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)

Molecular cloning of cytochrome P450(11 beta) cDNAs from the adrenal glands of Dahl's salt-sensitive hypertensive (DS) and salt-resistant normotensive (DR) rats was performed using a combined technique of the first strand cDNA synthesis by reverse transcriptase followed by polymerase chain reaction. The cDNA sequence of P450(11 beta)-DS was identical to that of wild type P450(11 beta). In contrast, the clone obtained from the DR rat contained six nucleotide substitutions causing five amino acid alterations (Arg-127-->Cys, Val-351-->Ala, Val-381-->Leu, Ile-384-->Leu, and Val-443-->Met). When the two cDNAs were expressed in COS-7 cells and steroid conversion rates of the transformed cells were determined, a ratio of 18-hydroxylation to 11 beta-hydroxylation of 11-deoxycorticosterone by P450(11 beta)-DS-expressed cells was 0.58, whereas that by P450(11 beta)-DR-expressed cells was 0.23. Plasma levels of 18-hydroxy-11-deoxycorticosterone and corticosterone (the 11 beta-hydroxylation product of 11-deoxycorticosterone) in DS and DR rats well reflected the steroidogenic activities of the two P450s. These results suggest that the characteristic plasma steroid level of the DR rat is caused by the mutations in P450(11 beta) gene and may act to maintain the normotensive blood pressure in this rat strain during sodium loading.
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PMID:Dahl's salt-resistant normotensive rat has mutations in cytochrome P450(11 beta), but the salt-sensitive hypertensive rat does not. 847 50

We have identified the tRNAs which are incorporated into both wild-type human immunodeficiency virus type 1 strain IIIB (HIV-1IIIB) produced in COS-7 cells transfected with HIV-1 proviral DNA and mutant, noninfectious HIV-1Lai particles produced in a genetically engineered Vero cell line. The mutant proviral DNA contains nucleotides 678 to 8944; i.e., both long terminal repeats and the primer binding site are absent. As analyzed by two-dimensional polyacrylamide gel electrophoresis, both mutant and wild-type HIV-1 contain four major-abundance tRNA species, which include tRNA(1,2Lys), tRNA(3Lys) (the putative primer for HIV-1 reverse transcriptase) and tRNA(Ile). Identification was accomplished by comparing the electrophoretic mobilities and RNase T1 digests with those of tRNA(3Lys) and tRNA(1,2Lys) purified from human placenta and comparing the partial nucleotide sequence at the 3' end of each viral tRNA species with published tRNA sequences. Thus, the absence of the primer binding site in the mutant virus does not affect tRNA(Lys) incorporation into HIV-1. However, only the wild-type virus contains tRNA(3Lys) tightly associated with the viral RNA genome. The identification of the tightly associated tRNA as tRNA(3Lys) is based upon an electrophoretic mobility identical to that of tRNA(3Lys) and the ability of this RNA to hybridize with a tRNA(3Lys)-specific DNA probe. In addition to the four wild-type tRNA species, the mutant HIV-1-like particle contains two tRNA(His) species and three tRNA-sized species that we have been unable to identify. Their absence in wild-type virus makes it unlikely that they are required for viral infectivity.
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PMID:Identification of tRNAs incorporated into wild-type and mutant human immunodeficiency virus type 1. 849 49

The characterization of naturally occurring mutations is one way to approach functionally significant domains of polypeptides. About 10 mutations have been reported in factor XIII (FXIII) A-subunit deficiency, but very little is known about the effects of the mutations on the expression or the structure of this enzyme. In this study, the recent crystallization of FXIII A-subunit and determination of the three-dimensional model were used for the first time to pursue the structural consequences of mutations in the A-subunit. The molecular analysis of four families from Sweden, Germany, and Denmark revealed four previously unreported point mutations. Three of the mutations were missense mutations, Arg326-->Gln, Arg252-->Ile, and Leu498-->Pro, and one was a nonsense mutation, a deletion of thymidine in codon for Phe8 resulting in early frameshift and premature termination of the polypeptide chain. In the case of the nonsense mutation, delT Phe8, the steady-state mRNA level of FXIII A-subunit was reduced, as quantitated by reverse transcriptase-polymerase chain reaction and solid-phase minisequencing. In contrast, none of the missense mutations affected mRNA levels, indicating the possible translation of the mutant polypeptides. However, by enzyme-linked immunosorbent analysis and immunofluorescence, all the patients demonstrated a complete lack of detectable factor XIIIA antigen in their platelets. In the structural analysis, we included the mutations described in this work and the Met242-->Thr mutation reported earlier by us. Interestingly, in the three-dimensional model, all four missense mutations are localized in the evolutionarily conserved catalytic core domain. The substitutions are at least 15 A away from the catalytic cleft and do not affect any of the residues known to be directly involved in the enzymatic reaction. The structural analyses suggest that the mutations are most likely interfering with proper folding and stability of the protein, which is in agreement with the observed absence of detectable FXIIIA antigen. Arg326, Arg252, and Met242 are all buried within the molecule. The Arg326-->Gln and Arg252-->Ile mutations are substitutions of smaller, neutral amino acids for large, charged residues. They disrupt the electrostatic balance and hydrogen-bonding interactions in structurally significant areas. The Met242-->Thr mutation is located in the same region of the core domain as the Arg252-->Ile site and is expected to have a destabilizing effect due to an introduction of a smaller, polar residue in a tightly packed hydrophobic pocket. The substitution of proline for Leu498 is predicted to cause unfavorable interatomic contacts and a disruption of the alpha-helix mainchain hydrogen-bonding pattern; it is likely to form a kink in the helix next to the dimer interface and is expected to impair proper dimerization of the A-subunits. In the case of all four missense mutations studied, the knowledge achieved from the three-dimensional model of crystallized FXIII A-subunit provides essential information about the structural significance of the specific residues and aids in understanding the biologic consequences of the mutations observed at the cellular level.
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PMID:Four novel mutations in deficiency of coagulation factor XIII: consequences to expression and structure of the A-subunit. 854 36

We attempted to determine whether HIV-1 developed resistance to (--)-2',3'-dideoxy-3'-thiacytidine ((--)-3TC or 3TC, lamivudine) in patients with advanced human immunodeficiency virus type 1 (HIV-1) infection during therapy with 3TC. Genotypic analysis of HIV-1 strains isolated from 6 patients receiving 3TC revealed that as early as 2 months of therapy, HIV-1 developed a Met to Val amino acid substitution at codon 184 (Met184-->Val) in the reverse transcriptase-coding region of the pol gene. A detailed study of a series of HIV-1 strains isolated from a patient demonstrated that Met at codon 184 was first substituted with Ile by 2 weeks of 3TC therapy, followed by the substitution with Val by 8 weeks. All HIV-1 strains with the Met184-->Val substitution were profoundly less susceptible to 3TC (1800- to 5500-fold decreased sensitivity) as compared to pretherapy virus strains. These strains were also moderately less sensitive to 2',3'-dideoxycytidine (4.5- to 9-fold), but more sensitive to 3'-azido-2',3'-dideoxythymidine (2- to 14-fold). A decrease in viremia levels and an increase in CD4 counts were observed early in therapy; however, these changes were only transient. Our data suggest that reversal of such beneficial changes is associated with the Met184-->Val substitution of the pol gene of HIV-1. The data also suggest that 3TC, as a single agent, may induce virologic and immunologic improvement in patients with advanced HIV-1 infection, but only transiently.
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PMID:Genotypic and phenotypic characterization of HIV-1 isolated from patients receiving (--)-2',3'-dideoxy-3'-thiacytidine. 858 67

Human immunodeficiency virus type 1 (HIV-1)-infected CEM cells were treated (as single agents or in combination) with (minus)-2', 3'-dideoxy-3'-thiacytidine (3TC) and the following HIV-1-specific non-nucleoside reverse transcriptase (RT) inhibitors (NNRTIs): 2', 5'-bis-O-(tert-butyldimethylsilyl)-3'-spiro-5'-(4'-amino-1',2'-oxathi ole)-2',2'-dioxide derivative of 3-methylthymidine (TSAO-m3T), the thiocarboxanilides UC10 and UC42, bis(heteroaryl)piperazine (BHAP) derivative U90152, and the 1-(2-hydroxyethoxymethyl)-6-(phenylthio)thymine (HEPT) derivative 5-isopropyl-1-ethoxymethyl-6-benzyluracil (MKC-442). When used individually, the compounds led to the emergence of HIV-1 strains containing the following mutations in the RT: Glu138 to lysine for TSAO-m3T, Met184 to valine for 3TC, Lys103 to threonine/asparagine for the thiocarboxanilides, and Tyr181 to cysteine for BHAP and MKC-442. When 3TC was combined with TSAO-m3T, UC10, UC42, BHAP, or MKC-442, breakthrough of virus was markedly delayed or even suppressed. For these drug combinations, the concentrations of the individual drugs could be lowered by > or = 25-50-fold to suppress virus breakthrough compared with the individual use of the compounds. The concomitant presence of the Lys138 and Ile/Val184 mutations was found in the RT of the mutant viruses that emerged with combination therapy of the lowest concentrations of 3TC with either the lowest concentrations of TSAO-m3T or UC10 (approximately 0.5-3-fold the EC50 value). These virus strains retained high sensitivity to other NNRTIs such as BHAP or HEPT. The virus mutants that arose in the presence of combinations of the lowest concentrations of 3TC with either BHAP or HEPT predominantly contained the Cys181 mutation in the RT. In one case, the Ile181 mutation was found. The latter mutations, particularly the Ile181 mutation, resulted in markedly decreased sensitivity to the NNRTIs but not to 3'-azido-2', 3'-dideoxythymidine or 3TC.
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PMID:Marked inhibitory activity of non-nucleoside reverse transcriptase inhibitors against human immunodeficiency virus type 1 when combined with (-)2',3'-dideoxy-3'-thiacytidine. 862 38

A new class of very potent and selective non-nucleoside inhibitors of HIV reverse transcriptase (RT) has recently been identified. The prototype compound trovirdine (LY 300046 HCl) and one analogue, MSC-127, have been studied with respect to inhibition of wild-type HIV-1 RT and RT with various mutations known to give rise to resistance to other non-nucleoside RT inhibitors, namely Leu100-->Ile (Ile100), Glu138-->Arg (Arg138), Tyr181-->Cys (Cys181) and Tyr188-->His (His188). The inhibition of HIV-1 RT by trovirdine and MSC-127 was reversible and template dependent. Trovirdine inhibited HIV-1 RT with an IC50 of 0.007 microM when employing heteropolymeric primer/template (oligo-DNA/ribosomal RNA) and dGTP as substrate. Enzyme kinetic studies showed that inhibition of RT by trovirdine was non-competitive with regard to deoxynucleoside triphosphates and uncompetitive with respect to varied primer/template under steady-state conditions. The amino acid changes Leu100, Tyr181 and Tyr188 gave rise to 25-, 147- and 12-fold decrease in inhibition by trovirdine. Enzyme-kinetic studies on trovirdine have been carried out using various RT mutants and compared to the properties of the earlier reported non-nucleoside RT inhibitors 9-Cl-TIBO, nevirapine and L-697,661.
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PMID:Inhibition of human immunodeficiency virus type 1 wild-type and mutant reverse transcriptases by the phenyl ethyl thiazolyl thiourea derivatives trovirdine and MSC-127. 866 92

Human immunodeficiency virus type 1 (HIV-1) variants with resistance mutations in the reverse transcriptase (RT) gene appear during drug therapy with the nucleoside analogue 2',3'-dideoxy-3'-thiacytidine (3TC). These resistance mutations alter the methionine (Met) residue of the conserved YMDD motif, which is part of the catalytic core of the RT enzyme. Isoleucine (Ile) variants are initially observed, followed by the appearance and eventual outgrowth of viruses encoding valine (Val). Similar replication kinetics were measured for wild-type and 3TC-resistant HIV-1 viruses in tissue culture infections of a T cell line, but we measured reduced polymerase activity for the two mutant RT enzymes compared with the wild-type enzyme (Ile = 43% and Val = 67%). Gel analysis of the reverse transcription products revealed that both 3TC-resistant RT mutants produce significantly shorter cDNA molecules than the wild-type enzyme [Met (wt)>Val>Ile], indicating that 3TC-resistant RT polymerases are less processive enzymes. Interestingly, these enzyme defects were more pronounced under limiting dNTP concentrations and we therefore assayed virus replication in primary cells that contain relatively low dNTP levels. Under these conditions, we measured significantly reduced replication kinetics for the 3TC-resistant HIV-1 variants [Met (wt)>Val>Ile]. If the level of virus replication can be similarly reduced in 3TC-treated patients that develop drug-resistant HIV-1 variants, this may be of considerable clinical benefit.
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PMID:Reduced replication of 3TC-resistant HIV-1 variants in primary cells due to a processivity defect of the reverse transcriptase enzyme. 867 Sep 8

The novel human immunodeficiency virus type 1-specific thiocarboxanilide derivatives that contain either a substituted furanyl (UC-781) or thienyl (UC-82) ring linked to the thiocarboxy group and a pentenyloxyether chain linked to the 4-chlorophenyl ring in meta position show highly favorable antiviral properties. Compounds UC-781 and UC-82 discovered by scientists at Uniroyal Chemical Ltd. proved to be > or = 5-10-fold more inhibitory to wild-type human immunodeficiency virus type 1 strains (EC50 approximately 0.002 microgram/ml) than the thiocarboxanilide oxime ether UC-10 and other non-nucleoside reverse transcriptase inhibitors such as nevirapine, bis(heteroaryl)piperazine, and tetrahydroimidazo[4,5,l-jk][1,4]-benzodiazepin-2(1H)-one. In addition, the compounds were able to knock out virus replication in cell culture at concentrations that were 20-50-fold lower than those of nevirapine or bis(heteroaryl)piperazine. They were also highly efficient (EC50 < or = 0.02 microgram/ml) in suppressing the replication of mutant virus strains that contained mutations in their reverse transcriptase that conferred resistance to other non-nucleoside reverse transcriptase inhibitors (i.e., Tyr181 to Cys, Lys103 to Asn, Val106 to Ala, and Leu100 to Ile). The compounds selected for virus mutants that were only marginally resistant to the thiocarboxanilides ( < 10-20-fold). The antiviral activity of the compounds was only slightly affected by the presence of high concentrations of human serum, and the compounds were shown to be highly stable in the presence of human serum for at least 24 hr at room temperature.
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PMID:Highly favorable antiviral activity and resistance profile of the novel thiocarboxanilide pentenyloxy ether derivatives UC-781 and UC-82 as inhibitors of human immunodeficiency virus type 1 replication. 870 Jan 48

A large variety of carboxanilide and thiocarboxanilide derivatives in which the original oxathiin or aliphatic moieties present in the prototype compounds UC84 and UC38 were replaced by an (un) substituted furanyl, thienyl, phenyl, or pyrrole entity have been evaluated for activity against wild-type human immunodeficiency virus type 1 strain IIIB [HIV-1 (IIIB)] and a series of mutant virus strains derived thereof. The mutant viruses contained either the Leu-100-->Ile, Lys-103-->Asn, Val-106-->Ala, Glu-138-->Lys, Tyr-181-->Cys, or Tyr-188-->Leu mutation in their reverse transcriptase. Several 3-(2-methylfuranyl)- and 3-(2-methylthienyl)-thiocarboxanilide ester, (thio)ether, and oxime ether derivatives showed exquisitely potent antiviral activity against wild-type HIV-1 (50% effective concentration, 0.009 to 0.021 microM). The pentenylethers of the 2-methylfuranyl and 2-methylthienyl derivatives (i.e., 313, N-[4-chloro-3-(3-methyl-2-butenyloxy)phenyl]- 2-methyl-3-furancarbothioamide or UC-781, and 314, N-[4-chloro-3-(3-methyl-2-butenyloxy)phenyl] -2-methyl-3-thiophenecarbothioamide or UC-82) proved virtually equally inhibitory for wild-type and the Ile-100, Ala-106, and Lys-138 mutant virus strains (50% effective concentration, 0.015 to 0.021 microM). Their inhibitory effect against the Asn-103 and Cys-181 reverse transcriptase mutant virus strains was decreased only four- to sevenfold compared with wildtype virus. UC-781 and UC-82 should be considered potential candidate drugs for the treatment of HIV-1-infected individuals.
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PMID:Identification of novel thiocarboxanilide derivatives that suppress a variety of drug-resistant mutant human immunodeficiency virus type 1 strains at a potency similar to that for wild-type virus. 872 19

A novel membrane-soluble prodrug of the 5'-monophosphate derivative of 3TC containing a phenyl group and the methyl ester of L-alanine linked to the phosphorus through a phosphoramidate bond with the primary amino moiety (designated Cf 1109) was prepared. The 3TC prodrug proved less potent an inhibitor of HIV-1 and HIV-2 replication in CEM cell cultures than 3TC, but lost only 20-fold antiviral potency in 2'-deoxycytidine kinase-deficient CEM/dCK- cells compared with a more than 2,000-fold decrease of activity of 3TC. In contrast, 3TC and Cf 1109 proved equally highly effective in inhibiting HBV release in supernatants of HBV-transfected Hep G2 2.2.15 cell cultures (50% effective concentration approximately 0.02 microM). Both compounds easily selected for highly resistant HIV-1 strains at a comparable speed of breakthrough. The mutant viruses contained an 184-Ile and/or 184-Val amino acid change in their reverse transcriptase. Our data are suggestive for a relatively poor delivery of 3TC-MP in the intact CEM cells but a remarkably high delivery of 3TC and/or 3TC-MP in the intact Hep G2 2.2.15 cells.
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PMID:Anti-HIV and anti-HBV activity and resistance profile of 2',3'-dideoxy-3'-thiacytidine (3TC) and its arylphosphoramidate derivative CF 1109. 875 70


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