The pharmacology of endosomal TLR agonists in viral disease.

The discovery of endosomal TLRs (Toll-like receptors) and their natural ligands has accelerated efforts to exploit them for therapeutic benefit. Importantly, this was preceded by clinical exploration of agents now known to be endosomal TLR agonists. Clinical effects in viral disease have been reported with agonists of TLR3, TLR7, TLR7/8 and TLR9, and the TLR7 agonist imiquimod is marketed for topical use against warts, a papillomavirus disease. The observed pre-clinical and clinical profiles of agonists of each of these TLRs suggest induction of a multifaceted innate immune response, with biomarker signatures indicative of type 1 interferon induction. However, these agents differ in both their pharmaceutical characteristics and the cellular distribution of their target TLRs, suggesting that drugs directed to these targets will display differences in their overall pharmacological profiles.

Regulation of both gene expression and protein stability provides genetically assisted target evaluation (GATE) for microbial target validation.

The attempt to develop novel antibiotics, active against organisms resistant to current therapies, has led researchers to seek and explore new drug targets. The rapid sequencing and analysis of entire microbial genomes has identified large numbers of genes that may be sufficiently different from their human counterparts to be exploited as targets for antimicrobial treatment. As a first step, the importance of the various putative targets for microbial growth and survival must be assessed. Emerging validation technologies are becoming increasingly sophisticated and, in certain cases, allow prioritisation of the best targets. In this paper, genetically assisted target evaluation (GATE) is introduced as a versatile target validation technology. GATE concomitantly manipulates both synthesis and stability of the targeted protein using copper ions as an effector. This technology allows rapid quantitation of the lethal consequences of inactivation of targeted gene products in Saccharomyces cerevisiae. Additional tools can then be applied to extend these results into pathogenic organisms, such as Candida albicans.

The ribavirin analog ICN 17261 demonstrates reduced toxicity and antiviral effects with retention of both immunomodulatory activity and reduction of hepatitis-induced serum alanine aminotransferase levels.

The demonstrated utility of the nucleoside analog ribavirin in the treatment of certain viral diseases can be ascribed to its multiple distinct properties. These properties may vary in relative importance in differing viral disease conditions and include the direct inhibition of viral replication, the promotion of T-cell-mediated immune responses via an enhanced type 1 cytokine response, and a reduction of circulating alanine aminotransferase (ALT) levels associated with hepatic injury. Ribavirin also has certain known toxicities, including the induction of anemia upon chronic administration. To determine if all these properties are linked, we compared the D-nucleoside ribavirin to its L-enantiomer (ICN 17261) with regard to these properties. Strong similarities were seen for these two compounds with respect to induction of type 1 cytokine bias in vitro, enhancement of type 1 cytokine responses in vivo, and the reduction of serum ALT levels in a murine hepatitis model. In contrast, ICN 17261 had no in vitro antiviral activity against a panel of RNA and DNA viruses, while ribavirin exhibited its characteristic activity profile. Importantly, the preliminary in vivo toxicology profile of ICN 17261 is significantly more favorable than that of ribavirin. Administration of 180 mg of ICN 17261 per kg of body weight to rats by oral gavage for 4 weeks generated substantial serum levels of drug but no observable clinical pathology, whereas equivalent doses of ribavirin induced a significant anemia and leukopenia. Thus, structural modification of ribavirin can dissociate its immunomodulatory properties from its antiviral and toxicologic properties, resulting in a compound (ICN 17261) with interesting therapeutic potential.

Monocyclic L-nucleosides with type 1 cytokine-inducing activity.

A series of 1,2,4-triazole L-nucleosides were synthesized and evaluated for their ability to stimulate type 1 cytokine production by activated human T cells in direct comparison to the known active agent ribavirin. Among the compounds prepared, 1-beta-L-ribofuranosyl-1,2,4-triazole-3-carboxamide (5, ICN 17261) was found to be the most uniformly potent compound. Conversion of the 3-carboxamide group of 5 to a carboxamidine functionality resulted in 1-beta-L-ribofuranosyl-1,2,4-triazole-3-carboxamidine hydrochloride (10), which induced cytokine levels comparable to 5 for two of the three type 1 cytokines examined. Modification of the carbohydrate moiety of 5 provided compounds of reduced activity. Significantly, ICN 17261 offers interesting immunomodulatory potential for the treatment of diseases where type 1 cytokines play an important role.

Inhibition of Borna disease virus replication by ribavirin.

The guanosine analogue ribavirin was tested for antiviral activity in two neural cell lines, human oligodendrocytes and rat glia, against Borna disease virus (BDV) strains V and He/80. Ribavirin treatment resulted in lower levels of virus and viral transcripts within 12 h. Addition of guanosine but not adenosine resulted in a profound reduction of the ribavirin effect. Ribavirin appears to be an effective antiviral agent for treatment of BDV infection in vitro. A likely mechanism for its activity is reduction of the intracellular GTP pool, resulting in inhibition of transcription and capping of BDV mRNAs.

Ribavirin polarizes human T cell responses towards a Type 1 cytokine profile.

BACKGROUND/AIMS: The therapeutic benefit of ribavirin, a nucleoside analog, in the treatment of chronic HCV infection is seen even in the absence of any apparent direct antiviral effect. We surmised that ribavirin may act by eliciting altered virus-specific immune responses. Because antiviral immunity is predominantly mediated by cytotoxic T cells and antiviral cytokines, we sought to determine whether ribavirin could promote antiviral (Type 1) cytokine expression in human T cells. METHODS: Isolated human T cells were activated in vitro with enterotoxin B or with phorbol ester plus ionomycin. Cytokine ELISAs were performed on culture supernatants, cytokine mRNA was detected following RT-polymerase chain reaction of T cell RNA, and T cell proliferation measured using MTT assay. RESULTS: Ribavirin enhanced a Type 1 (IL-2, IFNgamma, TNFalpha) while suppressing a Type 2 cytokine response (IL-4, IL-5 and IL-10), at the level of both protein and mRNA expression. Ribavirin mediated comparable effects on cytokine expression both following activation of specific T cell subpopulations with superantigen and following activation of a larger percentage of T cells via pharmacologic means. The in vitro effect on cytokine expression following ribavirin treatment was comparable in both CD4+ or CD8+ T cell subsets and was observed in a dose range that promoted T cell proliferation. CONCLUSIONS: These data support the view that ribavirin promotes a Type 1 cytokine-mediated immune response, a property which may account in part for its ability to enhance the antiviral activity of interferon-alpha in the treatment of chronic HCV infection.

1592U89, a novel carbocyclic nucleoside analog with potent, selective anti-human immunodeficiency virus activity.

1592U89, (-)-(1S,4R)-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclo pentene-1-methanol, is a carbocyclic nucleoside with a unique biological profile giving potent, selective anti-human immunodeficiency virus (HIV) activity. 1592U89 was selected after evaluation of a wide variety of analogs containing a cyclopentene substitution for the 2'-deoxyriboside of natural deoxynucleosides, optimizing in vitro anti-HIV potency, oral bioavailability, and central nervous system (CNS) penetration. 1592U89 was equivalent in potency to 3'-azido-3'-deoxythymidine (AZT) in human peripheral blood lymphocyte (PBL) cultures against clinical isolates of HIV type 1 (HIV-1) from antiretroviral drug-naive patients (average 50% inhibitory concentration [IC50], 0.26 microM for 1592U89 and 0.23 microM for AZT). 1592U89 showed minimal cross-resistance (approximately twofold) with AZT and other approved HIV reverse transcriptase (RT) inhibitors. 1592U89 was synergistic in combination with AZT, the nonnucleoside RT inhibitor nevirapine, and the protease inhibitor 141W94 in MT4 cells against HIV-1 (IIIB). 1592U89 was anabolized intracellularly to its 5'-monophosphate in CD4+ CEM cells and in PBLs, but the di- and triphosphates of 1592U89 were not detected. The only triphosphate found in cells incubated with 1592U89 was that of the guanine analog (-)-carbovir (CBV). However, the in vivo pharmacokinetic, distribution, and toxicological profiles of 1592U89 were distinct from and improved over those of CBV, probably because CBV itself was not appreciably formed from 1592U89 in cells or animals (<2%). The 5'-triphosphate of CBV was a potent, selective inhibitor of HIV-1 RT, with Ki values for DNA polymerases (alpha, beta, gamma, and epsilon which were 90-, 2,900-, 1,200-, and 1,900-fold greater, respectively, than for RT (Ki, 21 nM). 1592U89 was relatively nontoxic to human bone marrow progenitors erythroid burst-forming unit and granulocyte-macrophage CFU (IC50s, 110 microM) and human leukemic and liver tumor cell lines. 1592U89 had excellent oral bioavailability (105% in the rat) and penetrated the CNS (rat brain and monkey cerebrospinal fluid) as well as AZT. Having demonstrated an excellent preclinical profile, 1592U89 has progressed to clinical evaluation in HIV-infected patients.

A steady-state and pre-steady-state kinetic analysis of the NTPase activity associated with the hepatitis C virus NS3 helicase domain.

The helicase domain of hepatitis C virus NS3 (genotype 1b) was expressed in Escherichia coli and purified to homogeneity. The purified protein catalyzed the hydrolysis of nucleoside triphosphates (NTP) and the unwinding of duplex RNA in the presence of divalent metal ion. The enzyme was not selective for the NTP substrate. For example, UTP and acyclovir triphosphate were hydrolyzed efficiently by the enzyme. The rate of NTP hydrolysis was stimulated up to 27-fold by oligomeric nucleic acids (NA). Furthermore, NA bound to the enzyme with concomitant quenching of the intrinsic protein fluorescence. The dissociation constants of the enzyme for selected NA in the absence of NTP were between 10 and 35 microM at pH 7.0 and 25 degrees C. The enzyme had maximal affinity for NA with 12 or more nucleotides. A detailed steady-state and pre-steady-state kinetic analysis of ATP hydrolysis was made with (dU)18 as the effector. The kcat values for ATP hydrolysis in the presence and absence of (dU)18 were 80 s-1 and 2.7 s-1, respectively. The association (dissociation) rate constants for the enzyme and (dU)18 in the presence and absence of ATP were 5.7 microM-1 s-1 (3.9 s-1) and 290 microM-1 s-1 (2.27 s-1), respectively. The association (dissociation) rate constants for the enzyme and ATP in the presence and absence of (dU)18 were 0.4 microM-1 s-1 (<0.5 s-1) and 0.9 microM-1 s-1 (<10(-1) s-1), respectively. These data were consistent with a random kinetic mechanism.

(-)-cis-5-fluoro-1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]cytosine (524W91) inhibits hepatitis B virus replication in primary human hepatocytes.

The anti-hepatitis B virus (HBV) activity of (-)-cis-5-fluoro-1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]cytosine (524W91) in cultures of primary human hepatocytes was examined. 524W91 was anabolized to the active 5'-triphosphate in these cells. HBV replication was equally inhibited in cultures incubated with 524W91 when the drug was added 24 h preinfection, at infection, or 24 h postinfection. 524W91 inhibited HBV replication by 50% at less than 20 nM in human hepatocytes.

In vitro potency of inhibition by antiviral drugs of hematopoietic progenitor colony formation correlates with exposure at hemotoxic levels in human immunodeficiency virus-positive humans.

Inhibition of in vitro colony formation of human hematopoietic progenitors (CFU-granulocyte-macrophage, burst-forming unit-erythroid) by the antiviral nucleoside drugs alovudine, zalcitabine, zidovudine, ganciclovir, stavudine, didanosine, lamivudine, and acyclovir was measured. Significant correlations between in vitro 50% inhibitory concentrations and the daily human exposures (area under the concentration-time curve from 0 to 24 h; in micromolar.hour) of these chronically administered drugs in human immunodeficiency virus-positive patients that induced neutropenia or anemia were demonstrated by both linear regression and Spearman rank-order analyses. These quantitative correlations allow estimation of the exposure at which bone marrow toxicity may occur with candidate compounds.

2-Amino-6-methoxypurine arabinoside: an agent for T-cell malignancies.

Earlier studies have shown guanine arabinoside (ara-G) is an effective agent against growth of T-cell lines and freshly isolated human T-leukemic cells. However, poor water solubility of ara-G limits clinical use. 2-Amino-6-methoxypurine arabinoside (506U) is a water-soluble prodrug converted to ara-G by adenosine deaminase. 506U is not a substrate for deoxycytidine kinase, adenosine kinase, or purine nucleoside phosphorylase and is phosphorylated by mitochondrial deoxyguanosine kinase at a rate 4% that of ara-G phosphorylation. Mitochondrial DNA polymerase was the least sensitive to ara-GTP inhibition of the five human DNA polymerases tested. [3H]506U was anabolized to ara-G 5'-phosphates in CEM cells but not to phosphorylated metabolites of 506U. 506U was selective for transformed T over B cells and also inhibited growth in two of three monocytic lines tested. 506U given i.v. to cynomolgus monkeys was rapidly converted to ara-G; the ara-G had a half-life of approximately 2 h. 506U had in vivo dose-dependent efficacy against human T-cell tumors in immunodeficient mice. A Phase 1 trial of 506U against refractory hematological malignancies is now in progress at two study sites.

[[(Guaninylalkyl)phosphinico]methyl]phosphonic acids. Multisubstrate analogue inhibitors of human erythrocyte purine nucleoside phosphorylase.

A series of [[(guaninylalkyl)phosphinico]methyl]phosphonic acids, 2, was synthesized and tested as inhibitors of human erythrocyte purine nucleoside phosphorylase (PNPase). The target (phosphinicomethyl)phosphonic acids 2 were synthesized in six or seven steps from alkenylphosphonates 4. The latter were converted to the intermediate alkylmesylates 9 in a series of steps that included (1) conversion of the diethyl phosphonates 4 to the (phosphinoylmethyl)-phosphonates 7 and (2) conversion of the terminal double bond of [(alkenylphosphinoyl)methyl]-phosphonates 7 to the alkylmesylates 9. The pure 9-isomers 2 were obtained by alkylation of 2-amino-6-(2-methoxyethoxy)-9H-purine with alkylmesylates 9 followed by hydrolysis of the protecting groups with concentrated hydrochloric acid and ion exchange chromatography to give 2 as hydrated ammonium salts. The most potent inhibitor of human erythrocyte PNPase, [[[5-(2-amino-1,6-dihydro-6-oxo-9H-purin-9- yl)pentyl]phosphinico]methyl]phosphonic acid (2b), was a multisubstrate analogue inhibitor with a Ki' of 3.1 nM. Optimum PNPase inhibitory activity required the presence of zinc ions in the assay medium. These potent inhibitors of PNPase exhibited only weak activity against human leukemic T-cells in vitro.

Evidence that hepatocyte turnover is required for rapid clearance of duck hepatitis B virus during antiviral therapy of chronically infected ducks.

Duck hepatitis B virus (DHBV) DNA synthesis in congenitally infected ducks is inhibited by 2'-deoxycarbocyclic guanosine (2'-CDG). Three months of therapy reduces the number of infected hepatocytes at least 10-fold (W.S. Mason, J. Cullen, J. Saputelli, T.-T. Wu, C. Liu, W.T. London, E. Lustbader, P. Schaffer, A.P. O'Connell, I. Fourel, C.E. Aldrich, and A.R. Jilbert, Hepatology 19:393-411, 1994). The present study was performed to determine the kinetics of disappearance of infected hepatocytes and to evaluate the role of hepatocyte turnover in this process. Essentially all hepatocytes were infected before drug therapy. Oral treatment with 2'-CDG resulted in a prompt reduction in the number of infected hepatocytes. After 2 weeks, only 30 to 50% appeared to still be infected, and less than 10% were detectably infected after 5 weeks of therapy. To assess the possible role of hepatocyte turnover in these changes, 5-bromo-2'-deoxyuridine (BUdR) was administered 8 h before liver biopsy to label host DNA in hepatocytes passing through S phase, and stained nuclei were detected in tissue sections by using an antibody reactive to BUdR. The extent of nuclear labeling after 5 weeks was the same as that before therapy (ca. 1%). However, biopsies taken after 2 weeks of therapy showed a ca. 10-fold elevation in the number of nuclei labeled with BUdR. This result suggested that a rapid clearance of infected hepatocytes by 2'-CDG was caused not just by the inhibition of viral replication but also by an acceleration of the rate of hepatocyte turnover. To test this possibility further, antiviral therapy was carried out with another strong inhibitor of DHBV DNA synthesis, 5-fluoro-2',3'-dideoxy-3'-thiacytidine (524W), which did not accelerate hepatocyte turnover in ducks. 524W administration led to a strong inhibition of virus production but to a slower rate of decline in the number of infected hepatocytes, so that ca. 50% (and perhaps more) were still infected after 3 months of therapy. In addition, histopathologic evaluation of 2'-CDG-treated ducks revealed liver injury, especially at the start of therapy. No liver damage was observed during 524W therapy. These results imply that clearance of infected hepatocytes from the liver is correlated with hepatocyte turnover. Thus, in the absence of immune clearance or other sources for the accelerated elimination of infected hepatocytes, inhibitors of virus replication would have to be administered for a long period to substantially reduce the burden of infected hepatocytes in the liver.

Intracellular metabolism of (-)- and (+)-cis-5-fluoro-1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]cytosine in HepG2 derivative 2.2.15 (subclone P5A) cells.

The (-) and (+) enantiomers of the nucleoside analog cis-5-fluoro-1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]cytosine (2',3'-dideoxy-5-fluoro-3'-thiacytidine; FTC) have been shown to inhibit hepatitis B virus replication in vitro in HepG2 derivative 2.2.15 (subclone P5A) cells. (-)-FTC and (+)-FTC were anabolized to 5'-monophosphate, 5'-diphosphate, and 5'-triphosphate in this cell line. (-)-FTC was more efficiently phosphorylated to the 5'-triphosphate than (+)-FTC, and levels of 3.6 and 0.2 pmol/10(6) cells, respectively, were detected after incubation with 1 microM compound for 24 h. A time course study showed that nucleotides were formed rapidly in a dose-dependent manner and reached a steady-state intracellular concentration by 3 to 6 h. The intracellular half-life of (-)-FTC 5'-triphosphate was 2.4 h. Both (-)- and (+)-FTC were converted to diphosphocholine derivatives, analogous to CDP-choline, but only (+)-FTC was converted to the diphosphoethanolamine derivative, analogous to CDP-ethanolamine. (-)-FTC was not detectably deaminated at either the nucleoside or nucleotide level. (+)-FTC was partially deaminated by these cells. The transport of (-)-and (+)-FTC was examined in HepG2 cells. (+)-FTC enters these cells by way of the nitrobenzylthioinosine-susceptible, equilibrative nucleoside transporter. In contrast, the influx of (-)-FTC was only partially susceptible to inhibitors of nucleoside transport, indicating that (-)-FTC may have multiple transport mechanisms. These metabolic results are consistent with the conclusion that (-)-FTC 5'-triphosphate mediates the anti-hepatitis B virus activity of (-)-FTC.

An enantiospecific synthesis of the human cytomegalovirus antiviral agent [(R)-3-((2-amino-1,6-dihydro-6-oxo-9H-purin-9-yl)methoxy)-4- hydroxybutyl]phosphonic acid.

The racemic isosteric phosphonate of ganciclovir monophosphate (BW2482U89, SR3745, [3-((2-amino-1,6-dihydro-6-oxo-9H-purin-9-yl)methoxy)-4- hydroxybutyl]phosphonic acid, 1) has potent and selective in vitro activity against human cytomegalovirus. An enantiospecific synthesis of the R-enantiomer of compound 1 starting from L-arabinose was developed. The synthesis involved (1) the preparation of a chiral acyclic moiety, (2) the coupling of the chiral acyclic moiety to diacetylguanine, (3) the introduction of phosphorus, and (4) the final deprotection. The R-enantiomer, which has stereochemistry analogous to the natural compound GMP, was tested against human cytomegalovirus and had an IC50 of 1.7 microM, which was approximately 2-fold more active than the racemic material. Both racemic and chiral compounds were less toxic than ganciclovir to bone marrow progenitor cells in an in vitro assay.

Influence of stereochemistry on antiviral activities and resistance profiles of dideoxycytidine nucleosides.

beta-L-2',3'-Dideoxycytidine (beta-L-ddC) and beta-L-5-fluoro-2',3'-dideoxycytidine (5-F-beta-L-ddC) were prepared and shown to have potent activity against human immunodeficiency virus type 1 (HIV-1) and hepatitis B virus (HBV). These compounds were compared with beta-D-2',3'-dideoxycytidine (beta-D-ddC) and two beta-L-oxathiolane nucleosides (beta-L-3'-thio-2',3'-dideoxycytidine and beta-L-5-fluoro-3'-thio-2',3'-dideoxycytidine) in terms of anti-HIV and anti-HBV activity, cytotoxicity, and development of HIV-1 resistance. Compared with beta-D-ddC, the beta-L-dideoxycytidine nucleosides had similar anti-HIV-1 activities, significantly greater anti-HBV activities, and decreased toxicities to a B-cell line, T-cell lines, and human bone marrow progenitor cells. HIV-1 strains resistant to beta-D-ddC were susceptible to the beta-L-ddC analogs. Compared with the beta-L-oxathiolane nucleosides, beta-L-ddC and 5-F-beta-L-ddC had similar anti-HIV-1 activities, decreased anti-HBV activities, and greater toxicities to B- and T-cell lines and bone marrow progenitor cells. There were similarities between the beta-L-ddC and beta-L-oxathiolane nucleosides in the rate of development and pattern of resistant HIV-1 selection. While the in vitro activity and cytotoxicity profiles of the beta-L-ddC nucleosides differed from those of the beta-D-ddC and beta-L-oxathiolane nucleosides, the data presented herein suggest that the sugar configuration of a dideoxynucleoside analog may play a major role in the rate of development and the pattern of HIV-1 resistance.

High-capacity in vitro assessment of anti-hepatitis B virus compound selectivity by a virion-specific polymerase chain reaction assay.

An integrated assessment system specific for hepatitis B virus (HBV) Dane particle DNA was developed to examine the activity of potential anti-HBV compounds in chronic HBV-producing HepG2-derived 2.2.15 cells. Cell culture, immunoaffinity purification, polymerase chain reaction, and hybrid-capture detection were performed in the microtiter format to facilitate increased throughput by automation. The high sensitivity afforded by the assay provided quantitative detection of less than 0.5 fg of extracellular HBV DNA from 25 microliters of cell culture supernatants, and drug-induced reductions in HBV titers greater than 100-fold were easily measured. Fluorometric determination of total cellular DNA from the same 96-well proliferating cell cultures allowed simultaneous evaluation of inhibition of cell growth, thus providing the ability to assess the overall selectivities of candidate compounds in a single experiment. The potent activities of three anti-HBV compounds, the (+) and (-) enantiomers of cis-5-fluoro-1-[2-(hydroxymethyl)-1,3-oxythiolane-5-yl]cytosine (FTC) and D-carbocyclic-2'-deoxyguanosine (CDG), were confirmed by this method. (-)-FTC was more active than its (+) enantiomer (50% inhibitory concentrations, 0.033 +/- 0.006 and 0.723 +/- 0.160 microM [standard error of the mean; SEM], respectively), while both enantiomers demonstrated a lack of cytotoxicity at 200 microM. CDG was more potent (50% inhibitory concentration, 0.0063 +/- 0.0007 microM [SEM]) but was also significantly more toxic, inhibiting cell growth by 50% at 32 +/- 6 microM (SEM). These results demonstrate the usefulness of this immunoaffinity-based, quantitative polymerase chain reaction system as a high-capacity in vitro tool for assessment of anti-HBV compound selectivity.

Novel 6-alkoxypurine 2',3'-dideoxynucleosides as inhibitors of the cytopathic effect of the human immunodeficiency virus.

Twenty-one 6-alkoxypurine 2',3'-dideoxynucleosides were enzymatically synthesized with nucleoside phosphorylases purified from E. coli. Eighteen analogs exhibited anti-HIV-1 activity in MT4 cells. Two analogs, 6-(hexyloxy)-(17) and 6-(heptyloxy)-(18) purine 2',3'-dideoxynucleoside, were as potent as 2',3'-dideoxyinosine (ddI, didanosine, Videx). Although the antiviral activities of 17 and 18 were equivalent, 18 was more cytotoxic. Analogs containing less than four carbons in the 6-alkoxypurine substituent exhibited weak anti-HIV-1 activity. Analogs containing more than seven carbons in the 6-alkoxypurine substituent were too cytotoxic to be effectively evaluated for antiviral activity. Several 6-alkoxypurine 2',3'-dideoxynucleosides were evaluated for substrate activity with calf intestinal adenosine deaminase (ADA). Increasing the carbon chain length of the 6-alkoxypurine substituent decreased the rate of dealkoxylation. The best substrate in this series was 6-methoxypurine 2',3'-dideoxynucleaside (1); however, the rate of dealkoxylation of 100 microM 1 was 0.17% of the rate of deamination of 100 microM 2',3'-dideoxyadenosine. Compound 17, the most potent anti-HIV-1 analog, was not a substrate for ADA. EHNA (erthro-9-(2-hydroxy-3-nonyl)adenine), a potent inhibitor of ADA, had little effect on the antiviral activities of 17 and ddI. In contrast, coformycin, a potent inhibitor of both ADA and AMP deaminase, dramatically decreased the antiviral activity of 17, but not the antiviral activity of ddI. Thus, AMP deaminase appeared to be involved in the anabolism of 17. The pharmacokinetic profile of 17, the most promising analog in this series, was determined in the rat. At least seventeen metabolites of 17, including ddI, were detected in plasma samples. This analog also had poor oral bioavailability.

Anabolic pathway of 6-methoxypurine arabinoside in cells infected with varicella-zoster virus.

6-Methoxypurine arabinoside (ara-M) exhibits potent activity against varicella-zoster virus (VZV) as a result of ara-M's anabolism to the triphosphate of adenine arabinoside (ara-ATP) in VZV-infected cells. The adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) enhanced the formation of ara-ATP by inhibiting ara-M demethoxylation. In contrast, deoxycoformycin and coformycin, inhibitors of both adenosine deaminase and AMP deaminase, blocked the formation of ara-ATP and reversed the anti-VZV activity of ara-M. These results indicate that after the initial phosphorylation of ara-M by the VZV-coded thymidine kinase, the monophosphate is demethoxylated by AMP deaminase to form ara-IMP, which is converted to ara-ATP by the sequential actions of the cellular adenylosuccinate synthetase, adenylosuccinate lyase, and nucleotide kinases.

6-N-substituted derivatives of adenine arabinoside as selective inhibitors of varicella-zoster virus.

A series of 6-alkylaminopurine arabinosides were synthesized and found to inhibit varicella-zoster virus (VZV). The antiviral activities of these nucleosides were limited to VZV. None of the other viruses tested in the herpesvirus family were affected. The in vitro antiviral potencies of the 18 arabinosides correlated with their efficiencies as substrates of the VZV-encoded thymidine kinase in all but one case. The arabinosides of 6-methylaminopurine and 6-dimethylaminopurine were the most potent analogs, with 50% inhibitory concentrations against VZV of 3 and 1 microM, respectively. They were not cytotoxic to uninfected MRC-5 cells, human Detroit 98 cells, or mouse L cells (50% inhibitory concentration, greater than 100 microM). Neither 6-methylaminopurine arabinoside nor 6-dimethylaminopurine arabinoside was detectably phosphorylated by either adenosine kinase or 2'-deoxycytidine kinase. These two alkylaminopurine arabinosides were also resistant to deamination catalyzed by adenosine deaminase. The VZV-dependent phosphorylation of these nucleosides offers the possibility of a potent and highly selective therapy for VZV infection.