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PubMed2006

Structural insights into mechanisms of non-nucleoside drug resistance for HIV-1 reverse transcriptases mutated at codons 101 or 138.

Lys101Glu is a drug resistance mutation in reverse transcriptase clinically observed in HIV-1 from infected patients treated with the non-nucleoside inhibitor (NNRTI) drugs nevirapine and efavirenz. In contrast to many NNRTI resistance mutations, Lys101(p66 subunit) is positioned at the surface of the NNRTI pocket where it interacts across the reverse transcriptase (RT) subunit interface with Glu138(p51 subunit). However, nevirapine contacts Lys101 and Glu138 only indirectly, via water molecules, thus the structural basis of drug resistance induced by Lys101Glu is unclear. We have determined crystal structures of RT(Glu138Lys) and RT(Lys101Glu) in complexes with nevirapine to 2.5 A, allowing the determination of water structure within the NNRTI-binding pocket, essential for an understanding of nevirapine binding. Both RT(Glu138Lys) and RT(Lys101Glu) have remarkably similar protein conformations to wild-type RT, except for significant movement of the mutated side-chains away from the NNRTI pocket induced by charge inversion. There are also small shifts in the position of nevirapine for both mutant structures which may influence ring stacking interactions with Tyr181. However, the reduction in hydrogen bonds in the drug-water-side-chain network resulting from the mutated side-chain movement appears to be the most significant contribution to nevirapine resistance for RT(Lys101Glu). The movement of Glu101 away from the NNRTI pocket can also explain the resistance of RT(Lys101Glu) to efavirenz but in this case is due to a loss of side-chain contacts with the drug. RT(Lys101Glu) is thus a distinctive NNRTI resistance mutant in that it can give rise to both direct and indirect mechanisms of drug resistance, which are inhibitor-dependent.

Anti-HIV Agents↗

PubMed2000

Differential influence of nucleoside analog-resistance mutations K65R and L74V on the overall mutation rate and error specificity of human immunodeficiency virus type 1 reverse transcriptase.

Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) variants with the K65R or L74V substitution display resistance to several nucleoside analogs. An in vitro dNTP exclusion assay revealed an increased fidelity for K65R RT compared with wild-type RT, but little change for L74V RT. When the forward mutation rates were measured via a gap-filling assay, the K65R variant displayed an 8-fold decrease in the overall mutation rate (1.0 x 10(-3) versus 8.6 x 10(-3) for wild-type HIV-1 RT), whereas the rate for the L74V variant was closer to that for wild-type RT (5.0 x 10(-3)). The increase in overall fidelity observed for K65R RT is the largest reported for any drug-resistant HIV-1 RT variant. Nucleotide sequence analysis of lacZalpha mutants generated by variant RTs indicated that K65R RT displays uniform reduction in most types of errors, whereas L74V RT does not. Modeling the substitutions into the x-ray structure of the ternary complex revealed that the major influence of Leu(74) in stabilizing the templating base is unaffected by Val substitution, whereas the K65R substitution appears to increase the stringency of dNTP binding. It is speculated that the increased fidelity of K65R RT is due to an altered interaction with the dNTP substrate.

Anti-HIV Agents↗

PubMed1999

A new potent HIV-1 reverse transcriptase inhibitor. A synthetic peptide derived from the interface subunit domains.

The biologically relevant and active forms of human immunodeficiency viruses type 1 and 2 reverse transcriptase found in infectious virions are heterodimers produced in a two-step dimerization process. Dimerization involves first the rapid association of the two subunits, followed by a slow conformational change yielding a fully active form. We have shown that the dimeric nature of reverse transcriptase represents a important target for the design of a new class of antiviral agents. In this work, we propose a new strategy for its inhibition by targeting protein/protein interactions during viral formation in infected cells. From the screening of peptides derived from the tryptophan cluster at the interface of the connection subdomain, we have designed a short peptide (10 residues) corresponding to residues 395-404, which can block dimerization of reverse transcriptase in vitro and in infected cells. This peptide is highly efficient in abolishing the production of viral particles, without any adverse toxic side effects, when transduced into human immunodeficiency virus type 1-infected cells together with a new peptide carrier.

Amino Acid Sequence↗

PubMed1998

Implication of the tRNA initiation step for human immunodeficiency virus type 1 reverse transcriptase in the mechanism of 3'-azido-3'-deoxythymidine (AZT) resistance.

There is a lack of correlation between biochemical studies and the observed clinical resistance of AIDS patients on long-term AZT therapy. Mutant HIV-1 reverse transcriptase in the viral isolates from these patients shows a 100-fold decrease in sensitivity to AZT whereas little or no difference is observed in kinetic parameters in vitro using steady-state kinetic analysis. A pre-steady-state kinetic analysis was used to examine the binding and incorporation of 2'-deoxythymidine 5'-triphosphate (dTTP) and 3'-azido-3'-deoxythymidine 5'-triphosphate (AZTTP) by wild-type HIV-1 reverse transcriptase and a clinically important AZT-resistant mutant form of the enzyme (D67N, K70R, T215Y, K219Q) utilizing a physiologically relevant RNA 18-mer/RNA 36-mer primer-template substrate. It was determined that with this RNA/RNA substrate there is a 2.6-fold increase in the selection for incorporation of the natural nucleotide dTTP over the unnatural nucleoside analogue AZTTP by AZT-resistant reverse transcriptase as compared to its wild-type form. This observation indicates that the tRNALys initiation step plays an important role in the development of drug resistance. Furthermore, this result implies that the structural basis of AZT resistance in HIV-1 reverse transcriptase involves the conformation of the RNA-DNA junction (formed upon attachment of a deoxynucleotide to the RNA primer). Taken together, these observations suggest a new pharmacological basis for the development of more effective and novel AIDS drugs.

DNA Primers↗

PubMed1982

Inhibition of murine leukaemia virus reverse transcriptase by 2-halogenated polyadenylic acids.

Several new analogues of polyadenylic acid [(A)n], i.e. poly(2-fluoroadenylic acid) [(fl2A)n], poly(2-chloroadenylic acid [(cl2A)n], poly(2-bromoadenylic acid) [(br2A)n] and poly(2-iodoadenylic acid) [(io2A)n] have been synthesized and evaluated for their effects on the RNA-directed DNA polymerase (reverse transcriptase) activity of Moloney murine leukaemia virus. All (A)n analogues were found to be potent inhibitors of reverse transcriptase, the order of (decreasing) potency being (fl2A)n greater than (io2A)n greater than (br2A)n greater than (cl2A)n. For all four (A)n analogues the inhibition of reverse transcriptase was competitive with respect to the template-primer. (A)n . oligo(dT). The K1 values were 0.02 microgram/ml for (fl2A)n, 0.1 microgram/ml for (io2A)n, 0.5 microgram/ml for (br2A)n and 8 microgram/ml for (cl2A)n. With a Ki of 0.02 microgram/ml (approx. 0.04 microM), (fl2A)n can be considered as one of the most, if not the most, potent polynucleotide inhibitor of reverse transcriptase that has been described so far.

Halogens↗

PubMed1994

Mutational analysis of residue 190 of human immunodeficiency virus type 1 reverse transcriptase.

S-2720 and other members of the quinoline/quinoxaline class of HIV-1-specific nonnucleoside reverse transcriptase inhibitors (NNRTIs) select for a glycine to glutamate substitution at residue 190 (Gly 190 Glu) of the reverse transcriptase (RT), when drug-resistant viruses are generated in cell culture. This mutation has not been described to appear upon selection for resistant viral variants using derivatives of any other class of NNRTIs. Notably, the RNA-dependent DNA polymerase activity of the Gly 190 Glu mutant enzyme is drastically diminished with respect to the wild-type RT. We describe here the effects of other amino acid substitutions at position 190 of the RT that were introduced by using site-directed mutagenesis. Polymerase activities and sensitivities to inhibition by a number of NNRTIs were determined for the different RT mutants. In general, an inverse correlation was found between the enzymatic activity and increasing length of the side chain, whereas the size of the residue and the level of resistance to NNRTIs appeared to be positively related. Double mutants, which contain the Gly 190 Glu mutation together with substitutions that confer resistance to other RT inhibitors, were all shown to possess severely diminished polymerase activity.

Antiviral Agents↗

PubMed1992

The Mauriceville plasmid of Neurospora crassa: characterization of a novel reverse transcriptase that begins cDNA synthesis at the 3' end of template RNA.

The Mauriceville and Varkud plasmids are retroid elements that propagate in the mitochondria of some Neurospora spp. strains. Previous studies of endogenous reactions in ribonucleoprotein particle preparations suggested that the plasmids use a novel mechanism of reverse transcription that involves synthesis of a full-length minus-strand DNA beginning at the 3' end of the plasmid transcript, which has a 3' tRNA-like structure (M. T. R. Kuiper and A. M. Lambowitz, Cell 55:693-704, 1988). In this study, we developed procedures for releasing the Mauriceville plasmid reverse transcriptase from mitochondrial ribonucleoprotein particles and partially purifying it by heparin-Sepharose chromatography. By using these soluble preparations, we show directly that the Mauriceville plasmid reverse transcriptase synthesizes full-length cDNA copies of in vitro transcripts beginning at the 3' end and has a preference for transcripts having the 3' tRNA-like structure. Further, unlike retroviral reverse transcriptases, the Mauriceville plasmid reverse transcriptase begins cDNA synthesis directly opposite the 3'-terminal nucleotide of the template RNA. The ability to initiate cDNA synthesis directly at the 3' end of template RNAs may also be relevant to the mechanisms of reverse transcription used by LINEs, group II introns, and other non-long terminal repeat retroid elements.

Base Sequence↗

PubMed2004

Action of anti-HIV drugs and resistance: reverse transcriptase inhibitors and protease inhibitors.

Currently, 20 drugs have been approved for Human Immunodeficiency Virus type-1 (HIV-1) clinical therapy. These drugs inhibit HIV-1 reverse transcriptase, protease, or virus entry. Introduction of a combination therapy with reverse transcriptase inhibitors and protease inhibitors has resulted in a drastic decrease in HIV-1 related mortality. Although the combination therapy can suppress viral replication below detection levels in current available assays, low levels of on-going viral replication still persist in some patients. Long-term administration of the combination therapy may increase selective pressure against viruses, and subsequently induce emergence of multiple drug-resistant HIV-1 variants. Attempts have been made to design novel antiretroviral drugs that would be able to suppress replication of the resistant variants. At present, several investigational drugs are being tested in clinical trials. These drugs target not only the resistant variants, but also improvement in oral bioavilability or other viral proteins such as HIV-1 integrase, ribonuclease H, and HIV-1 entry (CD4 attachment inhibitors, chemokine receptors antagonists, and fusion inhibitors). Understanding mechanism(s) of action of the drugs and mechanisms of drug resistance is necessary for successful designs in the next generation of anti-HIV-1 drugs. In this review, the mechanisms of action of reverse transcriptase- and protease-inhibitors, and the mechanism of resistance to these inhibitors, are described.

Animals↗

PubMed1984

Optimal conditions for detection of reverse transcriptase activity in human placentas.

Optimal conditions for detecting reverse transcriptase activity in human placental extracts are described. They vary with the state of the placenta at birth and are influenced by relative amounts of detergent, monovalent cation, and protein in the reaction mixture. Demonstrating activity of the placental enzyme requires detergent, but the enzyme is sensitive to high detergent concentrations. This sensitivity can be be altered by lowering the monovalent cation concentration from 0.154 to 0.034 M and by adding protein to the reaction mixture. The detection of reverse transcriptase in Rauscher murine leukemia virus and baboon endogenous type C virus, but not in the Mason-Pfizer monkey type D virus, shows similar requirements.

Animals↗

PubMedDate not supplied

Effect of antimetabolite drugs of nucleotide metabolism on the anti-human immunodeficiency virus activity of nucleoside reverse transcriptase inhibitors.

A number of attempts are currently underway to combine antimetabolite drugs of nucleotide metabolism with a nucleoside reverse transcriptase inhibitor (NRTI) targeting human immunodeficiency virus (HIV) to improve the antiviral efficacy of the NRTIs and to better control HIV drug resistance. Hydroxyurea, a ribonucleotide reductase inhibitor, is currently combined with the NRTI didanosine (2',3'-dideoxyinosine) in clinical trials. However, other cellular target enzymes, including thymidylate synthase, inosinate dehydrogenase, cytidine-5'-triphosphate synthetase, and other enzymes from the de novo nucleotide biosynthesis pathway, can also be considered to potentiate the antiviral action of NRTIs. The underlying reasons for the potentiation of the antiviral activity of the NRTIs by antimetabolite drugs of nucleotide metabolism can be multiple. Decreased endogenous 2'-deoxynucleoside-5'-triphosphate (dNTP) pools result in a better competition of the NRTI (as its triphosphate derivative), with the dNTPs for the virus-encoded reverse transcriptase to be recognized as a substrate for the DNA polymerization reaction and subsequently to be incorporated into the growing viral DNA chain. Also, an increased metabolism (phosphorylation) of the NRTI by stimulatory enzyme feedback mechanisms may result in the production of higher levels of NRTI triphosphate. Thus, higher intracellular ratios of NRTI-triphosphate/dNTP created by well-defined combinations of NRTIs and antimetabolite drugs enable a more profound inhibitory effect of the NRTI against the reverse transcriptase (and thus, against the virus) and a better suppression of resistant (mutant) virus strains. A profound evaluation of this relatively new concept in the clinical setting will reveal whether this approach will establish a place in future treatment modalities of HIV infections.

Antimetabolites↗

PubMed1979

Phosphonoformate inhibits reverse transcriptase.

The new antiviral substance phosphonoformate (PFA) has been tested in a cell-free system for its effect on reverse transcriptases from an avian retrovirus (avian myeloblastosis virus, AMV) and from mammalian retroviruses (Rauscher leukaemia virus, RMuLV; bovine leukaemia virus; baboon endogenous virus; simian sarcoma virus; visna virus). The observed inhibitory effect of PFA has been compared with that of a structurally related substance, phosphonoacetate (PAA). Phosphonoformate, at a concentration of 100 microM, reduced the activities of all the above mentioned polymerases by 90% when (rA)n.(dT)10 was used as a template/primer. The dose-response curves for AMV and RMuLV polymerases primed with (rA)n.(dT)10 showed PFA to be a 1000-fold more active than PAA; the RMuLV polymerase activity was reduced to 50% after incubation with 0.7 microM-PFA and 0.7 mM-PAA, respectively. There was no difference in PFA inhibition of virus-associated and purified reverse transcriptase activity. Results with various synthetic templates showed that both the RNA- and the DNA-dependent polymerase activities of reverse transcriptase were inhibited by PFA. The endogenous polymerase activity of AMV was inhibited to 50% at 100 microM-PFA, while PAA had no effect. The PFA inhibition was dependent on whether Mg2+ or Mn2+ was used as divalent cation in the assay. Phosphonoformate arrested DNA synthesis immediately after being added to the assay system. The mechanism of inhibition of the AMV polymerase was non-competitive with respect to substrate and template and the apparent inhibition constants were 16 microM and 9 microM, respectively.

Animals↗

PubMed1995

2'-Fluoropolynucleotide-directed reverse transcriptase reactions. Effect of homologous polynucleotides.

Several homologous polynucleotides have been tested as inhibitors on the reactions catalyzed by avian myeloblastosis virus (AMV) reverse transcriptase, in the presence of polyribonucleotides and 2'-fluorinated polynucleotides as templates. Polynucleotides differentially inhibited the reactions catalyzed by reverse transcriptase in the presence of these synthetic templates. Polyriboadenylic acid (poly(rA), poly(2'-O-methyladenylic acid) (poly(Am)), poly(2'-fluoro-2'-deoxyadenylic acid) (poly(dAfl), polyinosinic acid (poly(rI)) and polyuridylic acid poly(rU)) inhibited the polyribonucleotide-, but not the 2'-fluorinated polynucleotide-directed reverse transcriptase activity.

Avian Myeloblastosis Virus↗

PubMed1998

Structure-based design of N-[2-(1-piperidinylethyl)]-N'-[2-(5-bromopyridyl)]-thiourea and N-[2-(1-piperazinylethyl)]-N'-[2-(5-bromopyridyl)]-thiourea as potent non-nucleoside inhibitors of HIV-1 reverse transcriptase.

A novel computer model of the HIV reverse transcriptase (RT) non-nucleoside inhibitor (NNI) binding pocket, which was generated using high resolution crystal structure information from 9 individual RT/NNI complexes, revealed previously unrecognized ligand derivatization sites for phenethylthiazolylthiourea (PETT) derivatives. Spatial gaps surrounding the pyridyl ring of the active PETT derivative trovirdine were discovered during modeling procedures. Docking studies using the computer-generated model of the binding pocket (composite binding pocket) suggested that the replacement of the planar pyridyl ring of trovirdine with a nonplanar piperidinyl or piperazinyl ring, which occupy larger volumes, would better fill the spacious Wing 2 region of the butterfly-shaped NNI binding pocket. The anti-HIV activity of the synthesized heterocyclic compounds N-[2-(1-piperidinylethyl)]-N'-[2-(5-bromopyridyl)]-thiourea and N-[2-(1-piperazinylethyl)]-N'-[2-(5-bromopyridyl)]-thiourea was examined in HTLVIIIB-infected peripheral blood mononuclear cells. Both compounds were more potent than trovirdine and abrogated HIV replication at nanomolar concentrations without any evidence of cytotoxicity.

Antiviral Agents↗

PubMed1988

Alpha-anomeric DNA: beta-RNA hybrids as new synthetic inhibitors of Escherichia coli RNase H, Drosophila embryo RNase H and M-MLV reverse transcriptase.

Nuclease-resistant alpha-anomeric DNA:beta-RNA hybrids are inhibitors of Escherichia coli RNase H, and Drosophila embryo RNase H. RNase H activities were measured by polyacrylamide gel electrophoresis, employing a short substrate, (A)12:d[G-G-(T)12-G-G], or by acid-solubility techniques, using a long substrate, poly(A):poly(dT). Strand exchanges which could be responsible for the observed inhibition have been ruled out by S1 nuclease experiments and by using inhibitors which do not allow strand exchange. Our results suggest that RNase H, for which DNA:RNA duplexes are the natural substrates, binds to non-physiological alpha-DNA:RNA hybrids and is consequently inhibited. These hybrids also inhibit the RNA-dependent DNA polymerase activity of M-MLV reverse transcriptase, therefore appearing as potential inhibitors of at least two reverse transcriptase activities. However, the inhibitory effect of these hybrids with respect to M-MLV reverse transcriptase is also observed with the single-stranded alpha-DNA itself. Unexpectedly, polymerase activity is highly stimulated by alpha-oligos, analogous in their sequence to the beta primer used at a concentration unable to generate a detectable synthesis. These results suggest that the inhibition of reverse transcriptase activity with the alpha:beta may occur at different levels.

Animals↗

PubMed2005

Functional characteristics of a reverse transcriptase encoded by an endogenous retrovirus from Drosophila melanogaster.

ZAM is an LTR-retrotransposon from Drosophila melanogaster that belongs to the genus errantivirus, viruses similar in structure and replication cycle to vertebrate retroviruses. A key component to its lifecycle is its reverse transcriptase which copies single-stranded genomic RNA into DNA. Here, we provide a detailed characterization of the enzymatic activities of the reverse transcriptase encoded by ZAM. When expressed in vitro, the reverse transcriptase domain associated with the RNase H domain encoded by the ZAM pol gene forms homodimers and displays an efficient RNA-dependent DNA-polymerase activity. It requires either Mg2+ or Mn2+ divalent cations, and works in basic pH, with a peak at around pH9. The so-called [RT-RH] polypeptide displays an optimal activity at 22 degrees C, a property that makes it well-adapted to the temperature of its host. This study contributes to our understanding of the general structures and functions of retroviral reverse transcriptases, a necessary process in the search for novel inhibitors.

Animals↗

PubMed1998

New alkenyldiarylmethanes with enhanced potencies as anti-HIV agents which act as non-nucleoside reverse transcriptase inhibitors.

Twenty-two new alkenyldiarylmethanes (ADAMs) were synthesized and evaluated for inhibition of HIV-1 replication. The most potent compound proved to be methyl 3',3"-dichloro-4',4"-dimethoxy-5', 5"-bis(methoxycarbonyl)-6,6-diphenyl-5-hexenoate (ADAM II), which displayed an EC50 of 13 nM for inhibition of the cytopathic effect of HIV-1RF in CEM-SS cells. ADAM II inhibited HIV-1 reverse transcriptase with an IC50 of 0.3 microM but was inactive as an inhibitor of HIV-1 attachment/fusion to cells, protease, integrase, and the nucleocapsid protein. Molecular target-based and cell-based assays revealed that ADAM II acted biologically as a nonnucleoside reverse transcriptase inhibitor (NNRTI). ADAM II inhibited replication of a wide variety of laboratory, clinical, and clade-representative isolates of HIV-1 in T cell lines and cultures of peripheral blood mononuclear cells or monocyte/macrophages. Mutations that conferred resistance to ADAM II clustered at residues 101, 103, 108, 139, 179, 181, and 188, which line the nonnucleoside binding pocket of HIV-1 reverse transcriptase. However, HIV-1 NL4-3 strain expressing a mutation at residue 100 of reverse transcriptase, and an AZT-resistant virus, displayed increased sensitivity to ADAM II. Thus, ADAM II could serve as an adjunct therapy to AZT and NNRTIs that select for L100I resistance mutations.

Alkanes↗

PubMed1993

Retroviral reverse transcriptase inhibitory activity in Thai herbs and spices: screening with Moloney murine leukemia viral enzyme.

Fifty-seven Thai herbs and spices were examined for their retroviral reverse transcriptase inhibitory activity. All herbs and spices were extracted with hot-water and methanol. Reverse transcriptase inhibitory activity of the extracts was determined by using Moloney Murine Leukemia Virus reverse transcriptase (M-MuLV-RT) reacted with 3H-dTTP and radioactivity measured with a scintillation counter. Eighty-one per cent (46/57) of hot-water extracts and 54% (31/57) of methanol extracts showed inhibitory activities. At a concentration of 125 micrograms/ml, 13% (6/46) of hot-water extracts, namely Eugenia caryophyllus Bullock et Harrison, Phyllanthus urinaria Linn., Terminalia belerica Roxb., Nelumbo nucifera Gaertn., Psidium guajava Linn. and Lawsonia inermis Linn., had a relative inhibitory ratio (IR) over 50%. They showed ratios of 100%, 91%, 75%, 74%, 61% and 60%, respectively. For methanol extracts, only 10% (3/31) had IR values over 50%. They were T. belerica, E. caryophyllus and N. nucifera which exhibited IR values of 83%, 54% and 54%, respectively.

Animals↗

PubMed1990

Dimerization of human immunodeficiency virus type 1 reverse transcriptase. A target for chemotherapeutic intervention.

Recombinant human immunodeficiency virus type 1 reverse transcriptase has been used to investigate the process of dimer formation and the properties of the different mono- and dimeric forms of the enzyme. The studies show that reverse transcriptase activity is exclusively confined to the dimeric forms. As we also demonstrate, the association rate constant between the monomers is relatively low so that the dimer-monomer equilibrium is very slowly established. This offers a new and potentially interesting target for antiviral chemotherapy with presumably higher specificity than the currently used nucleoside analogs (Yarchoan, R., Mitsuya, H., Myers, C.E., and Broder, S. (1989) N. Eng. J. Med. 321, 726-738), which in their active triphosphorylated form are also inhibitors of cellular polymerases.

Acquired Immunodeficiency Syndrome↗