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)

Hepatitis B virus (HBV) replication is initiated by binding of its reverse transcriptase (P) to the apical stem-loop (AL) and primer loop (PL) of epsilon, a highly conserved RNA element at the 5'-end of the RNA pregenome. Mutation studies on duck/heron and human in vitro systems have shown similarities but also differences between their P-epsilon interaction. Here, NMR and UV thermodynamic data on AL (and PL) from these three species are presented. The stabilities of the duck and heron ALs were found to be similar, and much lower than that of human. NMR data show that this low stability stems from an 11-nt internal bulge destabilizing the stem of heron AL. In duck, although structured at low temperature, this region also forms a weak point as its imino resonances broaden to disappearance between 30 and 35 degrees C well below the overall AL melting temperature. Surprisingly, the duck- and heron ALs were both found to be capped by a stable well-structured UGUU tetraloop. All avian ALs are expected to adhere to this because of their conserved sequence. Duck PL is stable and structured and, in view of sequence similarities, the same is expected for heron - and human PL.
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PMID:Thermodynamics and NMR studies on Duck, Heron and Human HBV encapsidation signals. 1743 Sep 68

High-throughput screening hit 1 was identified as a potent, broad-spectrum, non-nucleoside reverse transcriptase inhibitor (NNRTI) of HIV-1 replication. Analysis of the bound conformation of analogs of this inhibitor via molecular modeling and NMR contributed to the design of novel tertiary amide, carbamate, and thiocarbamate based NNRTIs.
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PMID:Scaffold hopping in the rational design of novel HIV-1 non-nucleoside reverse transcriptase inhibitors. 1745 54

The cerium Ce(III), lanthanum La(III), and neodymium Nd(III) complexes with 4-hydroxy-3-(3-oxo-1-phenylbutyl)-2H-1-benzopyran-2-one (warfarin) (W) and 3,3'-benzylidenebis[4-hydroxycoumarin] (1) were synthesized and studied for the first time for cytotoxicity (on MT-2 cells) and as anti-HIV agents under acute and chronic infection. The complexes were characterized by different physicochemical methods: mass spectrometry, (1)H NMR, (13)C NMR, and IR spectroscopy. The spectra of the complexes were interpreted on the basis of comparison with the spectrum of the free ligands. Anti-HIV effect of the complexes/ligands was measured in MT-2 cells by microtiter infection assay. Detection of endogenous reverse transcriptase (RT) activity and RT processivity by PCR indicative for proviral DNA synthesis demonstrated that anti-HIV activity has not been linked to early stages of viral replication. No effect on late steps of viral replication has been found using cells chronically producing HIV-1(LAI) virus. La(W) demonstrated anti-HIV activity (IC50=21.4 muM) close to maximal nontoxic concentration. Nd(W), Ce(1), and Nd(1) demonstrated limited anti-HIV potency, so none of the complexes seems appropriate to be used in clinic. Further targeting of HIV-1 inhibition by La(W) is under progress.
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PMID:Antihuman Immunodeficiency Virus Type 1 (HIV-1) Activity of Rare Earth Metal Complexes of 4-Hydroxycoumarins in Cell Culture. 1749 16

BMS-A78277 (1) is a 5,10-dihydrobenzo[beta][1,8]naphthyridine-N-oxide compound that resides in a class of novel non-nucleoside reverse transcriptase inhibitors (NNRTIs), displaying improved activity against clinically relevant mutants of HIV-1 and possessing pharmacokinetic profiles amenable to once-daily dosing. In the course of investigating the nonclinical metabolism of 1, a circulating metabolite specific to the cynomolgus monkey was identified and subsequently characterized as the carboxyindole metabolite 2. The present investigation describes the biotransformation of this NNRTI in cynomolgus monkey, one which results in a net ring contraction of 1. The use of mass spectrometry and high field NMR analysis aided in the structural characterization of metabolite 2, the source of which originated from the urine and bile of cynomolgus monkeys receiving oral doses of 1. Preparation of a synthetic standard of 2 not only provided ultimate structural confirmation but also afforded ample material for biological testing. The metabolism of 1 was investigated in monkey hepatocytes and hepatic subcellular fractions. While microsomes were incapable of generating metabolite 2, incubation of 1 in monkey S9 fractions as well as hepatocytes resulted in measurable levels of the carboxyindole metabolite. Consequently, incubation of 1 in monkey hepatocytes, which were suspended in media containing (18)O-labeled water, resulted in the incorporation of (18)O into the carboxyindole metabolite, 2. These data implicate a mechanism involving the bioactivation of 1 to an electrophilic intermediate that upon hydrolysis undergoes a concerted ring contraction, resulting in the formation of 2. Previously confined to discussions regarding the metabolism of natural products and select aliphatic heterocycles, the present investigation extends the discussion of metabolism-mediated ring contraction to aromatics such as the present naphthyridine compound, 1.
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PMID:Species-specific, P450- and sulfotransferase-mediated novel ring contraction of a naphthyridine-N-oxide compound in cynomolgus monkey. 1793 41

A purine-rich region of the plus-strand RNA genome of retroviruses and long terminal repeat (LTR)-containing retrotransposons, known as the polypurine tract (PPT), is resistant to hydrolysis by the RNase H domain of reverse transcriptase (RT) and ultimately serves as a primer for plus-strand DNA synthesis. The mechanisms underlying PPT resistance and selective processing remain largely unknown. Here, two RNA/DNA hybrids derived from the PPTs of HIV-1 and Ty3 were probed using high-resolution NMR for preexisting structural distortions in the absence of RT. The PPTs were selectively modified through base-pair changes or by incorporation of the thymine isostere, 2,4-difluoro-5-methylbenzene (dF), into the DNA strand. Although both wild-type (WT) and mutated hybrids adopted global A-form-like helical geometries, observed structural perturbations in the base-pair and dF-modified hybrids suggested that the PPT hybrids may function as structurally coupled domains.
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PMID:High-resolution NMR analysis of the conformations of native and base analog substituted retroviral and LTR-retrotransposon PPT primers. 1835 16

Nevirapine (NVP) is a non-nucleoside reverse transcriptase inhibitor used against the human immunodeficiency virus type 1 (HIV-1), mostly to prevent mother-to-child HIV transmission in developing countries. One of the limitations of nevirapine use is severe hepatotoxicity, which raises concerns about its administration, particularly in the perinatal and pediatric settings. Nevirapine metabolism involves oxidation of the 4-methyl substituent to 12-hydroxy-NVP and the formation of phenolic derivatives. Further metabolism of 12-hydroxy-NVP by phase II esterification may produce electrophilic derivatives capable of reacting with DNA to yield covalent adducts, which could potentially be involved in the initiation of mutagenic and carcinogenic events. In the present study, we have investigated the reactivity of the synthetic model electrophile, 12-mesyloxy-NVP, toward 2'-deoxynucleosides and DNA. Parallel synthetic studies were conducted with 12-bromo-NVP and 3',5'- O-bis( tert-butyldimethylsilyl)-2'-deoxynucleosides, using palladium(0) catalysis. Multiple adducts from deoxyguanosine, deoxyadenosine, and deoxycytidine were isolated in the reactions with 12-mesyloxy-NVP and characterized by NMR and MS. The adduct structures consistently involved binding through C12 of NVP and N7 or N9 of deoxyguanosine; N1, N3, or N9 of deoxyadenosine; and N3 of deoxycytidine. Reactions conducted under palladium(0) catalysis yielded adducts through O (6) and N1 of deoxyguanosine, N1 of deoxyadenosine, and N3 of deoxycytidine. At least seven deoxynucleoside-NVP adducts were present in DNA reacted with 12-mesyloxy-NVP and enzymatically hydrolyzed. Four of these adducts were identified as 12-(deoxyadenosin-N1-yl)nevirapine, 12-(deoxycytidin-N3-yl)nevirapine, 12-(deoxyguanosin- O(6)-yl)nevirapine, and 12-(deoxyadenosin- N(6)-yl)nevirapine. One depurinating adduct, 12-(guanin-N7-yl)nevirapine, was identified in the thermal neutral DNA hydrolysate. If formed in vivo, some of these adducts would have considerable mutagenic potential. Our results thus suggest that NVP metabolism to 12-hydroxy-NVP may be a factor in NVP hepatocarcinogenicity.
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PMID:Synthesis and characterization of DNA adducts from the HIV reverse transcriptase inhibitor nevirapine. 1859 99

In contrast to orthoretroviruses, foamy viruses (FVs) express their Pol polyprotein from a separate pol-specific transcript. Only the integrase domain is cleaved off, leading to a protease-reverse transcriptase (PR-RT) protein. We purified the separate PR domain (PRshort) of simian FV from macaques by expressing the recombinant gene in Escherichia coli. Sedimentation analyses and size exclusion chromatography indicate that PRshort is a stable monomer in solution. This allowed us to determine the structure of the PRshort monomer using 1426 experimental restraints derived from NMR spectroscopy. The superposition of 20 conformers resulted in a backbone atom rmsd of 0.55 A for residues Gln8-Leu93. Although the overall folds are similar, the macaque simian FV PRshort reveals significant differences in the dimerization interface relative to other retroviral PRs, such as HIV-1 (human immunodeficiency virus type 1) PR, which appear to be rather stable dimers. Especially the flap region and the N- and C-termini of PRshort are highly flexible. Neglecting these regions, the backbone atom rmsd drops to 0.32 A, highlighting the good definition of the central part of the protein. To exclude that the monomeric state of PRshort is due to cleaving off the RT, we purified the complete PR-RT and performed size exclusion chromatography. Our data show that PR-RT is also monomeric. We thus conclude adoption of a monomeric state of PR-RT to be a regulatory mechanism to inhibit PR activity before virus assembly in order to reduce packaging problems. Dimerization might therefore be triggered by additional viral or cellular factors.
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PMID:The solution structure of the simian foamy virus protease reveals a monomeric protein. 1859 83

Ecklonia cava (EC), which is an edible marine brown alga with a broad range of bioactivities, belongs to the family of Laminariaceae. The bioactive 6,6'-bieckol, one of the main phloroglucinol derivatives naturally occurred from this genus, was isolated and characterized by NMR techniques. For the first time, human immunodeficiency virus type-1 (HIV-1) inhibitory activity of 6,6'-bieckol showed wild inhibition against HIV-1 induced syncytia formation (EC(50) 1.72 microM), lytic effects (EC(50) 1.23 microM), and viral p24 antigen production (EC(50) 1.26 microM), respectively. This result was strongly and clearly supported by the further investigation also, which 6,6'-bieckol selectively inhibited the activity of HIV-1 reverse transcriptase (RT) enzyme with EC(50) of 1.07 microM, as well as HIV-1 entry. Moreover, unlike most of other tannins, 6,6'-bieckol exhibited no cytotoxicity at concentrations which inhibited HIV-1 replication almost completely. Thus, it can be suggested that the potentially effective 6,6'-bieckol might be employed as a drug candidate for development of new generation therapeutic agents against HIV.
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PMID:Anti-HIV-1 activity of phloroglucinol derivative, 6,6'-bieckol, from Ecklonia cava. 1869 22

The oral cavity is an environment challenged by a large variety of pathogens. Consequently, the antimicrobial peptides expressed in that environment are interesting as they evolved to defend against a broad spectrum of bacteria and fungi. Here we report the discovery of new alpha-defensins from rhesus macaque oral mucosa and determine the first alpha-defensin structure from that species. The new peptides were identified by sequencing of reverse transcriptase-PCR products obtained from oral mucosal tissues, disclosing three mucosal alpha-defensins, termed rhesus macaque oral alpha-defensins (ROADs). The peptide corresponding to fully processed ROAD-1 was synthesized, subjected to folding/oxidation conditions, and purified. ROAD-1 was active against Staphylococcus aureus, Escherichia coli, and Candida albicans in a concentration-dependent manner. We determined the structure of ROAD-1 using NMR spectroscopy and find that the synthetic peptide adopts the canonical disulfide pairing and alpha-defensin fold. The antimicrobial mechanism of defensins has been correlated with their ability to disrupt and permeabilize the cell envelope, activities that depend on the surface features of the folded peptide. Although ROAD-1 maintains the defensin fold, the oral defensin displays distinct surface features when compared with other alpha-defensin structures.
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PMID:Synthesis, structure, and activities of an oral mucosal alpha-defensin from rhesus macaque. 1893 Sep 22

The highly potent anti-HIV agent UC781 is being evaluated for use in topical microbicides to prevent HIV transmission. However, UC781 is extremely hydrophobic with poor water solubility, a property that may complicate appropriate formulation of the drug. In this study, we examined the ability of several cyclodextrins, beta-cyclodextrin (beta CD), methyl-beta-cyclodextrin (M beta CD), and 2-hydroxylpropyl-beta-cyclodextrin (HP beta CD), to enhance the aqueous solubility of UC781. Each of the cyclodextrins provided dramatic increases in UC781 aqueous solubility, the order being M beta CD>HP beta CD>beta CD. The complexation constants (K (1:1)) of the inclusion complexes were determined via a phase solubility technique using high-performance liquid chromatography and showed that UC781 solubility increased linearly as a function of cyclodextrin concentration. Ultraviolet spectroscopy, Fourier transform infrared spectroscopy, differential scanning calorimetry, and 2D (1)H ROESY NMR spectroscopy were used to further characterize these UC781/cyclodextrin complexes. The inhibitory potency of UC781 and its HP beta CD inclusion complex were evaluated using an in vitro HIV-1 reverse transcriptase inhibition assay The inhibitory potency of the UC781/HP beta CD complex was 30-fold greater than that of UC781 alone, showing that the complexed drug is able to provide substantial inhibition of its target. The enhancement of UC781 aqueous solubility is essential for the development of a useful vaginal microbicide dosage form, and our data suggest that UC781/cyclodextrin inclusion complexes may be useful in this context.
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PMID:Characterization of cyclodextrin inclusion complexes of the anti-HIV non-nucleoside reverse transcriptase inhibitor UC781. 1908 44


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