Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0019693 (HIV)
 170,526 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Among the clinically used nucleoside analogue inhibitors that target human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT), there is little detailed mechanistic information on the interactions of 2',3'-didehydro-2', 3'-dideoxythymidine-5'-triphosphate (d4TTP) with the enzyme. primer-template complex and how these interactions compare with those of the natural substrate, dTTP. Using a pre-steady-state kinetic analysis, we found that d4TTP was incorporated by HIV-1 RT just as efficiently as dTTP during both DNA- and RNA-dependent DNA synthesis. To our knowledge, these results represent the first observation of a 3'-modified nucleoside triphosphate analogue that has an incorporation efficiency comparable to that observed for the natural substrate during DNA synthesis by HIV-1 RT. This information provides a mechanistic basis for understanding the inhibition of HIV-1 RT by d4TTP as well as insight into the clinically observed lack of d4T resistance mutations in HIV-1 RT isolated from AIDS patients.
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PMID:Mechanism of inhibition of the human immunodeficiency virus type 1 reverse transcriptase by d4TTP: an equivalent incorporation efficiency relative to the natural substrate dTTP. 1060 55

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.
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PMID:Conformation and local environment of nucleotides bound to HIV type 1 reverse transcriptase (HIV-1 RT) in the ground state. 1077

Low levels of the CTP synthase inhibitor 3-deazauridine (3-DU) strongly potentiated the anti-HIV-1 activity of the 5'-triphosphates of the cytidine-based analogues [-]2'-deoxy-3'-thiacytidine (3TC; lamivudine) and 2',3'-dideoxycytidine (ddC). The potentiation was associated with a 3-DU-induced decrease in dCTP pool size; no changes were seen in cellular pool sizes of dATP, dGTP or dTTP.
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PMID:Potentiation of the anti-HIV activity of zalcitabine and lamivudine by a CTP synthase inhibitor, 3-deazauridine. 1077 21

The 5'-triphosphate of 4-thiothymidine (4S-TTP) is an excellent substrate for the Klenow fragment of Escherichia coli DNA polymerase 1 and HIV-1 reverse transcriptase with values of k(cat)/Km within a factor of approximately 3 of those for TTP. A large UV change (deltaepsilon= -9770 M(-1)cm(-1) at 340 nm) associated with incorporation of 4S-TMP into nucleic acid duplexes makes possible a rapid, continuous spectrophotometric assay of the reaction progress.
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PMID:Incorporation of 4-thiothymidine into DNA by the Klenow fragment and HIV-1 reverse transcriptase. 1085 57

Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) is a putative source of the genomic hypermutation that promotes rapid evolution of HIV-1. To understand the molecular strategies that create a highly mutagenic DNA polymerase active site in HIV-1 RT, we investigated the roles of four residues in the beta 8-alpha E loop. Gln151, which interacts with the sugar of the incoming dNTP, and Lys154, which interacts with the template, yielded site-directed mutants with increased fidelity, suggesting that these residues are directly involved in the mutagenic architecture of the active site. Mutations at Gln151 and Lys154 also reduced processivity. Q151N RT showed enhanced ability to discriminate between TTP and AZT triphosphate, consistent with the observation that the Q151M mutation confers AZT resistance in vivo. Mutations at Gly152 greatly decreased RT activity; molecular modeling suggests that Gly152 is critical for the proper geometric alignment that permits base-pairing of the incoming dNTP with the template. Mutations at Trp153 reduced the expression level, and presumably the stability, of RT proteins in bacteria. These observations support the conclusion that interactions of active site residues in the beta 8-alpha E loop with incoming dNTPs and the template are determinants of the accuracy, processivity, and substrate selectivity of HIV-1 RT.
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PMID:Molecular architecture of the mutagenic active site of human immunodeficiency virus type 1 reverse transcriptase: roles of the beta 8-alpha E loop in fidelity, processivity, and substrate interactions. 1097 52

Point mutations and inserts in the beta3-beta4 region of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) are associated with resistance to nucleoside analog inhibitors. This report describes HIV-1 strains from seven patients that were found to have a 3-bp deletion in the beta3-beta4 region of the RT gene. These patient strains also had a mean of 6.2 drug resistance-associated mutations in their RT genes (range, 3 to 10 mutations). The deletion was most frequently found in strains with the Q151M mutation. Nonnucleoside RT inhibitor mutations were found in six of seven strains. Culture-based drug sensitivity assays showed that deletion-containing isolates had reduced susceptibility to four to eight RT inhibitors. Site-directed mutagenesis experiments showed that the deletion alone conferred reduced susceptibility to nucleoside analogs. Changes in the three-dimensional models of the RT deletion mutants were consistently observed at the beta3-beta4 loop and at helices C and E in both the presence and the absence of dTTP. Loss of hydrogen bonds between the RT and dTTP were also observed in the RT deletion mutant. These results suggest that the deletion in the RT gene contributes to resistance to several nucleoside analogs through a complex interaction with other mutations in the RT gene.
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PMID:Genotypic, phenotypic, and modeling studies of a deletion in the beta3-beta4 region of the human immunodeficiency virus type 1 reverse transcriptase gene that is associated with resistance to nucleoside reverse transcriptase inhibitors. 1104 15

Telomerase is classified as one of the reverse transcriptases (RTs). To clarify whether l-enantiomers of natural 2'-deoxyribonucleoside 5'-triphosphates (dNTPs) are recognized by human telomerase, a quantitative telomerase assay based on the "stretch PCR" method was developed and used for kinetic analysis of the inhibitory effects of these compounds on the enzyme. Among the four l-enantiomers of dNTPs, l-dTTP and l-dGTP inhibited telomerase activity and the others showed slight or no inhibitory effect. Lineweaver-Burk plot analysis showed that the inhibition modes of l-dTTP and l-dGTP were partially competitive (mixed type) and competitive with the corresponding substrate dNTP, respectively. However, the K(i) values of l-dTTP and l-dGTP (21 and 15 microM) were several times larger than the K(m) values (3-6 microM). These results suggest that the active site of telomerase is not able to discriminate strictly the chirality of dNTPs, although it is more discriminatory than HIV-1 RT.
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PMID:Recognition of 2'-deoxy-l-ribonucleoside 5'-triphosphates by human telomerase. 1111 11

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

The association and dissociation of the homodimeric p66/p66 form of HIV-1 reverse transcriptase were investigated. The effects on the dimerization process of different salt concentrations, pH and the presence of a template/primer and nucleotide substrates were monitored by measuring polymerase activity and analytical size-exclusion HPLC. At submicromolar concentrations of enzyme and physiological salt concentrations, most of the enzyme exists in the inactive monomeric form. Increasing NaCl concentration from 0.05 to 1 M decreased the equilibrium dissociation constant from 2.0 to 0.34 microM. Analysis of the kinetics of the dimerization process indicated it followed a two-step mechanism, with rapid initial association of the two subunits to form an inactive homodimer followed by a slow isomerization step rendering the active enzyme form. The presence of poly(rA)/dT(20) decreased the equilibrium dissociation constant of the homodimer about 30-fold, while the addition of 5 microM dTTP had no effect. The kinetics of the process showed that the template/primer favored dimerization by binding to the inactive homodimer and promoting its isomerization to the active form. These results were confirmed by analyzing the reverse reaction, i.e. the dissociation of the enzyme, by dilution in a low-ionic-strength buffer. The results suggest that binding of immature HIV-1 reverse transcriptase to its natural template/primer may be relevant in both the dimerization process and the selection of its natural primer.
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PMID:Factors affecting the dimerization of the p66 form of HIV-1 reverse transcriptase. 1123 Dec 67


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