Elabela relaxes rat pulmonary artery and trachea via BKCa, KV, and KATP channels.

OBJECTIVE: Elabela is a newly discovered peptide hormone. This study aimed to determine the functional effects and mechanisms of action of elabela in rat pulmonary artery and trachea. MATERIALS AND METHODS: Vascular rings isolated from the pulmonary arteries of male Wistar Albino rats were placed in chambers in the isolated tissue bath system. The resting tension was set to 1 g. After the equilibration period, the pulmonary artery rings were contracted with 10-6 M phenylephrine. Once a stable contraction was achieved, elabela was applied cumulatively (10-10-10-6 M) to the vascular rings. To determine the vasoactive effect mechanisms of elabela, the specified experimental protocol was repeated after the incubation of signaling pathway inhibitors and potassium channel blockers. The effect and mechanisms of action of elabela on tracheal smooth muscle were also determined by a similar protocol. RESULTS: Elabela exhibited a concentration-dependent relaxation in the precontracted rat pulmonary artery rings (p < .001). Maximal relaxation level was 83% (pEC50: 7.947 CI95(7.824-8.069)). Removal of the endothelium, indomethacin incubation, and dideoxyadenosine incubation significantly decreased the vasorelaxant effect levels of elabela (p < .001). Elabela-induced vasorelaxation levels were significantly reduced after iberiotoxin, glyburide, and 4-Aminopyridine administrations (p < .001). L-NAME, methylene blue, apamin, TRAM-34, anandamide, and BaCl2 administrations did not cause a significant change in the vasorelaxant effect level of elabela (p = 1.000). Elabela showed a relaxing effect on precontracted tracheal rings (p < .001). Maximal relaxation level was 73% (pEC50: 6.978 CI95(6.791-7.153)). The relaxant effect of elabela on tracheal smooth muscle was decreased significantly after indomethacin, dideoxyadenosine, iberiotoxin, glyburide, and 4-Aminopyridine incubations (p < .001). CONCLUSIONS: Elabela exerted a prominent relaxant effect in the rat pulmonary artery and trachea. Intact endothelium, prostaglandins, cAMP signaling pathway, and potassium channels (BKCa, KV, and KATP channels) are involved in the vasorelaxant effect of elabela. Prostaglandins, cAMP signaling pathway, BKCa channels, KV channels, and KATP channels also contribute to elabela-induced tracheal smooth muscle relaxant effect.

Chemoenzymatic Preparation of Functional Click-Labeled Messenger RNA.

Synthetic mRNAs are promising candidates for a new class of transformative drugs that provide genetic information for patients' cells to develop their own cure. One key advancement to develop so-called druggable mRNAs was the preparation of chemically modified mRNAs, by replacing standard bases with modified bases, such as uridine with pseudouridine, which can ameliorate the immunogenic profile and translation efficiency of the mRNA. Thus the introduction of modified nucleobases was the foundation for the clinical use of such mRNAs. Herein we describe modular and simple methods to chemoenzymatically modify mRNA. Alkyne- and/or azide-modified nucleotides are enzymatically incorporated into mRNA and subsequently conjugated to fluorescent dyes using click chemistry. This allows visualization of the labeled mRNA inside cells. mRNA coding for the enhanced green fluorescent protein (eGFP) was chosen as a model system and the successful expression of eGFP demonstrated that our modified mRNA is accepted by the translation machinery.

Modulation of membrane permeability by carbon dioxide.

Promoting drug delivery across the biological membrane is a common strategy to improve bioavailability. Inspired by the observation that carbonated alcoholic beverages can increase the absorption rate of ethanol, we speculate that carbon dioxide (CO2 ) molecules could also enhance membrane permeability to drugs. In the present work, we have investigated the effect of CO2 on the permeability of a model membrane formed by 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine lipids to three drug-like molecules, namely, ethanol, 2',3'-dideoxyadenosine, and trimethoprim. The free-energy and fractional-diffusivity profiles underlying membrane translocation were obtained from µs-timescale simulations and combined in the framework of the fractional solubility-diffusion model. We find that addition of CO2 in the lipid environment results in an increase of the membrane permeability to the three substrates. Further analysis of the permeation events reveals that CO2 expands and loosens the membrane, which, in turn, facilitates permeation of the drug-like molecules. © 2019 Wiley Periodicals, Inc.

Link between Membrane Composition and Permeability to Drugs.

Prediction of membrane permeability to small molecules represents an important aspect of drug discovery. First-principles calculations of this quantity require an accurate description of both the thermodynamics and kinetics that underlie translocation of the permeant across the lipid bilayer. In this contribution, the membrane permeability to three drugs, or drug-like molecules, namely, 9-anthroic acid (ANA), 2',3'-dideoxyadenosine (DDA), and hydrocortisone (HYL), are estimated in a pure 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) and in a POPC:cholesterol (2:1) mixture. On the basis of independent 2-5-µs free-energy calculations combined with a time-fractional Smoluchowski determination of the diffusivity, the estimated membrane permeabilities to these chemically diverse permeants fall within an order of magnitude from the experimental values obtained in egg-lecithin bilayers, with the exception of HYL in pure POPC. This exception is particularly interesting because the calculated permeability of the sterol-rich bilayer to HYL, in close agreement with the experimental value, is about 600 times lower than that of the pure POPC bilayer to HYL. In contrast, the permeabilities to ANA and DDA differ by less than a factor of 10 between the pure POPC and POPC:cholesterol bilayers. The unusual behavior of HYL, a large, amphiphilic compound, may be linked with the longer range perturbation of the lipid bilayer it induces, compared to ANA and DDA, suggestive of a possibly different translocation mechanism. We find that the tendency of lower permeabilities of the POPC:cholesterol bilayer relative to those of the pure POPC one is a consequence of increased free-energy barriers. Beyond reporting accurate estimates of the membrane permeability, the present contribution also demonstrates that rigorous free-energy calculations and a fractional-diffusion model are key in revealing the molecular phenomena linking the composition of a membrane to its permeability to drugs.

Soluble adenylyl cyclase is an acid-base sensor in epithelial base-secreting cells.

Blood acid-base regulation by specialized epithelia, such as gills and kidney, requires the ability to sense blood acid-base status. Here, we developed primary cultures of ray (Urolophus halleri) gill cells to study mechanisms for acid-base sensing without the interference of whole animal hormonal regulation. Ray gills have abundant base-secreting cells, identified by their noticeable expression of vacuolar-type H(+)-ATPase (VHA), and also express the evolutionarily conserved acid-base sensor soluble adenylyl cyclase (sAC). Exposure of cultured cells to extracellular alkalosis (pH 8.0, 40 mM HCO3 (-)) triggered VHA translocation to the cell membrane, similar to previous reports in live animals experiencing blood alkalosis. VHA translocation was dependent on sAC, as it was blocked by the sAC-specific inhibitor KH7. Ray gill base-secreting cells also express transmembrane adenylyl cyclases (tmACs); however, tmAC inhibition by 2',5'-dideoxyadenosine did not prevent alkalosis-dependent VHA translocation, and tmAC activation by forskolin reduced the abundance of VHA at the cell membrane. This study demonstrates that sAC is a necessary and sufficient sensor of extracellular alkalosis in ray gill base-secreting cells. In addition, this study indicates that different sources of cAMP differentially modulate cell biology.

Effects of two novel nucleoside analogues on different hepatitis B virus promoters.

AIM: To explore the effects of the nucleoside analogues beta-L-D4A and beta-LPA on hepatitis B virus (HBV) promoters. METHODS: Four HBV promoters were amplified by polymerase chain reaction (PCR) and subcloned into the expression vector pEGFP-1. The four recombinants controlled by HBV promoters were confirmed by restriction analysis and sequencing. Human hepatoma HepG2 cells transfected with the recombinant plasmids were treated with various concentrations of beta-L-D4A and beta-LPA. Then, enhanced green fluorescent protein (EGFP)-positive cells were detected by fluorescence microscopy and using a fluorescence activated cell sorter (FACS). RESULTS: Four HBV promoters were separately obtained and successfully cloned into pEGFP-1. Expression of EGFP under the control of the surface promoter (Sp) and the X promoter (Xp) was inhibited by beta-L-D4A in a dose-dependent manner, while expression of EGFP under the control of the core promoter (Cp) and Xp was inhibited by beta-LPA in a dose-dependent manner. CONCLUSION: The two novel nucleoside analogues investigated here can inhibit the activities of HBV promoters in a dose-dependent manner. These findings may explain the mechanisms of action by which these two novel compounds inhibit HBV DNA replication.

Phosphorylation of paracellin-1 at Ser217 by protein kinase A is essential for localization in tight junctions.

Although paracellin-1 (PCLN-1) is known to have a crucial role in the control of Mg2+ reabsorption in the kidney, the molecular pathways involved in the regulation of PCLN-1 have not been clarified. We used FLAG-tagged PCLN-1 to investigate these pathways further, and found that PCLN-1 is phosphorylated at Ser217 by protein kinase A (PKA) under physiological conditions in Madin-Darby canine kidney (MDCK) cells. PCLN-1 expression decreased Na+ permeability, resulting in a decrease in the transepithelial electrical resistance (TER). By contrast, PCLN-1 enhanced transepithelial Mg2+ transport. PKA inhibitors, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H-89) and myristoylated protein kinase A inhibitor 14-22 amide PKI, and an adenylate cyclase inhibitor, 2',5'-dideoxy adenosine (DDA), reduced the phosphoserine level of PCLN-1. The inhibitory effect of DDA was rescued by 8-bromoadenosine-3',5'-cyclic monophosphate (8-Br-cAMP). PKA and adenylate cyclase inhibitors decreased transepithelial Mg2+ transport and TER. Dephosphorylated PCLN-1 moved from detergent-insoluble to soluble fractions and was dissociated from ZO-1. A fusion protein of PCLN-1 with glutathione-S-transferase revealed that Ser217 was phosphorylated by PKA. Phosphorylated PCLN-1 was localized in the tight junction (TJ) along with ZO-1, whereas dephosphorylated PCLN-1 and the S217A mutant were translocated into the lysosome. The degradation of dephosphorylated PCLN-1 and S217A mutant was inhibited by chloroquine, a specific lysosome inhibitor. Thus, the PKA-dependent phosphorylation of Ser217 in PCLN-1 is essential for its localization in the TJ and transepithelial Mg2+ transport.

Antiviral activity and intracellular metabolism of bis(tButylSATE) phosphotriester of beta-L-2',3'dideoxyadenosine, a potent inhibitor of HIV and HBV replication.

The beta-L-nucleoside analogue beta-L-2',3'-dideoxy adenosine (beta-L-ddA) has been shown to exhibit limited antiviral activities. This was attributed to its rapid catabolism through cleavage of the glycosidic bond and poor phosphorylation to the nucleotide beta-L-2',3'-dideoxyadenosine-5'-mono phosphate (beta-L-ddAMP) (Placidi et al., 2000). However, the nucleotide beta-L-2',3'-dideoxyadenosine-5'-triphosphate (beta-L-ddATP) inhibited the activity of both HIV-1 reverse transcriptase (RT) and viral DNA polymerase isolated from woodchuck hepatitis virus-infected serum (a model of hepatitis B) with an inhibitory concentration (IC50) of 2.0 microM without inhibiting human DNA polymerases alpha, beta, or gamma up to a concentration of 100 microM. These results suggested that prodrugs of beta-L-ddAMP may bypass the poor metabolic activation of beta-L-ddA and lead to more potent and selective antiviral activity. Therefore, the mononucleoside phosphotriester derivative of beta-L-ddAMP incorporating the S-pivaloyl-2-thioethyl (tButylSATE) groups, beta-L-ddAMP-bis(tButylSATE) was synthesized. Beta-L-ddAMP-bis(tButylSATE) inhibited HIV replication in human peripheral blood mononuclear cells (PBMCs) and HBV replication in 2.2.15 cells with effective concentrations (EC50s) of 2 and 80 nM, respectively. Intracellular metabolism of beta-L-ddAMP-bis(tButylSATE) demonstrated that beta-L-ddATP was the predominant intracellular metabolite in PBMC and liver cells. The intracellular half-life of beta-L-ddATP was 5.4 and 9.2 h in HepG2 and PBMCs, respectively. The intracellular concentrations of beta-L-ddATP were maintained above the EC50 for the inhibition of HIV RT and hepatitis B virus (HBV) for as long as 24 h after removal of the drug.

Fluorometric determination of 2'-beta-fluoro-2',3'-dideoxyadenosine 5'-triphosphate, the active metabolite of a new anti-human immunodeficiency virus drug, in human lymphocytes.

A sensitive precolumn derivatization method has been developed to measure the 5'-triphosphate of 2'-beta-fluoro-2',3'-dideoxyadenosine (F-ddA, lodenosine), a new anti-HIV drug, in human lymphocytes by HPLC using fluorescence detection. Reaction of chloroacetaldehyde with F-ddA triphosphate in extracts from human lymphocytes produces a highly fluorescent etheno adduct. This derivative is then separated and quantitated by reverse-phase paired-ion chromatography. Degradation of natural nucleic acid ribosides, such as ATP, using periodate oxidation simplifies the chromatogram and minimizes interference with detection of the target analyte. This method, modeled using cultured MOLT-4 T-lymphocytes, achieves a linear detector response for peak area measurements over the range 2.5 to 22.5 pmol (50-450 nM using 50 microl sample). Analyte recovery is greater than 90%, and the method achieves a limit of detection and limit of quantitation of 1.4 and 2.5 pmol per HPLC injection (50 microl sample containing cellular extract from 2.5 x 10(6) cells), respectively. Application of this method to measure F-ddATP in peripheral blood mononuclear cells from HIV-infected patients treated with F-ddA at 3.2 mg/kg twice daily for 22 days shows F-ddATP levels which range from 1.5 to 3.5 pmol/10(6) cells.

Determination of lodenosine and its major metabolite in human plasma by liquid chromatography/electrospray ionization tandem mass spectrometry.

A sensitive and selective method for the determination of 2'-beta-fluoro-2',3'-dideoxyadenosine (lodenosine, F-ddA), an experimental anti-AIDS drug, and its major metabolite, 2'-beta-fluoro-2',3'-dideoxyinosine (F-ddI), in human plasma was developed and validated. The procedure employs two internal standards and a simple ultrafiltration step followed by chromatography on a Betasil C(18) minibore column. An in-line valve is used to remove salts before reaching the ion source. Detection is by electrospray ionization tandem mass spectrometry with selected reaction monitoring. The method has a limit of quantitation of 4 ng ml(-1) (16 nM) for F-ddA and 8 ng ml(-1) (32 nM) for F-ddI with a linear range up to 2000 ng ml(-1) (7.9 microM) for each. Predicted concentrations from a three-day validation study were within 5% of the nominal values for F-ddA and 16% for F-ddI. Intra- and inter-assay precision, as measured by relative standard deviation, was 13% or better for both compounds. To achieve good reproducibility, many variables related to the electrospray ionization were optimized for both precision and sensitivity. The method was successfully employed to analyze samples and evaluate plasma pharmacokinetics from a Phase I clinical trial.

Phosphorylation of the anti-HIV compound (S,S)-isodideoxyadenosine by human recombinant deoxycytidine kinase.

(S,S)-Isodideoxyadenosine [(S,S)-isoddA] is an anti-HIV active compound discovered in our laboratory. However, its cellular mechanism of action, particularly the critical first stage of phosphorylation, is not understood. IsoddA is not phosphorylated by adenosine kinase. Also, because it is not a substrate for adenosine deaminase, it would not be activated by the pathway taken by ddA, i. e. via 5'-nucleotidase phosphorylation of ddI and conversion of ddIMP to ddAMP. However, we have discovered that human recombinant 2'-deoxycytidine kinase (dCK) phosphorylates (S,S)-isoddA. The enzyme kinetic data revealed that the extent of monophosphorylation of this L-related nucleoside was comparable to that found with ddA. (S,S)-IsoddATP is among the most potent inhibitors of HIV reverse transcriptase known, which suggests that the observed low efficiency of phosphorylation of this compound by dCK is a key factor that limits the capacity of human lymphocytes to make (S,S)-isoddA an exceptionally active anti-HIV agent.

Identification of residues involved in the specificity and regulation of the highly efficient multisubstrate deoxyribonucleoside kinase from Drosophila melanogaster.

In contrast to all known deoxyribonucleoside kinases, a single highly efficient deoxyribonucleoside kinase from Drosophila melanogaster (Dm-dNK) is able to phosphorylate all precursor nucleosides for DNA synthesis. Dm-dNK was mutated in vitro by high-frequency random mutagenesis, expressed in the thymidine kinase-deficient Escherichia coli strain KY895 and clones were selected for sensitivity to the nucleoside analogs 1-beta-d-arabinofuranosylcytosine (AraC, Cytarabine), 3'-azido-2', 3'-dideoxythymidine (AZT, Zidovudine, Retrovir, 2', 3'-dideoxyadenosine (ddA) and 2',3'-dideoxycytidine (ddC, Zalcitabine, Hivid. Thirteen mutants with increased sensitivity compared to the wild-type Dm-dNK were isolated from a relatively small pool of less than 10,000 clones. Eight mutant Dm-dNKs increased the sensitivity of KY895 to more than one analog, and two of these mutants even to all four nucleoside analogs. Surprisingly, the mutations did not map to the five regions which are highly conserved among deoxyribonucleoside kinases. The molecular background of improved sensitivity was characterized for the double-mutant MuD (N45D, N64D), where the LD(100) value of transformed KY895 decreased 316-fold for AZT and more than 11-fold for ddC when compared to wild-type Dm-dNK. Purified recombinant MuD displayed higher K(m) values for the native substrates than wild-type Dm-dNK and the V(max) values were substantially lower. On the other hand, the K(m) and V(max) values for AZT and the K(m) value for ddC were nearly unchanged between MuD and wild-type Dm-dNK. Additionally, a decrease in feedback inhibition of MuD by thymidine triphosphate (TTP) was found. This study demonstrates how high-frequency mutagenesis combined with a parallel selection for desired properties provides an insight into the structure-function relationships of the multisubstrate kinase from D. melanogaster. At the same time these mutant enzymes exhibit properties useful in biotechnological and medical applications.

Interactions of conformationally biased north and south 2'-fluoro-2', 3'-dideoxynucleoside 5'-triphosphates with the active site of HIV-1 reverse transcriptase.

Molecular dynamics simulations of a ternary complex of HIV-1 reverse transcriptase (RT), double-stranded DNA, and bound dideoxynucleoside-5'-triphosphate (RT-DNA-ddNTP), utilizing the ddNTPs ddATP, betaFddATP, and alphaFddATP, explain the experimentally observed order of potency of these 5'-triphosphates as inhibitors of RT: ddATP > betaFddATP > alphaFddATP. On the basis of RT's known preference to bind the incoming dNTP (or ddNTP) with a north conformation at the polymerase site, alphaFddATP, which in solution prefers almost exclusively a north conformation, was predicted to be the most potent inhibitor. However, Tyr115, which appears to function as a steric gate to preclude the binding of ribonucleoside 5'-triphosphates, prevents the effective binding of alphaFddATP in its preferred north conformation. The south-biased betaFddATP, while able to bind to RT without hindrance by Tyr115, has to pay a high energy penalty to be flipped to the active north conformation at the polymerase site. Finally, the more flexible and less conformationally biased ddATP is able to switch to a north conformation at the RT site with a smaller energy penalty than betaFddATP. These results highlight the opposite conformational preferences of HIV-1 RT for alphaFddATP and betaFddATP and help establish conformational guidelines for optimal binding at the polymerase site of this enzyme.

Intracellular metabolism of beta-L-2',3'-dideoxyadenosine: relevance to its limited antiviral activity.

The intracellular metabolism of the beta-L- enantiomer of 2', 3'-dideoxyadenosine (beta-L-ddA) was investigated in HepG2 cells, human peripheral blood mononuclear cells (PBMC), and primary cultured human hepatocytes in an effort to understand the metabolic basis of its limited activity on the replication of human immunodeficiency virus and hepatitis B virus. Incubation of cells with 10 microM [2',3',8-(3)H]-beta-L-ddA resulted in an increased intracellular concentration of beta-L-ddA with time, demonstrating that these cells were able to transport beta-L-ddA. However, it did not result in the phosphorylation of beta-L-ddA to its pharmacologically active 5'-triphosphate (beta-L-ddATP). Five other intracellular metabolites were detected and identified as beta-L-2', 3'-dideoxyribonolactone, hypoxanthine, inosine, ADP, and ATP, with the last being the predominant metabolite, reaching levels as high as 5.14 +/- 0.95, 8.15 +/- 2.64, and 15.60 +/- 1.74 pmol/10(6) cells at 8, 4, and 2 h in HepG2 cells, PBMC, and hepatocytes, respectively. In addition, a beta-glucuronic derivative of beta-L-ddA was detected in cultured hepatocytes, accounting for 12.5% of the total metabolite pool. Coincubation of hepatocytes in primary culture with beta-L-ddA in the presence of increasing concentrations of 5'-methylthioadenosine resulted in decreased phosphorolysis of beta-L-ddA and formation of associated metabolites. These results indicate that the limited antiviral activity of beta-L-ddA is the result of its inadequate phosphorylation to the nucleotide level due to phosphorolysis and catabolism of beta-L-ddA by methylthioadenosine phosphorylase (EC 2.4.2.28).

Determination of 2'-beta-fluoro-2',3'-dideoxyadenosine, an experimental anti-AIDS drug, in human plasma by high-performance liquid chromatography.

2'-Beta-fluoro-2',3'-dideoxyadenosine (F-ddA, lodenosine) is an experimental anti-AIDS drug currently being evaluated in a Phase I clinical trial. A simple and specific HPLC method with UV detection, suitable for use in clinical studies, has been developed to determine both F-ddA and its deaminated catabolite, 2'-beta-fluoro-2',3'-dideoxyinosine (F-ddI) in human plasma. After inactivation of plasma HIV by 0.5% Triton X-100, the compounds of interest are isolated and concentrated using solid-phase extraction. Processed samples are separated by use of a pH 4.8 buffered methanol gradient on a reversed-phase phenyl column. The method has a linear range of 0.05-5 microg/ml (0.2-20 microM) and intra-assay precision is better than 8%. Analyte recovery is quantitative and plasma protein binding is minimal. In addition, drug and metabolite levels measured in Triton-treated human plasma remain stable for at least 5 months when samples are stored frozen without further treatment. Compound concentrations determined after samples are processed and then frozen for up to 1 month before analysis are also unchanged.

Enzymatic properties of the unnatural beta-L-enantiomers of 2',3'-dideoxyadenosine and 2',3'-didehydro-2',3'-dideoxyadenosine.

The beta-L-enantiomers of 2',3'-dideoxyadenosine and 2',3'-didehydro-2',3'-dideoxyadenosine have been stereospecifically synthesized. In an attempt to explain the previously reported antiviral activities of these compounds, their enzymatic properties were studied with respect to adenosine kinase, deoxycytidine kinase, adenosine deaminase, and purine nucleoside phosphorylase. Adenosine deaminase was strictly enantioselective and favored beta-D-ddA and beta-D-d4A, whereas adenosine kinase and purine nucleoside phosphorylase had no apparent substrate properties for the D- or L-enantiomers of beta-ddA or beta-d4A. Human deoxycytidine kinase showed a remarkable inversion of the expected enantioselectivity, with beta-L-ddA and beta-L-d4A having better substrate efficiencies than their corresponding beta-D-enantiomers. Our results demonstrate the potential of beta-L-adenosine analogues as antiviral agents and suggest that deoxycytidine kinase has a strategic importance in their cellular activation.

Cellular phosphorylation of anti-HIV nucleosides. Role of nucleoside diphosphate kinase.

Nucleotide analogs are widely used in antiviral therapy and particularly against AIDS. Delivered to the cell as nucleosides, they are phosphorylated into their active triphospho derivative form by cellular kinases from the host. The last step in this series of phosphorylations is performed by nucleoside diphosphate (NDP) kinase, an enzyme that can use both purine or pyrimidine and oxy- or deoxynucleotides as substrates. Using pure recombinant human NDP kinase type B (product of the gene nm23-H2), we have characterized the kinetic parameters of several nucleotide analogs for this enzyme. Contrary to what is generally assumed, diphospho- and triphospho- derivatives of azidothymidine as well as of dideoxyadenosine and dideoxythymidine are very poor substrates for NDP kinase. The rate of phosphorylation of these analogs varies between 0.05% and 0.5%, as compared to the corresponding natural nucleotide, a result that is not due to the inability of the analogs to bind to the enzyme. Using the data from the high resolution crystal structure of NDP kinase, we provide an interpretation of these results based on the crucial role played by the 3'-OH moiety of the nucleotide in catalysis.

Comparison of the DNA incorporation in human MOLT-4 cells of two 2'-beta-fluoronucleosides, 2'-beta-fluoro-2',3'-dideoxyadenosine and fialuridine.

Incorporation of 2'-beta-fluoro-2',3'-dideoxyadenosine (F-ddA), a recently developed anti-HIV agent, into the cellular DNA of human MOLT-4 cells has been compared with the DNA incorporation seen with fialuridine (FIAU; 1-[2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl]-5-iodouracil), a potent anti-hepatitis B (anti-HBV) nucleoside analogue recently found to cause severe hepatic toxicity in human subjects. At equimolar concentrations (10 microM), incorporation of F-ddA was less than 1% of that for FIAU, a difference attributable to the lack of a 3'-hydroxyl group in the former compound and a consequent inability of F-ddA, unlike FIAU, to form DNA internucleotide linkages.

Antiviral, metabolic, and pharmacokinetic properties of the isomeric dideoxynucleoside 4(S)-(6-amino-9H-purin-9-yl)tetrahydro-2(S)-furanmethanol.

4(S)-(6-Amino-9H-purin-9-yl)tetrahydro-2(S)-furanmethanol (IsoddA) is the most antivirally active member of a novel class of optically active isomeric dideoxynucleosides in which the base has been transposed from the natural 1' position to the 2' position and the absolute configuration is (S,S). IsoddA was active against human immunodeficiency virus type 1 (HIV-1) (strain IIIB), HIV-2 (strain ZY), and HIV-1 clinical isolates. Combinations of the compound with zidovudine (3'-azido-3'-deoxythymidine), 2',3'-dideoxyinosine, or 5-fluoro-2'-deoxy-3'-thiacytidine showed synergistic inhibition of HIV. A moderate reduction of activity was observed with clinical isolates resistant to zidovudine. An IsoddA-resistant virus (eightfold-increased 50% inhibitory concentration) was selected in vitro by repeated passage of HIV-1 (HXB2) in the presence of increasing concentrations of IsoddA. The reverse transcriptase-coding region of the mutant virus contained a single base change resulting in a change at codon 184 from Met to Val. IsoddA was also active against hepatitis B virus (HBV) in vitro; however, it lacked substantial selective activity in an in vivo HBV model. IsoddA was inefficiently phosphorylated in CEM cells; however, the half-life of the triphosphate was 9.4 h, and IsoddATP was a potent inhibitor of HIV-1 reverse transcriptase, with a Ki of 16 nM. The cytotoxicity 50% inhibitory concentrations of IsoddA were greater than 100 microM for CEM, MOLT-4, IM9, and the HepG2-derived HBV-infected 2.2.15 (subclone P5A) cell lines but were 12 and 11 microM for human granulocyte-macrophage (CFU-GM) and erythroid (BFU-E) progenitor cells, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Substrate specificity of Ty1 integrase.

Integration of the Saccharomyces cerevisiae retrotransposon Ty1 requires the element-encoded integrase (IN) protein, which is a component of cytoplasmic virus-like particles (VLPs). Using purified recombinant Ty1 IN and an oligonucleotide integration assay based on Ty1 long terminal repeat sequences, we have compared IN activity on substrates having either wild-type or altered donor ends. IN showed a marked preference for blunt-end substrates terminating in an A:T pair over substrates ending in a G:C pair or a 3' dideoxyadenosine. VLP activity on representative substrates also showed preference for donor strands which have an adenosine terminus. Staggered-end substrates showed little activity when nucleotides were removed from the end of the wild-type donor strand, but removal of one nucleotide from the complementary strand did not significantly diminish activity. Removal of additional nucleotides from the complementary strand reduced activity to minimal detection levels. These results suggest that the sequence specificity of Ty1 IN is not stringent in vitro. The absence of Ty1 IN-mediated 3' dinucleotide cleavage, a characteristic of retroviral integrases, was demonstrated by using selected substrates. In addition to the forward reaction, both recombinant IN and VLP-associated IN carry out the reverse disintegration reaction with long terminal repeat-based dumbbell substrates. Disintegration activity exhibits sequence preferences similar to those observed for the forward reaction.