Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.49 (reverse transcriptase)
31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

2',3'-Dideoxy-2',3'-didehydro-beta-L(-)-5-fluorocytidine [L(-)Fd4C] has been reported to be a potent inhibitor of the human immunodeficiency virus (HIV) in cell culture. In the present study the antiviral activity of this compound in two-drug combinations and its intracellular metabolism are addressed. The two-drug combination of L(-)Fd4C plus 2',3'-didehydro-2'-3'-dideoxythymidine (D4T, or stavudine) or 3'-azido-3'-deoxythymidine (AZT, or zidovudine) synergistically inhibited replication of HIV in vitro. Additive antiviral activity was observed with L(-)Fd4C in combination with 2',3'-dideoxycytidine (ddC, or zalcitabine) or 2',3'-dideoxyinosine (ddI, or didanosine). This beta-L(-) nucleoside analog has no activity against mitochondrial DNA synthesis at concentrations up to 10 microM. As we previously reported for other beta-L(-) nucleoside analogs, L(-)Fd4C could protect against mitochondrial toxicity associated with D4T, ddC, and ddI. Metabolism studies showed that this drug is converted intracellularly to its mono-, di-, and triphosphate metabolites. The enzyme responsible for monophosphate formation was identified as cytoplasmic deoxycytidine kinase, and the K(m) is 100 microM. L(-)Fd4C was not recognized in vitro by human mitochondrial deoxypyrimidine nucleoside kinase. Also, L(-)Fd4C was not a substrate for deoxycytidine deaminase. L(-)Fd4C 5'-triphosphate served as an alternative substrate to dCTP for incorporation into DNA by HIV reverse transcriptase. The favorable anti-HIV activity and protection from mitochondrial toxicity by L(-)Fd4C in two-drug combinations favors the further development of L(-)Fd4C as an anti-HIV agent.
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PMID:Metabolism of 2',3'-dideoxy-2',3'-didehydro-beta-L(-)-5-fluorocytidine and its activity in combination with clinically approved anti-human immunodeficiency virus beta-D(+) nucleoside analogs in vitro. 966 Oct 24

2-Chloro-2'-deoxyadenine (2CldA) is used for treatment of several lymphoid malignancies. Since this drug is incorporated into DNA, we have undertaken studies on base pairing of 2-chloroadenine (2ClA). 2CldA phosphoramidite was synthesized and used for preparation of 25-mer templates with 2ClA located at site 21 from the 3'-end. Kinetic parameters (Km and Vmax) for the incorporation of deoxynucleoside-5'-triphosphates by AMV reverse transcriptase opposite the 2ClA template, as well as for the extension of 2ClA.T pair, were determined. The efficiency (Vmax/Km) of incorporation of dGTP, dCTP, and dATP opposite 2ClA is at least one order of magnitude lower than opposite unmodified A. The efficiency of incorporation of dTTP opposite 2ClA is about 30-fold lower than opposite A and extension of 2ClA.T pair is 3-fold lower than of A.T pair. From the analysis of the parameters of dTTP incorporation we conclude that formation of 2ClA.T pair is thermodynamically, but not kinetically controlled. The difference in binding energy (deltadeltaG) between 2ClA.T and A.T pairs in the environment of the polymerase active site is 2 kcal/mol. Our results indicate that the presence of 2ClA in DNA slows down replication, but does not lead to base-substitution mutations.
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PMID:Template-directed base pairing of 2-chloro-2'-deoxyadenosine catalyzed by AMV reverse transcriptase. 982 87

Among the nucleoside inhibitors used clinically as anti-HIV drugs which target HIV-1 reverse transcriptase (RT), (-)-2', 3'-dideoxy-3'-thiacytidine [(-)SddC or 3TC] is the only analogue with the unnatural L(-) nucleoside configuration. 3TC has been shown to be more potent and less toxic than the D(+) isomer, (+)SddC, which has the natural nucleoside configuration. The mechanistic basis for the stereochemical selectivity and differential toxicity of the isomeric SddC compounds is not completely understood although a number of factors may clearly come into play including differences in uptake, metabolic activation, degradation, and transport. We used a pre-steady-state kinetic analysis to determine the maximum rate of incorporation, kpol, nucleotide-binding affinity, Kd, and efficiency of incorporation, kpol/Kd, for the (-) and (+) isomeric SddCTP compounds as well as the corresponding dideoxy and natural nucleoside triphosphates into a primer-template complex using HIV-1 reverse transcriptase. The affinity (Kd) of the dNTP was much tighter and the efficiency (kpol/Kd) of incorporation by enzyme into the primer-template complex was much higher for the DNA/RNA primer-template compared to DNA/DNA. The maximum rate of incorporation, kpol, followed the trend of dCTP > ddCTP > (+)SddCTP > (-)SddCTP while the Kd values determined for the DNA/RNA primer-template followed the order (-)SddCTP congruent with (+)SddCTP congruent with ddCTP > dCTP. The corresponding efficiency of incorporation followed the trend dCTP > ddCTP > (+)SddCTP > (-)SddCTP. These data suggest that perturbations on the ribose ring of cytidine analogues (C --> S) decrease the rate and efficiency of incorporation but enhance the binding affinity. These results are discussed in the context of a computer modeled structure of the ternary complexes of RT, DNA/RNA primer-template, and SddCTP analogues as well as implications for structure-activity relationships and further drug design. This information provides a mechanistic basis for understanding the inhibition of HIV-1 reverse transcriptase by 3TC.
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PMID:Mechanistic studies comparing the incorporation of (+) and (-) isomers of 3TCTP by HIV-1 reverse transcriptase. 989 Aug 82

The presence of progesterone receptors (PR) in the human placenta has been demonstrated using the reverse transcriptase-polymerase chain reaction technique. It was observed that the amount of PR in the human placenta is less during late gestation. Electrophoretic mobility shift assays with nuclear extract isolated from the first trimester and term revealed three complexes when incubated with [32P]dCTP-labelled progesterone response element, and, in competition with unlabelled progesterone response element, the formation of all three complexes was inhibited. When supershift analysis of these complexes was carried out using antibodies which cross-react with both the A and B types of the PR or only with the B type receptor, only the A-form of PR was detected in the human placenta.
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PMID:Progesterone receptor expression in the human placenta. 1033 72

A single amino acid substitution from methionine-184 to valine (M184V) of HIV-1 reverse transcriptase (RT) evokes the 1000-fold 3TC (Lamivudine) resistance by the HIV-1 virus observed in the clinic. The M184V mutant HIV-1 RT was studied to assess its catalytic efficiency during single nucleotide incorporation using a transient kinetic approach. The maximum rate of polymerization (k(pol)), binding affinity (K(d)), and incorporation efficiency (k(pol)/K(d)) were determined for incorporating dCTP and 3TC-TP by wild-type and 3TC-resistant HIV-1 RT. The 3TC-resistant HIV-1 RT showed a similar efficiency of incorporation compared with the wild-type enzyme during DNA-dependent DNA polymerization; however, the incorporation efficiency is reduced 3.5-fold during RNA-dependent polymerization. A dramatic 146- and 117-fold decrease in incorporation efficiency was observed for 3TC-MP incorporation by M184V RT for DNA- and RNA-dependent DNA polymerization, respectively, as compared with wild-type HIV-1 RT. While the k(pol) was slower and the K(d) was weaker for 3TC-TP incorporation by the M184V RT, the decrease in the efficiency of incorporation is primarily due to a substantially reduced binding affinity for the 3TC-TP to the enzyme.DNA (or RNA) complex poised for DNA elongation. The fidelity of M184V RT was also examined to evaluate mispair formation since this mutant has been suggested to exhibit a higher level of fidelity. The results of our studies indicate that there is a maximum 2.4-fold increase in fidelity for M184V RT as compared with wild-type HIV-1 RT. Both the wild-type and 3TC-resistant mutant RT showed higher fidelity using an RNA template as contrasted with the corresponding DNA template. This mechanistic information provides insight into our understanding of the molecular mechanism of 3TC-drug resistance and supports suggestions that increased RT fidelity and decreased fitness of the M184V HIV-1 virus may be factors contributing to the strong antiviral effect of AZT-3TC combination therapy.
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PMID:Mechanistic studies examining the efficiency and fidelity of DNA synthesis by the 3TC-resistant mutant (184V) of HIV-1 reverse transcriptase. 1041 20

A novel L-nucleoside analog of deoxycytidine, 2',3'-dideoxy-2', 3'-didehydro-beta-L-5-fluorocytidine (beta-L-Fd4C), was recently shown to strongly inhibit hepatitis B virus (HBV) replication in the 2.2.15 cell line. Therefore, its antiviral activity was evaluated in the duck HBV (DHBV) infection model. Using a cell-free system for the expression of the DHBV polymerase, beta-L-Fd4C-TP exhibited a concentration-dependent inhibition of dCTP incorporation into viral minus-strand DNA with a 50% inhibitory concentration of 0.2 microM which was lower than that of other tested deoxycytidine analogs, i.e. , lamivudine-TP, ddC-TP, and beta-L-FddC-TP. Further analysis showed that beta-L-Fd4C-TP is likely to be a competitive inhibitor of dCTP incorporation and to cause premature DNA chain termination. In primary duck hepatocyte cultures infected in vitro, beta-L-Fd4C administration exhibited a long-lasting inhibitory effect on viral DNA synthesis but could not clear viral covalently closed circular DNA (CCC DNA). Results of short-term antiviral treatment in experimentally infected ducklings showed that beta-L-Fd4C exhibited the most potent antiviral effect, followed by beta-L-FddC, lamivudine, and ddC. Longer administration of beta-L-Fd4C induced a sustained suppression of viremia (>95% of controls) and of viral DNA synthesis within the liver. However, the persistence of trace amounts of viral CCC DNA detected only by PCR was associated with a recurrence of viral replication after drug withdrawal. In parallel, beta-L-Fd4C treatment suppressed viral antigen expression within the liver and decreased intrahepatic inflammation and was not associated with any sign of toxicity. Our data, therefore, demonstrate that in the duck model of HBV infection, beta-L-Fd4C is a potent inhibitor of DHBV reverse transcriptase activity in vitro and suppresses viral replication in the liver in vivo.
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PMID:Characterization of the antiviral effect of 2',3'-dideoxy-2', 3'-didehydro-beta-L-5-fluorocytidine in the duck hepatitis B virus infection model. 1060 31

The initiation of (-) strand DNA synthesis by HIV-1 reverse transcriptase was examined using a transient kinetic approach and a physiologically relevant RNA 18-mer/RNA 36-mer primer-template substrate. HIV-1 reverse transcriptase (RT) was found to bind with reasonably high affinity to the RNA/RNA substrate (K(d) = 90 nM), although the affinity for DNA/RNA and DNA/DNA substrates is higher (K(d) approximately 5 nM). A pre-steady-state burst of deoxynucleotide incorporation (k(obsd) = 1.0 s(-)(1)) into the RNA duplex was observed followed by a slower steady-state release of the elongated primer-template product (k(ss) = 0.58 s(-)(1)). The observation of a burst provides evidence that the release of the product is most likely the rate-limiting step in the overall kinetic pathway for the enzymatic reaction during a single deoxynucleotide incorporation event. Furthermore, the release of this product was 5-fold faster than that for elongated DNA/RNA and DNA/DNA products. Single-turnover experiments showed that there is a hyperbolic dependence of the rate of deoxynucleotide incorporation on the concentration of dCTP and demonstrated that the maximum rate of dCTP incorporation (k(pol) = 1.4 s(-)(1)) is 33- and 12-fold slower than the values for DNA/RNA and DNA/DNA primer-template substrates, respectively, while the affinity of dCTP (K(d) = 780 microM) for the HIV-1 RT.RNA/RNA complex is 56- and 71-fold weaker than the affinities for HIV-1 RT.DNA/RNA and HIV-1 RT.DNA/DNA complexes, respectively. Consequently, the overall efficiency of dCTP incorporation (k(pol)/K(d)) into the RNA/RNA substrate is approximately 1800- and 800-fold less than that for DNA/RNA and DNA/DNA substrates, respectively. These findings provide evidence which suggests that the HIV-1 RT.RNA/RNA.dCTP ternary complex exists in a significantly different conformation compared to ternary complexes involving DNA/RNA and DNA/DNA substrates. A model summarizing these results is presented, and implications for the molecular mechanism of initiation of (-) strand DNA synthesis by RT are discussed.
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PMID:Initiation of minus-strand DNA synthesis by human immunodeficiency virus type 1 reverse transcriptase. 1062 65

A series of unnatural L-nucleosides such as 3TC, FTC and L-FMAU have been found to be potent antiviral agents. The mode of action of L-nucleosides has been found to be similar to that of D-nucleosides as antiviral agents, despite their unnatural stereochemistry, that is, nucleotide formation by kinases followed by interaction with the reverse transcriptase (RT) of HIV or DNA polymerase. To date, the mode of action of nucleoside inhibitors at the molecular level with respect to the active conformations of the 5'-triphosphates as well as the interaction with the RT is not known. Recently, the X-ray crystal structure of the RT-DNA-dTTP catalytic complex has been reported. Computer modeling has been performed for several pairs of D- and L-nucleoside inhibitors using the HIV-1 RT model and crystal coordinate data from a subset of the protein surrounding the deoxynucleoside triphosphate (dNTP) binding pocket region. Results from our modeling studies of D-/L-zidovudine, D-/L-3TC, D-/L-dideoxycytosine triphosphates, dTTP and dCTP show that their binding energies correlate with the reported 50% effective concentrations. Modeling results are also discussed with respect to favorable conformations of each inhibitor at the dNTP site in the polymerization process. Additionally, the clinically important M184V mutation, which confers resistance against 3TC and FTC, was studied with our modeling system. The binding energy patterns of nucleoside inhibitors at the M184V mutation site correlate with the reported antiviral data.
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PMID:Molecular modeling approach to understanding the mode of action of L-nucleosides as antiviral agents. 1112 Sep 56

Following intracellular activation of HIV nucleoside analogue reverse transcriptase inhibitors, their triphosphates (ddNTPs) compete with endogenous nucleoside triphosphates (dNTPs) for incorporation into proviral DNA. In this study we have examined the effect of combinations of two thymidine analogues, stavudine (d4T) and zidovudine (ZDV), and two cytidine analogues, lamivudine (3TC) and zalcitabine (ddC) on intracellular drug activation and on the relevant competing dNTP in uninfected and persistently HIV-infected cells. Endogenous triphosphates of deoxycytidine (dCTP) and deoxythymidine (dTTP) were measured using a template primer assay and the ratio of ddNTP:dNTP was calculated. Antiviral activity of two-drug combinations was also assayed by p24 ELISA. A significant reduction in d4T triphosphate (d4TTP) [0.11+/-0.09 pmol/10(6) cells to undetectable (<0.01); P=0.039] in the presence of equimolar concentrations of ZDV and d4T, resulted in a decrease in the d4TTP/dTTP ratio of 90%. ZDVTP/dTTP was not significantly altered in the presence of d4T. 3TC (10 microM) reduced total ddC phosphates by 57% and ddCTP/dCTP by 27%. 3TC phosphorylation was comparatively unaffected by ddC, up to a concentration of 10 microM ddC (>100 times the plasma concentration achieved following standard dosing). 3TC plus ddC resulted in greater p24 inhibition than 3TC or ddC alone (P<0.001). Combining one thymidine analogue (ZDV or d4T) with one cytidine analogue (3TC or ddC) resulted in greater inhibition of p24 inhibition than with any single agent. From a pharmacological viewpoint, the combination of ZDV plus d4T should be avoided, but in vitro the combination of 3TC plus ddC confers modest benefit over either drug alone. This in vitro study illustrates that decreases in ddNTP/dNTP are consistent with a reduction in antiviral effect.
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PMID:Correlation between intracellular pharmacological activation of nucleoside analogues and HIV suppression in vitro. 1122 92

Success in treating hepatitis B virus (HBV) infection with nucleoside analog drugs like lamivudine is limited by the emergence of drug-resistant viral strains upon prolonged therapy. The predominant lamivudine resistance mutations in HBV-infected patients are Met552IIe and Met552Val (Met552Ile/Val), frequently in association with a second mutation, Leu528Met. The effects of Leu528Met, Met552Ile, and Met552Val mutations on the binding of HBV polymerase inhibitors and the natural substrate dCTP were evaluated using an in vitro HBV polymerase assay. Susceptibility to lamivudine triphosphate (3TCTP), emtricitabine triphosphate (FTCTP), adefovir diphosphate, penciclovir triphosphate, and lobucavir triphosphate was assessed by determination of inhibition constants (K(i)). Recognition of the natural substrate, dCTP, was assessed by determination of Km values. The results from the in vitro studies were as follows: (i) dCTP substrate binding was largely unaffected by the mutations, with Km changing moderately, only in a range of 0.6 to 2.6-fold; (ii) K(i)s for 3TCTP and FTCTP against Met552Ile/Val mutant HBV polymerases were increased 8- to 30-fold; and (iii) the Leu528Met mutation had a modest effect on direct binding of these beta-L-oxathiolane ring-containing nucleotide analogs. A three-dimensional homology model of the catalytic core of HBV polymerase was constructed via extrapolation from retroviral reverse transcriptase structures. Molecular modeling studies using the HBV polymerase homology model suggested that steric hindrance between the mutant amino acid side chain and lamivudine or emtricitabine could account for the resistance phenotype. Specifically, steric conflict between the Cgamma2-methyl group of Ile or Val at position 552 in HBV polymerase and the sulfur atom in the oxathiolane ring (common to both beta-L-nucleoside analogs lamivudine and emtricitabine) is proposed to account for the resistance observed upon Met552Ile/Val mutation. The effects of the Leu528Met mutation, which also occurs near the HBV polymerase active site, appeared to be less direct, potentially involving rearrangement of the deoxynucleoside triphosphate-binding pocket residues. These modeling results suggest that nucleotide analogs that are beta-D-enantiomers, that have the sulfur replaced by a smaller atom, or that have modified or acyclic ring systems may retain activity against lamivudine-resistant mutants, consistent with the observed susceptibility of these mutants to adefovir, lobucavir, and penciclovir in vitro and adefovir in vivo.
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PMID:Molecular modeling and biochemical characterization reveal the mechanism of hepatitis B virus polymerase resistance to lamivudine (3TC) and emtricitabine (FTC). 1131 49


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