Kinetics of hepadnavirus loss from the liver during inhibition of viral DNA synthesis.

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-beta-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 approximately 33 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.

Mechanism of action of 1-beta-D-2,6-diaminopurine dioxolane, a prodrug of the human immunodeficiency virus type 1 inhibitor 1-beta-D-dioxolane guanosine.

(-)-beta-D-2,6-Diaminopurine dioxolane (DAPD), is a nucleoside reverse transcriptase (RT) inhibitor with activity against human immunodeficiency virus type 1 (HIV-1). DAPD, which was designed as a water-soluble prodrug, is deaminated by adenosine deaminase to give (-)-beta-D-dioxolane guanine (DXG). By using calf adenosine deaminase a K(m) value of 15 +/- 0.7 microM was determined for DAPD, which was similar to the K(m) value for adenosine. However, the k(cat) for DAPD was 540-fold slower than the k(cat) for adenosine. In CEM cells and peripheral blood mononuclear cells exposed to DAPD or DXG, only the 5'-triphosphate of DXG (DXG-TP) was detected. DXG-TP is a potent alternative substrate inhibitor of HIV-1 RT. Rapid transient kinetic studies show the efficiency of incorporation for DXG-TP to be lower than that measured for the natural substrate, 2'-deoxyguanosine 5'-triphosphate. DXG-TP is a weak inhibitor of human DNA polymerases alpha and beta. Against the large subunit of human DNA polymerase gamma a K(i) value of 4.3 +/- 0.4 microM was determined for DXG-TP. DXG showed little or no cytotoxicity and no mitochondrial toxicity at the concentrations tested.

An analysis of the catalytic cycle of HIV-1 reverse transcriptase: opportunities for chemotherapeutic intervention based on enzyme inhibition.

This review describes each of the steps in the HIV-1 reverse transcriptase catalytic cycle and evaluates each of these steps as a potential point of inhibition of the enzyme and consequently viral replication. To date, two classes of approved drugs act on the reverse transcriptase. They are: (1) the nucleoside reverse transcriptase inhibitors which either directly inhibit the enzyme or serve as alternative substrates for catalysis (resulting in chain termination) and (2) the non-nucleoside reverse transcriptase inhibitors which bind to an allosteric site and adversely affect the function of the enzyme by slowing the rate of chemical catalysis. In order to provide the best possible analysis of the potential of each of the steps in the catalytic cycle as a site of inhibition, the molecular forces which determine the intrinsic binding affinities and specificity of natural components of the catalytic complex will be described in as much detail as possible.

Conformation and local environment of nucleotides bound to HIV type 1 reverse transcriptase (HIV-1 RT) in the ground state.

Nuclear Overhauser effect experiments were used to characterize the protein environment and conformations of dTTP, dATP and AZTTP bound to HIV-RT in the ground state. The results show the initial binding sites for the nucleotides overlap but are not completely coincident. All of the bound nucleotides assume the same anti C4'-exo conformation.

Screening for potential toxicity of antiviral therapies.

Preclinical screening for the toxicity of antiviral drugs has thus far proven quite successful. Twenty-three antivirals spanning a variety of chemical classes and including a combination product, have been safely developed and are listed in the 1999 Physician's Desk Reference. Several of these antivirals have been administered for many years and in various combinations and we are currently unaware of any being withdrawn for safety-related reasons. Progress in protecting this record includes advances in rational drug design, the development of in vitro and in vivo models available to study both efficacy and safety, and improved bioanalytical capabilities. Coupled with approximately 25 years of experience since vidarabine was approved for the treatment of herpes encephalitis, these advances set the stage for even more precise and focused development of pharmaceuticals for the treatment of life-threatening viral infections, including those caused by HIV, HBV and emerging viruses. Experience to date suggests that each antiviral, particularly the nucleoside analogs, may have unique attributes of efficacy and safety. This brief review is an attempt to highlight a combination of established and recent methods that may be helpful in screening antivirals for potential toxicity. The earliest phases of drug development are mentioned in the introduction, followed by contributions provided by in vitro and in vivo testing in models of viral disease. Finally, current and new methods applicable to screening for toxicity are discussed.

Human immunodeficiency virus type-1 reverse transcriptase. Contribution of Met-184 to binding of nucleoside 5'-triphosphate.

Mutations were made in recombinant human immunodeficiency virus type-1 reverse transcriptase (RT) by substituting methionine 184 with alanine (M184A) or valine (M184V), and steady-state and pre-steady-state kinetic constants were determined. The Km values of M184A RT for dNTPs were larger than those of wt RT for RNA-directed synthesis; the kcat values of M184A RT for processive or distributive synthesis were similar. In contrast to M184A RT, the Km and kcat values of M184V RT for dNTP substrates were similar to those of wt RT. The Ki values of M184V RT for 1-beta-L-nucleoside analogs were increased 30-500-fold relative to wt RT for both RNA- and DNA-directed synthesis. The Kd and kp values of wt RT and M184V RT for dCTP and cis-5-fluoro-1-[2-(hydroxymethyl)-1, 3-oxathiolan-5-yl]cytosine 5'-triphosphate (1-beta-L-FTCTP) were estimated from pre-steady-state kinetics for single nucleotide incorporation. The Kd value of M184V RT for 1-beta-L-FTCTP was 19-fold greater than that of wt RT; the kpvalues of the two enzymes were similar. These results support the hypothesis that methionine 184 in the highly conserved YMDD region of wt RT participates in the binding of the nucleoside (analog) 5'-triphosphate.

Recombinant human immunodeficiency virus type 1 reverse transcriptase is heterogeneous.

Recombinant wild type (wt) and T215Y HIV-1 reverse transcriptase (RT) were isolated using three methods designated A, B, and C. The three samples of wt RT were kinetically indistinguishable with respect to dTTP turnover on poly(rA).p(dT)10. However, whereas the kinetic constants for dTTP and AZTTP for both T215Y B and T215Y C were similar to those of wt protein, T215Y A exhibited a twofold increase in Km value for dTTP and a 13-fold increase in Ki value for AZTTP with respect to wt protein purified in the same manner. We further investigated this observation by studying the denaturation of wt RT by urea. The urea denaturation curves monitored by fluorescence and circular dichroism spectroscopy were not coincident with the denaturation curve monitored by enzyme activity and yielded Cm values (the concentration of urea at which 50% of the protein is denatured) of 4.1 and 2.0 M urea, respectively. The noncoincidence of the transition curves reflects two separable, sequential, noncooperative conformational changes in the molecule: (a) from a catalytically active to an inactive conformation, and (b) from a catalytically inactive to a denatured, unfolded conformation. We therefore used denaturation as detected by changes in enzyme activity to compare the conformational stability of the three samples of wt and T215Y RT A, B, and C. The Cm values for T215Y RT did not differ from those of the respective wt; however, differences in Cm values were noted depending on how the protein was isolated. This suggested that the heterogeneity of the recombinant RT was due to small differences in conformation at or near the active site.

Evaluation of the potent anti-hepatitis B virus agent (-) cis-5-fluoro-1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]cytosine in a novel in vivo model.

A murine model was developed to investigate the in vivo activity of anti-hepatitis B virus (HBV) agents. Mice with subcutaneous tumors of HBV-producing 2.2.15 cells showed reductions in levels of HBV in serum and in intracellular levels of HBV when the mice were orally dosed with (-) cis-5-fluoro-1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]cytosine (FTC). No effects on tumor size or alpha-fetoprotein levels were observed. FTC can selectively inhibit HBV replication at nontoxic doses.

Pharmacokinetics, oral bioavailability, and metabolic disposition in rats of (-)-cis-5-fluoro-1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl] cytosine, a nucleoside analog active against human immunodeficiency virus and hepatitis B virus.

The pharmacokinetics and metabolism of the potent anti-human immunodeficiency virus and anti-hepatitis B virus compound, (-)-cis-5-fluoro-1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl] cytosine (FTC), were investigated in male CD rats. Plasma clearance of 10 mg of FTC per kg of body weight was biexponential in rats, with a half-life at alpha phase of 4.7 +/- 1.1 min (mean +/- standard deviation) and a half-life at beta phase of 44 +/- 8.8 min (n = 5). The total body clearance of FTC was 1.8 +/- 0.1 liters/h/kg, and the oral bioavailability was 90% +/- 8%. The volume of distribution at steady state (Vss) was 1.5 +/- 0.1 liters/kg. Increasing the dose to 100 mg/kg slowed clearance to 1.5 +/- 0.2 liters/kg/h, lowered the Vss to 1.2 +/- 0.2 liters/kg, and reduced the oral bioavailability to 65% +/- 15%. FTC in the brains of rats was initially less than 2% of the plasma concentration but increased to 6% by 2 h postdose. Probenecid elevated levels of FTC in plasma as well as in brains but did not alter the brain-to-plasma ratio. The urinary and fecal recoveries of unchanged FTC after a 10-mg/kg intravenous dose were 87% +/- 3% and 5% +/- 1.6%, respectively. After a 10-mg/kg oral dose, respective urinary and fecal recoveries were 70% +/- 2.5% and 25% +/- 1.6%. Two sulfoxides of FTC were observed in the urine, accounting for 0.4% +/- 0.03% and 2.7% +/- 0.2% of the intravenous dose and 0.4% +/- 0.06% and 2.5% +/- 0.3% of the oral dose. Also observed were 5-fluorocytosine, representing 0.4% +/- 0.06% of the intravenous dose and 0.4% +/- 0.07% of the oral dose, and FTC glucuronide, representing 0.7% +/- 0.2% of the oral dose and 0.4% +/- 0.2% of the intravenous dose. Neither deaminated FTC nor 5-fluorouracil was observed in the urine (less than 0.2% of dose). The high oral availability and minimal metabolism of FTC encourage its further preclinical development.

Identification of the nucleotide binding site of HIV-1 reverse transcriptase using dTTP as a photoaffinity label.

We have utilized UV-induced cross-linking of [methyl-3H]dTTP to identify the nucleotide binding site on heterodimeric HIV-1 reverse transcriptase (RT). RT was derivatized by irradiating a solution containing [methyl-3H]dTTP and purified recombinant RT for 10 min. The UV-induced cross-linking reaction between dTTP and RT is linear with time of UV exposure up to 10 min, and it has been determined previously that dTTP cross-linking is half-maximal at 90 microM [Cheng, N., Painter, G. R., & Furmann, P.A. (1991) Biochem. Biophys. Res. Commun. 174, 785-789]. Under these reaction conditions, only the 66-kDa subunit of the 66-kDa/51-kDa RT heterodimer was labeled with dTTP. The [methyl-3H]dTTP-labeled RT was fragmented with trypsin and endoproteinase Asp-N, and peptides were purified on reversed phase HPLC. The peptide covalently linked to [methyl-3H]dTTP was subjected to amino acid sequence analysis. The sequencing data localized the nucleotide binding site of RT to Lys-73 in the vicinity of several mutation sites linked to antiviral drug resistance. Since most effective anti-AIDS compounds are inhibitors of RT, information about its dNTP binding site may make it possible to understand the basis for the antiviral activity of nucleoside analogs such as AZT, ddI, and ddC. This information may also be useful for a more rationally based design of anti-HIV agents.

The 5'-triphosphates of the (-) and (+) enantiomers of cis-5-fluoro-1-[2-(hydroxymethyl)-1,3-oxathiolane-5-yl]cytosine equally inhibit human immunodeficiency virus type 1 reverse transcriptase.

The (-) enantiomer of cis-5-fluoro-1-[2-(hydroxymethyl)-1,3-oxathiolane-5-yl]cytosine is a potent inhibitor of human immunodeficiency virus type 1 in vitro. The 5'-triphosphates of the (-) and (+) enantiomers equally inhibited the production of full-length minus-strand DNA in an endogenous reverse transcriptase reaction, each competitively inhibited DNA synthesis, and each was used as a chain-terminating substrate.

Mechanism of resistance of human immunodeficiency virus type 1 to 2',3'-dideoxyinosine.

A molecular clone containing the wild-type reverse transcriptase (RT) coding region of human immunodeficiency virus type 1 (HIV-1) was constructed, and site-directed mutagenesis was used to introduce mutations--Leu74-->Val (L74V), T215Y, and the combination L74V/T215Y--into the RT coding region. The proteins were purified by immunoaffinity chromatography. Assays were performed with mutant and wild-type RT to determine substrate and inhibitor specificity. All three mutant enzymes catalyzed the incorporation of substrate 2'-deoxynucleoside 5'-triphosphates (dNTPs) as efficiently as wild-type HIV-1 RT. Small changes were observed in the Km values for dNTPs with all three mutant enzymes, while more significant changes were noted in sensitivity to nucleoside 5'-triphosphate analogues that inhibit the enzyme activity. Results suggest that altered substrate recognition by the HIV-1 RT is involved in the mechanism of resistance.

Biochemical analysis of human immunodeficiency virus-1 reverse transcriptase containing a mutation at position lysine 263.

Site-directed mutagenesis has been used to assess the importance of lysine 263 in substrate binding of human immunodeficiency virus-1 (HIV-1) reverse transcriptase. Previous studies have indicated that lysine 263 functions in the binding of 2'-deoxynucleoside 5'-triphosphate (dNTP) substrates (Basu, A., Tirumalai, R. S., and Modak, M. J. (1989) J. Biol. Chem. 264, 8746-8752). We studied this interaction directly by using site-specific mutagenesis to change lysine 263 to a serine. Highly purified mutant enzyme K263S bound natural dNTP substrates and primed polynucleic acid substrates with equal affinity when compared to the wild type reverse transcriptase. No difference was observed in the binding of 3'-azido-2',3'-dideoxythymidine 5'-triphosphate to the mutant reverse transcriptase on the basis of Km and Ki determinations. The serine substitution had no effect on RNase H activity. These results indicate that lysine 263 is not essential in the binding of substrates to HIV-1 reverse transcriptase.

Conformational changes induced in herpes simplex virus DNA polymerase upon DNA binding.

Herpesvirus DNA polymerases are prototypes for alpha-like DNA polymerases and important targets for antiherpesvirus drugs. We have investigated changes in the catalytic subunit of herpes simplex virus DNA polymerase following DNA binding by using the techniques of endogeneous fluorescence quenching and limited proteolysis. The fluorescence studies revealed a reduction in the rate of quenching by acrylamide in the presence of DNA without changes in the wavelength of the emission peak or in the lifetime of the fluorophore, consistent with the possibility of conformational changes. Strikingly, the proteolysis studies revealed that binding to a variety of natural and synthetic DNA and RNA molecules induced the appearance of a new cleavage site for trypsin near residue 1060 of the protein and increased cleavage by trypsin near the center of the protein. The extent of these cleavages correlated with the affinity of the polymerase for these ligands. These data provide strong evidence that binding to nucleic acid polymers induces substantial localized conformational changes in the polymerase. The locations of enhanced tryptic cleavage near sites implicated in substrate recognition and interaction with a processivity factor suggest that the conformational changes are important for catalysis and processivity of this prototype alpha-like DNA polymerase. Inhibition of these changes may provide a mechanism for antiherpesvirus drugs.

The anti-hepatitis B virus activities, cytotoxicities, and anabolic profiles of the (-) and (+) enantiomers of cis-5-fluoro-1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]cytosine.

The anti-hepatitis B (anti-HBV) activities of the (-) and (+) enantiomers of cis-5-fluoro-1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]cytosine (2'-deoxy-3'-thia-5-fluorocytosine [FTC]) were studied by using an HBV-transfected cell line (HepG2 derivative 2.2.15, subclone P5A). The (-) isomer was found to be a potent inhibitor of viral replication, with an apparent 50% inhibitory concentration of 10 nM, while the (+) isomer was found to be considerably less active. Both isomers showed minimal toxicity to HepG2 cells (50% inhibitory concentration, > 200 microM) and showed minimal toxicity in the human bone marrow progenitor cell assay. In accord with the cellular antiviral activity data, the 5'-triphosphate of (-)-FTC inhibited viral DNA synthesis in an endogenous HBV DNA polymerase assay, while the 5'-triphosphate of the (+) isomer was inactive. Unphosphorylated (-)-FTC did not inhibit product formation in the endogenous assay, suggesting that the antiviral activity of the compound is dependent on anabolism to the 5'-triphosphate. Both (-)- and (+)-FTC were anabolized to the corresponding 5'-triphosphates in chronically HBV-infected HepG2 cells. The rate of accumulation and the steady-state concentration of the 5'-triphosphate of (-)-FTC were greater. Also, (-)-FTC was not a substrate for cytidine deaminase and, therefore, is not subject to deamination and conversion to an inactive uridine analog. The (+) isomer is, however, a good substrate for cytidine deaminase.

Selective inhibition of human immunodeficiency viruses by racemates and enantiomers of cis-5-fluoro-1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]cytosine.

2',3'-Dideoxy-5-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 of FTC were separated. The (-)-beta-enantiomer of FTC was about 20-fold more potent than the (+)-beta-enantiomer against human immunodeficiency virus type 1 in peripheral blood mononuclear cells and was also effective in thymidine kinase-deficient CEM cells. Racemic FTC and its enantiomers were nontoxic to human lymphocytes and other cell lines at concentrations of up to 100 microM. Studies with human bone marrow cells indicated that racemic FTC and its (-)-enantiomer had a median inhibitory concentration of > 30 microM. 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 microM, when a poly(I)n.oligo(dC)19-24 template primer was used. These results suggest that further development of the (-)-Beta-enantiomer of FTC is warranted as an antiviral agent for infections caused by human immunodeficiency viruses.

Purification and kinetic characterization of equine infectious anemia virus reverse transcriptase.

The reverse transcriptase of Equine Infectious Anemia Virus (EIAV) was partially purified from virus particles and appeared to be a heterodimer with subunit molecular masses of 70 kdal and 59 kdal. The polymerase activity of this enzyme had an absolute requirement for a divalent cation, preferring Mg++ over Mn++. Addition of a monovalent cation to the reaction mixture enhanced, but was not required for enzyme activity. Kinetically, the reverse transcriptase of EIAV is similar to the reverse transcriptase of Human Imunodeficiency Virus Type 1 (HIV-1). Both enzymes have similar Km values for 2'-deoxynucleoside-5'-triphosphates on the synthetic template/primers tested, both exhibit substrate inhibition, and both are inhibited to similar extents by most nucleoside-triphosphate analogs. The results of this study suggest that the reverse transcriptase of EIAV may be a good model for studying structure/function relationships of retroviral reverse transcriptases.

Initial binding of 2'-deoxynucleoside 5'-triphosphates to human immunodeficiency virus type 1 reverse transcriptase.

Human immunodeficiency virus type 1 reverse transcriptase (EC 2.7.7.49), a heterodimer consisting of two polypeptide chains of molecular weights 66,000 and 51,000, fluoresces due to the presence of 36 tryptophan residues with an emission peak centered at 338 nm. The association of 2'-deoxynucleoside 5'-triphosphates with the enzyme results in a decrease in the intensity of the tryptophan emission spectrum, which can be used to calculate apparent dissociation constants. The Kd values determined for binding of the four natural 2'-deoxynucleoside 5'-triphosphates to the free enzyme range from 36.7 +/- 1.8 microM for dTTP to 47.3 +/- 3.9 microM for dATP. The 5'-triphosphate of zidovudine has a Kd of 54.1 +/- 1.3 microM. The enzyme shows no preference for purine or pyrimidine nucleotides. Hill coefficients and the results of dual ligand titration experiments demonstrate that the free enzyme possesses a single dNTP binding site for which the four natural substrates and the 5'-triphosphate of zidovudine compete. The presence of homopolymeric template-primers does not result in selective binding of the complementary 2'-deoxynucleoside 5'-triphosphate, indicating that Watson-Crick base pairing is not involved in the initial binding reaction. The major force driving the association of the ligands with the binding site is hydrophobic. Approximately 14% of the binding energy is derived from electrostatic interactions. Although Mg2+ is required for catalytic activity, it is not absolutely required for initial binding.

Substrate inhibition of the human immunodeficiency virus type 1 reverse transcriptase.

Substrate inhibition was observed with the heterodimeric (p66/p51) and the homodimeric (p66/p66, p51/p51) forms of human immunodeficiency virus type 1 reverse transcriptase (RNA-dependent DNA polymerase, EC 2.7.7.49). An apparent Ki value of 195 +/- 37 microM was determined for dTTP using the bacterial cloned and expressed heterodimer. Similar values were obtained with the homodimeric and the virus-encoded enzymes. When poly-(rC).p(dG)10 was used as template-primer, dGTP exhibited substrate inhibition with an apparent Ki value of 189 +/- 32 microM. Substrate inhibition was not observed with dTTP when DNA.DNA template-primers were used. Hill coefficients for substrate binding determined in the presence of saturating concentrations of template-primer were equal to 1.0, suggesting that substrate inhibition of the heterodimer is not the result of an allosteric mechanism involving the p51 subunit. Furthermore, UV crosslinking experiments with [gamma-32P]dTTP showed crosslinking only to the p66 subunit. Substrate inhibition was not as pronounced with other retroviral reverse transcriptases as it was with human immunodeficiency type 1 reverse transcriptase.