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.

Safety assessment, in vitro and in vivo, and pharmacokinetics of emivirine, a potent and selective nonnucleoside reverse transcriptase inhibitor of human immunodeficiency virus type 1.

Emivirine (EMV), formerly known as MKC-442, is 6-benzyl-1-(ethoxymethyl)-5-isopropyl-uracil, a novel nonnucleoside reverse transcriptase inhibitor that displays potent and selective anti-human immunodeficiency virus type 1 (HIV-1) activity in vivo. EMV showed little or no toxicity towards human mitochondria or human bone marrow progenitor cells. Pharmacokinetics were linear for both rats and monkeys, and oral absorption was 68% in rats. Whole-body autoradiography showed widespread distribution in tissue 30 min after rats were given an oral dose of [(14)C]EMV at 10 mg/kg of body weight. In rats given an oral dose of 250 mg/kg, there were equal levels of EMV in the plasma and the brain. In vitro experiments using liver microsomes demonstrated that the metabolism of EMV by human microsomes is approximately a third of that encountered with rat and monkey microsomes. In 1-month, 3-month, and chronic toxicology experiments (6 months with rats and 1 year with cynomolgus monkeys), toxicity was limited to readily reversible effects on the kidney consisting of vacuolation of kidney tubular epithelial cells and mild increases in blood urea nitrogen. Liver weights increased at the higher doses in rats and monkeys and were attributed to the induction of drug-metabolizing enzymes. EMV tested negative for genotoxic activity, and except for decreased feed consumption at the high dose (160 mg/kg/day), with resultant decreases in maternal and fetal body weights, EMV produced no adverse effects in a complete range of reproductive toxicology experiments performed on rats and rabbits. These results support the clinical development of EMV as a treatment for HIV-1 infection in adult and pediatric patient populations.

DNA polymerase activity of hepatitis B virus particles: differential inhibition by L-enantiomers of nucleotide analogs.

DNA polymerase activity was assayed in hepatitis B virus (HBV) and core particles isolated from chronic producer lines. The particle-associated DNA polymerase activity, which was found to be limited to incorporation of only a few nucleotides, was inhibited by the 5'-triphosphates of nucleoside analogs. The 1-beta-L (1S,4R) and 1-beta-D (1R,4S) enantiomers of antiviral nucleoside analogs were compared for the ability to inhibit incorporation of natural nucleoside triphosphates into the viral DNA. Previously, both enantiomers of several analogs were found to be substrates for human immunodeficiency virus type 1 reverse transcriptase (HIV RT); the 1-beta-D enantiomers of some pairs were preferred as substrates. In contrast, the 1-beta-L enantiomers of all pairs tested were the more potent inhibitors of labeled substrate incorporation into hepatitis B virus DNA; the concentration required to inhibit the incorporation of the natural substrate by 50% was 6-fold to several hundred-fold lower than the concentration of the 1-beta-D enantiomer required for the same inhibitory effect. This preference for the 1-beta-L enantiomers was observed for both RNA-directed synthesis in core particles and DNA-directed synthesis in viral particles. The observed antiviral effect of the nucleoside analogs in cell culture seemed to be limited chiefly by their phosphorylation in cells.

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.

Weak binding of VX-478 to human plasma proteins and implications for anti-human immunodeficiency virus therapy.

VX-478 is a potent inhibitor of human immunodeficiency virus type 1 (HIV-1) protease (Ki, 0.6 nM) and of HIV-1 replication in antiviral assays (IC90, 80 nM). The fractional binding of VX-478 to human plasma and to purified plasma proteins was determined by equilibrium dialysis and difference UV spectrophotometry. Binding to alpha 1-acid glycoprotein (89% at 2 microM total drug concentration, Kd of 4 microM) accounts for its fractional binding in plasma (93%). Stopped-flow spectrophotometry methods showed that binding is a reversible two-step process. The measured dissociation rate constant approaches 100 s-1. The antiviral effect of VX-478 was determined in the presence of 45% human plasma, in which the IC90 increased by 1.5-fold compared with control experiments in the presence of 15% fetal bovine serum. The effects of protein binding on the antiviral activity of VX-478 are minor, as expected for a weak drug-protein interaction.

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.

Structural and functional characterization of the HPV16 E7 protein expressed in bacteria.

The E7 gene of the human papillomaviruses (HPV) encodes a 98-amino acid, multifunctional nuclear phosphoprotein with functional and structural similarities to adenovirus E1A and the papovavirus T antigens. E7 is a viral oncoprotein, which will cooperate with an activated ras oncogene to transform primary rodent cells, and can cooperate with the HPV E6 protein for the efficient immortalization of primary human keratinocytes. Due to the compelling epidemiological and experimental association between HPV infection and cervical cancer, we have undertaken a detailed study of the structure of the HPV16 E7 protein. The E7 protein was expressed in Escherichia coli as a native, unfused polypeptide, and soluble protein was purified by conventional chromatographic techniques. The purified protein was assessed for various biochemical and biophysical properties. Purified E7 binds the retinoblastoma protein avidly and specifically, and it can dissociate the E2F transcription factor when assayed in vitro. Circular dichroism spectroscopy indicated that E7 reversibly binds Zn2+ and Cd2+, resulting in a substantial increase in the alpha-helical content of the metal-bound E7 consistent with the stabilization of a hydrophobic core in the COOH terminus of the protein.

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.

A comparison of the conformations of the 5'-triphosphates of zidovudine (AZT) and thymidine bound to HIV-1 reverse transcriptase.

Nuclear Overhauser effect experiments were performed at 500 MHz to determine the conformations of AZTTP and dTTP when bound to HIV-1 reverse transcriptase. The conformations of both ligands were found to be similar in the bound state. The orientation of the glycosidic angle is anti (chi = -120 degrees +/- 12 for AZTTP and -110 degrees +/- 12 for dTTP), gamma is +sc and the pucker of the 3'-azido-2',3'-dideoxy- and 2'-deoxyribose rings is predominantly C4' exo (P = 60 degrees +/- 10 for AZTTP and 55 degrees +/- 8 for dTTP). These results indicate that the unusual C4'endo/C3'exo pucker (P = 215 degrees) reported for the dideoxyribose ring of AZT in the solid state does not play a role in the interaction of HIV-1 reverse transcriptase with AZTTP.

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.

Role of desolvation energy in the nonfacilitated membrane permeability of dideoxyribose analogs of thymidine.

The ability of thymidine and four dideoxyribose analogs of thymidine to cross phase barriers, such as those encountered at the exo and endo faces of a membrane, has been studied in a two-phase water/chloroform system. The rate constants for entering, ka, and leaving, kb, the organic phase and the partition coefficient, Kp (ka/kb), were determined for each compound. The values of Kp were found to be proportional to the values of the rate constants for nonfacilitated diffusion, c, into human erythrocytes reported in the preceding paper [Mol. Pharmacol. 41:950-956 (1992)]. This linear relationship suggests that, in accord with the solubility-diffusion model of nonmediated membrane permeation, movement of the thymidine analogs across the potential energy barriers at the membrane interfaces is fast, relative to the rate of transbilayer diffusion. Because the compounds have similar molecular volumes and would, therefore, have similar rates of transbilayer diffusion, the differences in c reflect the differences in the distribution of the compounds across the interfaces, i.e., the Kp values. The magnitudes of the Kp values are dependent not only on the pi value of each substituent on the dideoxyribose ring but also on the position of the substituent. Analogs having a hydroxyl group in the 5'-position have a higher Kp and a higher c than the corresponding analogs with the hydroxyl group in the 3'-position. This increased lipophilicity is attributed to a decrease in desolvation energy, resulting from the ability of the 5'-hydroxyl analogs to assume a syn configuration in which a bifurcated, intramolecular, hydrogen bond can be formed to the O-4' and the C-2 carbonyl groups.

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.

Crosslinking of substrates occurs exclusively to the p66 subunit of heterodimeric HIV-1 reverse transcriptase.

Photoaffinity labeling of the hetero- and homodimeric forms of HIV-1 reverse transcriptase has been carried out using [32P]rA12-18.dT10 as a representative template-primer and [alpha-32P]dTTP as a representative 2'-deoxynucleoside-5'-triphosphate. UV irradiation produces stable, covalent crosslinks between each of the reactants and both the hetero-(p66/p51) and homodimeric (p66/p66, p51/p51) forms of the enzyme. In the case of the p66/p51 heterodimer, the form of the enzyme believed to be involved in viral replication, crosslinking occurs exclusively to the p66 subunit. These results suggest that the polymerase activity of the heterodimer residues on p66.

The effects of lipid composition on the binding of lasalocid A to small unilamellar vesicles.

The binding of the carboxylic ionophore lasalocid A (X537A) to small unilamellar phospholipid vesicles of varying composition was examined in an effort to determine what structural features of the phospholipid membrane influence the ionophore-membrane interaction. Apparent dissociation constants (Kapp) were calculated for both the acidic and anionic forms of the ionophore using the change in fluorescence intensity observed for lasalocid A upon addition of phospholipid vesicles. The Kapp for binding to fluid phase dimyristoylphosphatidylcholine (DMPC) vesicles is 46 microM for the anion and 14 microM for the acid. While the phase transition of DMPC had no effect on the Kapp of the anion, an increase was observed in the Kapp of the acid below the phase transition temperature. The Kapp of the anion was not affected by the incorporation of 10% dimyristoylphosphatidylethanolamine (DMPE), but increased slightly upon incorporation of cholesterol. The pKa values of the ionophore were the same in DMPC and DMPC/DMPE membranes. Incorporation of the negative lipids phosphatidylglycerol, phosphatidic acid, or phosphatidylethanolamine (at pH 9.4 where PE carries a negative charge) decreases binding of the anion in accord with the increase in surface potential estimated from Gouy-Chapman theory. The CD spectrum of membrane-bound lasalocid A anion indicated the ionophore to be in an extended acyclic conformation on the membrane surface with the C-1 carboxylate rotated out of the plane of the salicylate ring. The out-of-plane rotation of the carboxylate may be the result of facial binding by the amphiphilic ionophore on the membrane surface or of weak ion pairing to the polar lipid head groups. These results suggest that the primary determinants of binding of the anionic ionophore on the membrane surface are packing density of the polar head groups and membrane surface potential. There is no evidence of strong hydrogen bond formation between the lipid polar head groups and the ionophore as has previously been suggested.

Carrier-independent entry of 1-methyl-4-phenylpyridinium (MPP+) into adrenal chromaffin cells as a consequence of charge delocalization.

The administration of 1-methyl-4-phenylpyridinium (MPP+) to cultures of adrenal medullary chromaffin cells resulted in time and concentration-dependent increases in the cellular content of MPP+. Co-incubation of cells with MPP+, in the presence of desmethylimipramine (DMI), reduced but did not prevent the accumulation of the pyridinium in these cells. Similarly, DMI and MPP+ co-administration reduced but did not prevent the neurotoxicant-induced release of a cytosolic marker, lactate dehydrogenase, into the media. Molecular orbital calculations reveal that the positive charge of MPP+ is highly delocalized throughout the pyridinium ring and consequently MPP+ may be able to diffuse down concentration or charge gradients. Thus, these data provide a basis for the entry of MPP+ into cells and subcellular organelles that lack a catecholamine transporter, e.g. mitochondria.

Investigation of the stereochemical course of DNA synthesis catalysed by human immunodeficiency virus type 1 reverse transcriptase.

The Sp-isomer of thymidine 5'-O-(1-thiotriphosphate) is used as substrate by HIV-1 reverse transcriptase. The absolute configuration of the internucleotide linkage in the oligonucleotide product was identified by 31p NMR spectroscopy to be the Rp-isomer, indicating that incorporation of dTTP alpha S into the oligonucleotide proceeded with inversion of configuration at the alpha-phosphorus. The mechanistic implications of these observations are discussed.

31P nuclear magnetic resonance study of the metabolic pools of adenosine triphosphate in cultured bovine adrenal medullary chromaffin cells.

31P NMR was used to resolve and determine the relative quantity and mobility of ATP in the cytosolic and vesicular compartments of isolated adrenomedullary chromaffin cells. The cells were cultured on microcarrier beads and superfused with an oxygenated medium--thereby permitting dense suspensions of viable cells to be maintained in the NMR probe for extended time periods. Under these conditions, distinct 31P signals could be seen for ATP within the vesicular and the cytosolic pools. Comparison of the integrated areas of the beta-phosphate resonances from the two ATP pools indicated 77% of the endogenous ATP was in the vesicular pool. From this observation and the assumption that the concentration of ATP in the vesicle is 87.5 mM, the concentration of ATP calculated to be in the cytoplasmic pool was approximately 4 mM. The pH in the vesicle determined from the chemical shift of the gamma-phosphate resonance of vesicular ATP was 5.84 +/- 0.17 (n = 6), slightly higher than the intragranular pH measured in hypoxic cells (5.57 +/- 0.15, n = 8). Spin-lattice relaxation times of ATP 31P resonances in the vesicular pool were from 12 to 14 times shorter than the ATP resonances in the cytosol, corresponding to a decrease in molecular mobility due to incorporation of ATP within a catecholamine-storage complex.