated death rate of hepatocytes in these woodchucks, suggesting that death of infected cells was one of the major routes for elimination of cccDNA. However, the decline in the actual number of infected hepatocytes lagged behind the decline in cccDNA, so that the average cccDNA copy number in infected cells dropped during the early phase of therapy. This observation was consistent with the possibility that some fraction of cccDNA was distributed to daughter cells in those infected hepatocytes that passed through mitosis.

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. Hepatitis C virus (HCV) currently infects more than 170 million people worldwide and is the leading cause of chronic liver disease and liver transplantation in the United States. HCV is a single plus-strand RNA virus about 9.6 kb in genome size and contains one open reading frame that encodes 10 structural and nonstructural (NS) proteins. The structural proteins (core, E1, and E2), which constitute the viral capsid and envelope proteins, are located at the amino terminus, followed by p7, which oligomerizes to form an ion channel critical for viral assembly, and the nonstructural proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B), which are responsible for viral replication (35). Recently, two antiviral agents have been approved, in combination with pegylated alpha interferon and ribavirin (Peg-IFN/RBV), to treat patients infected with genotype (GT) 1 HCV. Both of these compounds, boceprevir (Victrelis) and telaprevir (Incivek), target the NS3/4A protease and inhibit HCV replication by blocking processing of NS3, NS4A/B, and NS5A/B (25, 33). These treatments showed improvement over Peg-IFN/RBV; however, patients may develop additional side effects corresponding to each of these antiviral compounds (4). Furthermore, viral resistance against these compounds has emerged both in vitro and in vivo as a result of the high genetic diversity of HCV and error-prone nature of the HCV NS5B RNA-dependent RNA polymerase (12). Therefore, research...