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)

The replicative cycle of the human immunodeficiency virus (HIV) is reviewed, and currently used and investigational agents directed against the virus are discussed. The first step in the replication of HIV is selective binding of the envelope glycoprotein to CD4 receptors located on T lymphocytes. The virion is then uncoated within the cytoplasm, yielding viral genomic RNA. Reverse transcriptase uses the viral RNA as a template to form single-stranded DNA, which is duplicated to form proviral DNA through the activity of ribonuclease H. Host RNA polymerases transcribe the integrated proviral DNA into messenger RNA, and there is subsequent translation to viral proteins. After translation, further modification of precursor polyproteins is necessary to produce functional peptides. The assembled virus then buds from the cell surface and invades other cells. Targets of drug intervention in the replicative cycle include (1) binding and entry, (2) reverse transcriptase, (3) transcription and translation, and (4) viral maturation and budding. Inhibitors of binding and entry include recombinant soluble CD4, immunoadhesins, peptide T, and hypericin. Nucleoside reverse-transcriptase inhibitors include zidovudine, didanosine, zalcitabine, and stavudine. Foscarnet, tetrahydroimidazobenzo-diazepinthione compounds, and nevirapine are some nonnucleoside reverse-transcriptase inhibitors. Inhibitors of transcription and translation include antagonists of the tat gene and GLQ223. Castanospermine, N-butyldeoxynojirimycin, and protease inhibitors interfere with viral maturation and budding. Drug combinations that have been or are being investigated include zidovudine plus interferon alfa, zidovudine plus zalcitabine, and zidovudine plus didanosine. Four agents currently have approved labeling for use against HIV infection: zidovudine, didanosine, zalcitabine, and stavudine. Monotherapy with zidovudine remains the treatment of first choice. Although progress has been made in developing drug therapies for HIV infection, more selective and more potent drugs are urgently needed. The best approach at present is to optimize the use of available agents, continue to investigate new therapies, and educate the public about prevention.
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PMID:Agents for treating human immunodeficiency virus infection. 775 75

Both foscarnet (PFA) and zidovudine (AZT) select for drug-resistant variants of human immunodeficiency virus type 1 (HIV-1), but the interactions between the mutations causing such resistance are unknown. The introduction of the previously identified PFA resistance mutation W to G at codon 88 (W88G), E89K, L92I, or Q161L into an HIV-1 strain having the four known AZT resistance mutations completely reversed high-level AZT resistance. Two additional PFA resistance mutations, W88S and S156A, partially suppressed AZT resistance. Phenotypic reversion of AZT resistance by W88S, W88G, E89K, L921, and S156A was associated with a concomitant suppression of PFA resistance. The degree to which PFA resistance mutations reversed AZT resistance was directly correlated with each mutation's ability to confer high-level PFA resistance (> or = 5.0-fold) and AZT hypersusceptibility in a wild-type genetic background. Highly PFA-resistant HIV- 1 strains were hypersusceptible to AZT; conversely, AZT-resistant strains with M41L and T215Y; M41L, L210W, and T215Y; or M41L, D67N, K70R, and T215Y mutations were 2.2- to 2.5-fold hypersusceptible to PFA. Prolonged in vitro selection of wild-type or AZT-resistant HIV-1 strains with the combination AZT and PFA failed to generate coresistant virus, indicating that dual resistance was relatively difficult to achieve. Strains selected by passage in PFA plus AZT were phenotypically PFA resistant and AZT susceptible despite multiple reverse transcriptase mutations known to confer AZT resistance. These data show that PFA resistance mutations can phenotypically reverse AZT resistance and that AZT and PFA resistance might be mutually exclusive. The reciprocal interactions between AZT and PFA resistance-conferring mutations have implications for structure-function studies of the HIV-1 reverse transcriptase.
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PMID:Zidovudine resistance is suppressed by mutations conferring resistance of human immunodeficiency virus type 1 to foscarnet. 879 64

Foscarnet (trisodium phosphonoformate, PFA) is an effective inhibitor of retroviral reverse transcriptase (RT) and is known to block the replication of human immunodeficiency virus type 1 (HIV-1). In this article we analyzed the evolutionary process in generating HIV-1 strains related to drug resistance, using PFA as a selective pressure. PFA inhibited virus replication and protected the virus-induced cell killing, but it did not completely eliminate HIV-1 during the course of 7 weeks of treatment. The nucleotide sequence of the 859-bp DNA fragment spanning the core region of the HIV-1 pol gene was determined for 51 clones obtained from genomic DNA of the HIV-1-infected cells at different time points during PFA treatment. The nucleotide sequence analysis documented the presence of a minor HIV-1 variant prior to the PFA treatment. Molecular evolutionary techniques were utilized to analyze how the minor HIV-1 clones became predominant during this evolutionary process under the selective pressure of PFA. A phylogenetic tree analysis divided these 51 HIV-1 clones into 3 groups. One of the groups consisted of the clones associated with the resistance to PFA. The clones belonging to this group became predominant over time during the course of PFA treatment. Thus, the acquisition of PFA resistance by HIV-1 was considered to be due to clonal selection. Furthermore, among the various amino acid substitutions observed, the substitution of arginine at position 172 by lysine (Arg172Lys) clearly distinguished this group from the others. Since the consistent amino acid substitution observed here has not been identified in the HIV-1 strains resistant to other RT inhibitors, PFA in combination with other RT inhibitors is considered to be a feasible candidate for a convergent combined chemotherapy against HIV-1 in the treatment of patients with AIDS and related conditions.
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PMID:Clonal selection of HIV type 1 variants associated with resistance to foscarnet in vitro: confirmation by molecular evolutionary analysis. 913 74

Lipophilic esters of 3'-azido-3'-deoxy-5'-O-(carboxyphosphinyl)thymidine (PFA-AZT) were synthesized and tested for antiretroviral activity in CD4+ HT4-6C cells infected with either wild-type HIV-1LAI, a PFA-resistant strain encoding a single-point mutation in reverse transcriptase (E89K), or an AZT-resistant clinical isolate (A018-post). Arbuzov condensation of 1-octadecyl, 1-eicosanyl, and 1-docosanyl chloroformate with trimethyl phosphite yielded the corresponding dimethyl long-chain alkyl triesters of PFA. Selective removal of one methyl group from the triesters with sodium iodide yielded monosodium salts, whereas treatment with bromotrimethylsilane cleaved both methyl groups while leaving the long-chain alkyl group intact. Neutralization of the resulting [(alkyloxy)carbonyl]phosphonic acids with 2 equiv of sodium methoxide afforded disodium salts of the phosphonic acid moiety. Similar chemistry was used to obtain the mono- and disodium salts of the cholesterol ester of PFA. Reaction of the triesters with phosphorous pentachloride, followed by coupling with AZT and O-demethylation with sodium iodide, afforded 3'-azido-3'-deoxy-5'-O-[[(1-octadecyloxy)carbonyl]phosphinyl ]thymidine (9a), 3'-azido-3'-deoxy-5'-O-[[(1-eicosanyloxy)carbonyl]phosphinyl ]thymidine (9b), 3'-azido-3'-deoxy-5'-O-[[(1-docosanyloxy)carbonyl]phosphinyl ]thymidine (9c), and 3'-azido-3'-deoxy-5'-O-[[(3 beta-cholest-5-enyloxy)carbonyl]phosphinyl]thymidine (9d). Concentrations of 9a-d found to inhibit replication of wild-type HIV-1LAI by 50% (EC50 values) as measured in a plaque reduction assay were in the 0.1-0.3 microM range as compared with 0.013 microM for AZT and 133 microM for PFA. The concentration at which toxicity was observed in 50% of the host cells (TC50 values) as measured by a visual grading scale of cellular morphology was 10 microM for 9a and 9d, 32 microM for 9b, and 320 microM for 9c. Thus, the TC50/EC50 ratio or selectivity index (SI) was 100 for 9a, 230 for 9b, and 1000 for 9c but only 33 for 9d, suggesting that the straight-chained fatty alcohol esters were more therapeutically selective. Similar TC50 and SI values were obtained for rapidly dividing CEM lymphoblasts as for HT4-6C cells. In assays against E89K, 9a-c had mean EC50 values of 0.13, 0.009, and 0.17 microM, whereas the EC50 of PFA was > 1000 microM and that of AZT was 0.009 microM; thus, E89K was highly resistant to PFA but not cross-resistant to either AZT or the lipophilic PFA-AZT conjugates. In viral replication assays against the A018C-post isolate, the mean EC50 values of 9a-c were 0.30, 0.53, and 0.77 microM as compared with 2.9 microM for AZT and 65 microM for PFA; thus, the virus recovered from a patient pretreated with AZT was not cross-resistant to either PFA or 9a-c. A notable feature of these results was that, in addition to being > 1000-fold more potent than PFA against the PFA-resistant mutant, the lipophilic PFA-AZT conjugates were more potent than PFA, as well as AZT, against AZT-resistant HIV-1.
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PMID:Synthesis and in vitro activity of long-chain 5'-O-[(alkoxycarbonyl)phosphinyl]-3'-azido-3'-deoxythymidines against wild-type and AZT- and foscarnet-resistant strains of HIV-1. 925 55

Many antiviral drugs must be metabolized to their active form by cellular enzymes. Their antiviral activity may therefore be limited by an inefficient metabolism, leading to low intracellular concentration of the active form or to the accumulation of toxic intermediate metabolites. Gene transfer might be used to overcome such limitations by transducing a gene able to increase intracellular drug metabolism. To prove such a concept, we chose the well-studied paradigm of zidovudine (AZT) metabolism and anti-HIV activity. AZT-triphosphate is the active form of AZT, acting through inhibition of HIV reverse transcription. In human cells, the rate-limiting step for AZT phosphorylation is catalyzed by the thymidylate kinase. We thus tested the capacity of herpes simplex virus type 1 thymidine kinase, which possesses a thymidylate kinase activity, to improve AZT metabolism and antiviral activity. Our results show enhanced AZT phosphorylation in HSV-1 TK-expressing lymphoid and monoblastoid cells, which correlated with significantly improved antiviral activity against different strains of HIV-1. The antiviral activity of Foscarnet, another reverse transcriptase inhibitor that does not require phosphorylation, remained unchanged. These results suggest that gene transfer might be envisioned for genetic pharmacomodulation of antiviral drugs.
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PMID:Use of herpes simplex virus thymidine kinase to improve the antiviral activity of zidovudine. 928 20

Combined therapy using reverse transcriptase (RT) and protease inhibitors is the current established treatment for HIV-1 infection. Foscarnet is an RT inhibitor that is a product analogue, in contrast to the widely used nucleoside analogues. In this study, the anti-HIV-1 effect of foscarnet, 50 mg three times per day administered intravenously for 4 weeks, was evaluated in 10 patients with minor or no symptoms. Serious adverse events developed in 2 patients, although most patients experienced some side effects. The levels of HIV-1 RNA decreased from a median value of 4.7 to 2.6 10log copies/ml. The effect was sustained through 4 weeks. One week after cessation of treatment, HIV-1 RNA levels increased to baseline. In contrast, no increase in the number of CD4+ cells was observed. The anti-HIV-1 effect was considered to be a direct effect on HIV-1 replication because no patient had concomitant cytomegalovirus (CMV) infection.
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PMID:Pronounced anti-HIV-1 activity of foscarnet in patients without cytomegalovirus infection. 959 58

Foscarnet (PFA) is a pyrophosphate analogue antiviral active against human immunodeficiency virus (HIV-1) and herpesviruses. Strains of HIV-1 resistant to PFA have mutations in the HIV-1 reverse transcriptase (RT). We examined the influence of PFA resistance mutations, in different genetic backgrounds, on HIV-1 replication competency in both replication kinetics and growth competition assays. In replication kinetics assays, the recombinant strains HX89K, HX92I, and HX156A (encoding RT mutations E89K, L92I, and S156A, respectively, in the HXB2-D genetic background) replicated to lower titers than the wild-type parent in the absence of drug, and the degree of replication impairment increased as PFA resistance increased. PFA-resistant strains LAI 92I and LAI 156A (encoding RT mutations L92I and S156A, respectively) were replication impaired in comparison to the wild-type parent LAI to a similar degree as observed for strains in the HXB2D background. In growth competition assays with wild-type LAI, strains LAI 92I and LAI 156A had relative fitness values of 0.5 and 0.8, respectively. These results show that the RT mutations E89K, L92I and S156A, observed in PFA-resistant strains selected in cell culture, reduce replication competence. Furthermore, these data show a correlation of increasing PFA resistance and decreasing replication competence mediated by single amino acid substitutions in the RT.
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PMID:Impaired fitness of foscarnet-resistant strains of human immunodeficiency virus type 1. 971 21

Casein kinase II (CKII) phosphorylates wild-type (WT) recombinant reverse transcriptase (RT) mainly in the p66 subunit in vitro. Phosphorylation of T215F RT and D67N/K70R/T215F/K219Q RT (AZT-resistant RT) in vitro increases discrimination against AZTTP 2. 5- and 3.6-fold, respectively. This in vitro resistance can be reversed by treatment of phosphorylated AZT-resistant RT with phosphatase. Phosphorylation has no effect on WT RT. Terminal transferase activity of RT is selectively suppressed on phosphorylated AZT-resistant RT. Resistance to phosphonoformic acid (PFA, foscarnet) increases 3-fold upon phosphorylation of AZT-resistant RT. Although T215, the most important residue for AZT-resistance, is part of a CKII consensus target site, serines are primarily phosphorylated relative to threonines. Mutational analysis shows that phosphorylation can be reduced to 10% that of WT when amino-acid changes are introduced both in the "fingers" subdomain and motif D. These results suggest that phosphorylation of RT might be one factor involved in drug resistance in vivo.
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PMID:Phosphorylation of AZT-resistant human immunodeficiency virus type 1 reverse transcriptase by casein kinase II in vitro: effects on inhibitor sensitivity. 1094 35

Phosphonoformate (foscarnet; PFA) is a potent inhibitor of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT), but its use for the treatment of HIV-1 infection is limited by toxicity and the lack of an orally bioavailable formulation. Alkylglycerol-conjugated prodrugs of PFA (1-O-octadecyl-sn-glycero-3-PFA [B-PFA]) having sn-2 substituents of hydrogen (deoxybatyl-PFA [DB-PFA]), methyl (MB-PFA), or ethyl (EB-PFA) are more-potent inhibitors of wild-type HIV-1 in vitro than unmodified PFA and are orally bioavailable in mice. We have evaluated the activities of these compounds against a panel of nucleoside-resistant HIV-1 variants and have characterized the resistant variants that emerge following in vitro selection with the prodrugs. Except for an HIV-1 variant encoding the K65R mutation in RT that exhibited 3.3- to 8.2-fold resistance, the nucleoside-resistant viruses included in the panel were sensitive to the PFA prodrugs (<3-fold increase in 50% inhibitory concentration), including multinucleoside-resistant variants encoding the Q151M complex of mutations or the T69S[SA] insert. Viruses resistant to the PFA prodrugs (>10-fold) were selected in vitro after 15 or more serial passages of HIV-1 in MT-2 cells in escalating prodrug concentrations. Mutations detected in the resistant viruses were S117T, F160Y, and L214F (DB-PFA); M164I and L214F (MB-PFA); and W88G and L214F (EB-PFA). The S117T, F160Y, and M164I mutations have not been previously identified. Generation of recombinant viruses encoding the single and double mutations confirmed their roles in prodrug resistance, including 214F, which generally increased the level of resistance. When introduced into a zidovudine (AZT)-resistant background (67N 70R 215F 219Q), the W88G, S117T, F160Y, and M164I mutations reversed AZT resistance. This suppression of AZT resistance is consistent with the effects of other foscarnet resistance mutations that reduce ATP-dependent removal of AZT monophosphate from terminated template primers. The favorable activity and resistance profiles of these PFA prodrugs warrant their further evaluation as clinical candidates.
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PMID:Alkylglycerol prodrugs of phosphonoformate are potent in vitro inhibitors of nucleoside-resistant human immunodeficiency virus type 1 and select for resistance mutations that suppress zidovudine resistance. 1135 3

Two different enzyme assays, both based on the interaction of native reverse transcriptase (RT) and 3'-azido-2',3'-deoxythymidine triphosphate (AZT-TP), were used to characterize the enzymes from 18 HIV-1 isolates with decreased sensitivity to AZT in cell culture. The first assay, which measures the balance between incorporation and excision of AZT monophosphate in the presence of dNTP substrate (in terms of IC(50)), gave an approx. 9-fold variation in sensitivity to AZT-TP. There was a correlation between the IC(50) values and the sensitivity of the corresponding virus to AZT in cell culture (r=0.60, P<0.01). The second assay, which was designed specifically for measurement of chain termination in the absence of dNTP substrate (as the concentration of AZT-TP giving 50% residual primer function, or CT(50)), revealed a more than 600-fold difference between the different isolate RTs. For the majority of enzymes there was a strict correlation between the results from the two assays; however, four isolates exhibited significantly higher CT(50)/IC(50) ratios than the other isolates. These differences were not related to sensitivity of the corresponding viruses to AZT but to the occurrence of certain mutations in their pol gene. The four deviating isolates contained either a minimum of four AZT-specific substitutions, including Thr-215-->Tyr (isolates 134 and 143), or some of the known specific substitutions combined with Thr-39-->Ala (isolates 80 and 157). The Thr-39-->Ala substitution has previously been recorded in connection with AZT/Foscarnet combination therapy.
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PMID:Application of a colorimetric chain-termination assay for characterization of reverse transcriptase from 3'-azido-2',3'-deoxythymidine-resistant HIV isolates. 1207 93


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