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PubMed2001

Incidence of pancreatitis in HIV-infected patients receiving nucleoside reverse transcriptase inhibitor drugs.

BACKGROUND: Pancreatitis is a known adverse effect of the nucleoside reverse transcriptase inhibitors, particularly didanosine. Hydroxyurea has been used to potentiate the antiviral efficacy of didanosine, but recently there has been concern that severe and even fatal pancreatitis may be more likely to occur when hydroxyurea is used in combination with didanosine. We investigated the incidence of pancreatitis in patients using nucleoside analogues with or without hydroxyurea. METHODS: Data were obtained from patients followed longitudinally on the Johns Hopkins HIV Clinic. Incidence rates of pancreatitis were calculated for each antiretroviral regimen that included zidovudine, stavudine, didanosine (+ hydroxyurea), and didanosine + stavudine (+ hydroxyurea). Poisson regression was used to compare the relative rate of pancreatitis for each regimen adjusting for other covariates. RESULTS: A total of 2613 patients received at least one of the nucleoside reverse transcriptase inhibitor-containing regimens. There were 33 cases of pancreatitis. The crude incidence rate of pancreatitis ranged from 0.18 cases per 100 person-years on therapy for zidovudine to 6.25 cases per 100 person-years for didanosine + hydroxyurea. Compared to didanosine alone, and adjusting for CD4 cell count and other variables, the relative risk (RR) of pancreatitis was 8.56 [95% confidence interval) CI, 1.85-35.59] for didanosine + hydroxyurea, and 2.35 (95% CI, 0.46-11.89) for didanosine + stavudine + hydroxyurea. For any use of hydroxyurea, the RR = 4.01 (95% CI, 1.02-15.89). Other risk factors for pancreatitis included a CD4 cell count < 200 x 106 cells/l, female sex, and a history of pancreatitis. CONCLUSIONS: Our data show that the risk of pancreatitis is four-fold higher when hydroxyurea is used. The use of hydroxyurea with didanosine should probably be discouraged if other treatment options are available.

Adult↗
PubMed1992

Unequal human immunodeficiency virus type 1 reverse transcriptase error rates with RNA and DNA templates.

Sequence variation in the type 1 human immunodeficiency virus (HIV-1) results, in part, from inaccurate replication by reverse transcriptase. Although this enzyme is error-prone during synthesis in vitro with DNA templates, the fidelity of RNA-dependent DNA synthesis relevant to minus-strand replication in the virus life cycle has not been examined extensively. In the present study, we have developed a system to determine the fidelity of transcription and reverse transcription and have used it to compare the fidelity of DNA synthesis by the HIV-1 reverse transcriptase with RNA and DNA templates of the same sequence. Overall, fidelity was several-fold higher with RNA than with DNA. Sequence analysis of mutants generated with the two substrates revealed that differences in error rates were substantial for specific errors. Fidelity with RNA was greater than 10-fold higher for substitution and minus-one nucleotide errors at five different homopolymeric positions. Because such errors likely result from template-primer slippage, this result suggests that misaligned intermediates are formed and/or used less frequently with an RNA template-DNA primer than with a DNA template-DNA primer. The results also suggest that HIV-1 reverse transcriptase synthesis with an RNA template-DNA primer was error-prone during incorporation of the first two nucleotides, perhaps due to aberrant enzyme-substrate interactions as synthesis initiates. The unequal error rates with RNA and DNA templates suggest that mistakes during minus- and plus-strand DNA synthesis may not contribute equally to the mutation rate of HIV-1. The data also provide estimates of substitution and frameshift error rates during transcription by T7 RNA polymerase.

Avian Myeloblastosis Virus↗
PubMed2001

Novel inhibitors of HIV-reverse transcriptase catalyzed DNA strand transfer: can we alter the infidelity of the enzyme?

Reverse transcription of viral RNA by the enzyme HIV1-reverse transcriptase (HIV1-RT) involves two DNA-strand transfers (DNA-ST). We report here a time-dependent inactivation of DNA-strand transfer by specific DNA-strand transfer inhibitors that do not affect the polymerase activity of the enzyme. Irreversible inhibitors of this type may be useful in characterizing the sites on the enzyme responsible for DNA-strand transfer.

Base Sequence↗
PubMed2002

Induction of apoptosis by a nonnucleoside human immunodeficiency virus type 1 reverse transcriptase inhibitor.

Inhibition of human immunodeficiency virus type 1 reverse transcriptase (RT) by both nucleoside and nonnucleoside RT inhibitors profoundly inhibits virus replication. Nucleoside RT inhibitors are known to be toxic, but there is little information regarding the toxicities of nonnucleoside RT inhibitors (NNRTI). We demonstrate that efavirenz (an NNRTI) induces caspase- and mitochondrion-dependent apoptosis of Jurkat T cells and human peripheral blood mononuclear cells. The clinical relevance of these observations is not yet clear.

Alkynes↗
PubMed1989

Expression of an enzymatically active murine retroviral reverse transcriptase in human cells.

The region of the pol gene of the Moloney murine leukemia virus (M-MuLV) encoding the reverse transcriptase and RNase H activities was inserted in an eukaryotic expression vector and transiently expressed in human cultured cells. This results in the expression of high levels of reverse transcriptase activity. This enzyme, partially purified, also carries a RNase H activity, has the biochemical requirements of the viral enzyme and is recognized and inhibited by antibodies directed against a M-MuLV reverse transcriptase expressed in Escherichia coli.

Animals↗
PubMed1992

Template. Phosphorothioate oligonucleotides duplexes as inhibitors of HIV-1 reverse transcriptase.

We have investigated the interaction between a number of 14 mers phosphorothioate oligonucleotides and HIV-1 reverse transcriptase. Two methods were used to measure the affinity of the analogs for the enzyme. In the first, the oligonucleotide or its duplex with Poly(rl) were used as inhibitors of the enzyme using Poly(rA).(dT)14 as template primer. In the second, the oligonucleotides or their duplexes were used to displace a fluorescent template primer complex of known affinity from its binding site on reverse transcriptase. The two methods gave the same relative order of affinity. Phosphorothioate oligodeoxyribonucleotides had a much higher affinity than oligo(dC)14 and it was increased on hybridization. Quantitatively similar results were obtained for S(dC)14 or its analog with bases in the alpha-configuration. Of the analogs tested, only S(dC)14 showed priming activity.

Antiviral Agents↗
PubMed1994

Effect of template secondary structure on the inhibition of HIV-1 reverse transcriptase by a pyridinone non-nucleoside inhibitor.

The importance of RNA secondary structure on HIV-1 reverse transcriptase catalyzed polymerization and on the potency of the pyridin-2-one inhibitor 3-(4,7-dichlorobenzoxazol-2-ylmethylamino)-5-ethyl-6-meth ylpyridin-2(1H)-one, L-697,661, were investigated by employing heteromeric primer-template systems. Our data revealed that a stem-loop hairpin secondary structure in the RNA template could lead to strong hindrance of reverse transcription in the reaction catalyzed by HIV-1 reverse transcriptase resulting in the build up of intermediate-length (pause) polymerization products. The presence of L-697,661 greatly enhanced the accumulation of the pause products suggesting that the rate of enzyme translocation from the pause product might be more potently inhibited than polymerization up to the pause site. Model experiments using a synthetic RNA template containing a stem-loop hairpin revealed that the inhibitory potency of L-697, 661 increased 2-fold upon polymerization to within four bases of the secondary structure. Inhibitor potency was enhanced over 6-fold when primer-extension proceeded through the duplex region of the stem-loop.

Animals↗
PubMed1978

Resolution and characterization of intracytoplasmic forms of reverse transcriptase from Rauscher leukemia virus-producing cells.

The microsomal supernatant fraction obtained from a murine cell line chronically infected with and producing Rauscher leukemia virus (JLSV-10) was found to contain two forms of RNA-directed DNA polymerase (reverse transcriptase). The two enzyme forms, neither of which is detectable in uninfected cells (JLSV-9), were initially partially purified by poly(C)-agarose chromatography, and their separation was achieved by phosphocellulose chromatography. The enzyme form eluting first from phosphocellulose (0.3 M KCl), designated PC I, was found to be identical in all parameters tested to that form isolated directly from purified virions. The second enzyme peak, designated PC II, eluted from phosphocellulose at 0.5 M KCl and was not detectable in purified virions. The PC II enzyme has a molecular weight, determined by velocity sedimentation, of approximately 109,000, as compared with 70,000 for the PC I enzyme, and could not be further dissociated by exposure to high salt or nonionic detergent. Mixing purified virion or PC I DNA polymerase with uninfected cells followed by fractionation did not produce the PC II form, suggesting that it is neither an artifact of purification nor the result of fortuitous complexing of reverse transcriptase with normal cellular component(s). Both PC I and PC II enzyme forms appeared antigenically similar to virion DNA polymerase, demonstrated identical divalent cation requirements for various template-primers, and were capable of copying heteropolymeric regions of rabbit globin mRNA. However, kinetic studies of heat inactivation revealed that the PC II enzyme was far more heat labile than the PC I form, which appeared identical to the virion enzyme in this respect. Furthermore, whereas the PC I and virion-derived reverse transcriptase copied poly(C).(dG)12-18 most efficiently at a template-to-primer molar nucleotide ratio of 25:1, the PC II enzyme preferred a ratio of 5:1 for optimal rates of poly(dG) synthesis. Therefore, by these criteria, there appear to exist two intracellular forms of reverse transcriptase in the JLSV-10 Rauscher leukemia virus-producing murine cell line.

Cell Line↗
PubMed1993

The quinoline U-78036 is a potent inhibitor of HIV-1 reverse transcriptase.

The quinoline U-78036 represents a new class of non-nucleoside human immunodeficiency virus (HIV)-1 reverse transcriptase inhibitors. The agent possesses excellent antiviral activity at nontoxic doses in HIV-1-infected lymphocytes grown in tissue culture. Enzymatic kinetic studies of the HIV-1 reverse transcriptase (RT)-catalyzed RNA-directed DNA polymerase function were carried out in order to determine whether the inhibitor interacts with the template-primer or deoxyribonucleotide triphosphate (dNTP) binding sites of the polymerase. The data were analyzed using steady-state or Briggs-Haldane kinetics assuming that the template-primer binds to the enzyme first followed by the dNTP and that the polymerase functions processively. The calculated rate constants are in agreement with this model. The results show that the inhibitor acts as a mixed to noncompetitive inhibitor with respect to both the template-primer and the dNTP binding sites of the enzyme. Hence, U-78036 inhibits the RNA-directed DNA polymerase activity of RT by interacting with a site distinct from the template-primer and dNTP binding sites. Moreover, the potency of U-78036 is dependent on the base composition of the template-primer. The equilibrium constants for various enzyme-substrate-inhibitor complexes were at least seven times lower for the poly(rC).(dG)10-catalyzed system than the one catalyzed by poly(rA).(dT)10. In addition, the inhibitor does not impair the DNA-dependent DNA polymerase activity and the RNase H function of HIV-1 RT nor does it inhibit the RNA-directed DNA polymerase activity of the HIV-2, avian myoblastoma virus, and murine leukemia virus RT enzymes.

Antiviral Agents↗
PubMed2001

Non-nucleoside reverse transcriptase inhibitors: perspectives on novel therapeutic compounds and strategies for the treatment of HIV infection.

At present, the nucleoside reverse transcriptase (RT) inhibitors and protease inhibitors (PI) have dominated the therapeutic options for the treatment of human immunodeficiency virus (HIV) infection. From the initial monotherapeutic strategies, to the widely accepted multi-drug cocktails of today, the use of these two classes of compounds has successfully prolonged patient survival following infection with HIV. The efficacy of the multi-drug cocktails has delayed the onset of disease and generated hope that long-term therapy might allow the natural immune response to HIV infection to control both virus replication and pathogenesis within the context of an intact immune system despite the continuing presence of virus in various reservoirs within the body and the inability of these therapies to completely eradicate virus. However, the use of antiretroviral compounds for prolonged periods of time has also resulted in the appearance of significant drug-induced toxicity and metabolic abnormalities, as well as drug-induced variations in disease progression. Thus, continued research and development to identify new and improved antiretroviral agents will be a critical requirement in the foreseeable future. This ongoing research and development should also consider the challenges of defining more effective use of existing therapeutic agents, including the non-nucleoside reverse transcriptase inhibitors (NNRTIs).

Antiviral Agents↗
PubMed2000

Structural basis for activation of alpha-boranophosphate nucleotide analogues targeting drug-resistant reverse transcriptase.

AIDS chemotherapy is limited by inadequate intracellular concentrations of the active triphosphate form of nucleoside analogues, leading to incomplete inhibition of viral replication and the appearance of drug-resistant virus. Drug activation by nucleoside diphosphate kinase and inhibition of HIV-1 reverse transcriptase were studied comparatively. We synthesized analogues with a borano (BH(3)(-)) group on the alpha-phosphate, and found that they are substrates for both enzymes. X-ray structures of complexes with nucleotide diphosphate kinase provided a structural basis for their activation. The complex with d4T triphosphate displayed an intramolecular CH.O bond contributing to catalysis, and the R(p) diastereoisomer of thymidine alpha-boranotriphosphate bound like a normal substrate. Using alpha-(R(p))-boranophosphate derivatives of the clinically relevant compounds AZT and d4T, the presence of the alpha-borano group improved both phosphorylation by nucleotide diphosphate kinase and inhibition of reverse transcription. Moreover, repair of blocked DNA chains by pyrophosphorolysis was reduced significantly in variant reverse transcriptases bearing substitutions found in drug-resistant viruses. Thus, the alpha-borano modification of analogues targeting reverse transcriptase may be of generic value in fighting viral drug resistance.

Anti-HIV Agents↗
PubMed1989

Exoribonuclease activity of purified reverse transcriptase preparations from retroviruses.

Highly purified and commercially available preparations of reverse transcriptases from retroviruses contain a 3' to 5' exoribonuclease activity capable of hydrolyzing synthetic homopolyribonucleotides having a 3'-OH end. The exoribonuclease activity of reverse transcriptase preparations from Rous associated virus-2 was further characterized. This exoribonuclease activity cleaves poly(C) and poly(U) exonucleolytically from the 3'-OH end to produce nucleoside 5'-phosphates. Poly(A), poly(G), circular polyribonucleotide, and double-stranded polyribonucleotide were not hydrolyzed by the activity. This is a novel type of exoribonuclease activity.

Buffers↗
PubMed2004

K65R-associated virologic failure in HIV-infected patients receiving tenofovir-containing triple nucleoside/nucleotide reverse transcriptase inhibitor regimens.

High rates of early virologic failure associated with the emergence of the K65R mutation in HIV-1 reverse transcriptase (RT) have been reported among HIV-infected patients who received novel, tenofovir-containing, triple-nucleoside/nucleotide reverse transcriptase inhibitor (NRTI/NtRTI) regimens as their initial therapy. This review surveys the findings of prospective and retrospective studies in this regard, examines the significance of the K65R mutation and other factors associated with reports of early virologic failure among patients receiving tenofovir-containing NRTI/NtRTI regimens, and discusses clinical approaches to preventing and managing HIV drug resistance and treatment failure associated with the K65R mutation.

Adenine↗
PubMed1997

Mode of inhibition of HIV reverse transcriptase by 2-hexaprenylhydroquinone, a novel general inhibitor of RNA-and DNA-directed DNA polymerases.

A natural compound from the Red Sea sponge Ircinia sp., 2-hexaprenylhydroquinone (HPH), has been shown to be a general inhibitor of retroviral reverse transcriptases (from HIV-1, HIV-2 and murine leukaemia virus) as well as of cellular DNA polymerases (Escherichia coli DNA polymerase I, and DNA polymerases alpha and beta). The pattern of inhibition was found to be similar for all DNA polymerases tested. Thus the mode of inhibition was studied in detail for HIV-1 reverse transcriptase. HPH is a non-competitive inhibitor and binds the enzyme irreversibly with high affinity (Ki=0. 62 microM). The polar hydroxy groups have been shown to be of key importance. A methylated derivative, mHPH, which is devoid of these polar moieties, showed a significantly decreased capacity to inhibit all DNA polymerases tested. Like the natural product, mHPH binds the enzyme independently at an allosteric site, but with reduced affinity (Ki=7.4 microM). We show that HPH does not interfere with the first step of the polymerization process, i.e. the physical formation of the reverse-transcriptase-DNA complex. Consequently, we suggest that the natural inhibitor interferes with the subsequent steps of the overall reaction. Since HPH seems not to affect the affinity of dNTP for the enzyme (the Km is unchanged under conditions where the HPH concentration is increased), we speculate that its inhibitory capacity is derived from its effect on the nucleotidyl-transfer catalytic reaction. We suggest that such a mechanism of inhibition is typical of an inhibitor whose mode of inhibition should be common to all RNA- and DNA-directed polymerases.

Animals↗
PubMed2007

Small-molecule c-Myc inhibitor, 10058-F4, inhibits proliferation, downregulates human telomerase reverse transcriptase and enhances chemosensitivity in human hepatocellular carcinoma cells.

c-Myc oncogene is critical for the development of hepatocellular carcinoma. Given the successful use of small-molecule inhibitors on cancers, targeting c-Myc with small-molecule inhibitors represents a promising approach. The potential of using small-molecule c-Myc inhibitor, 10058-F4, was evaluated on hepatocellular carcinoma cell lines, HepG2 and Hep3B cells. HepG2 cells were more sensitive to 10058-F4 than Hep3B cells, as demonstrated by reduced cell viability, marked morphological changes and decreased c-Myc levels. 10058-F4 arrested the cell cycle (at G0/G1 phase) and induced apoptosis upon extended treatment. These observations might be attributable to the increased cyclin-dependent kinase inhibitor, p21, and decreased cyclin D3 levels. Besides, 10058-F4 also significantly decreased the alpha-fetoprotein levels, an indicator for hepatocellular carcinoma differentiation. We further found that 10058-F4 inhibited the transactivation of human telomerase reverse transcriptase, downregulated human telomerase reverse transcriptase expression and abrogated telomerase activity. In addition, pretreatment with 10058-F4 increased the chemosensitivity of HepG2 cells to low-dose doxorubicin, 5-fluorouracil and cisplatin. Therefore, small-molecule c-Myc inhibitors might represent a novel agent, alone or in combination with conventional chemotherapeutic agents, for anti-hepatocellular carcinoma therapy.

Antineoplastic Agents↗
PubMed1997

Phosphorothioate oligonucleotides derived from human immunodeficiency virus type 1 (HIV-1) primer tRNALys3 are strong inhibitors of HIV-1 reverse transcriptase and arrest viral replication in infected cells.

Retroviral reverse transcriptase (RT) is involved in the selection of a specific tRNA primer which initiates proviral DNA minus-strand synthesis. Studies of the interactions between human immunodeficiency virus type 1 (HIV-1) RT and primer tRNALys3 have shown that the dihydrouridine (diHU), anticodon, and pseudouridine regions of tRNA are highly protected in the RT-tRNA complex. The CCA 3' end of tRNA is also in close contact with the enzyme during the cDNA initiation step. Using synthetic oligoribonucleotides corresponding to the anticodon and diHU regions, we have previously shown a low but significant inhibition of HIV-1 RT activity. We extend this observation and show that primer tRNA-derived oligodeoxynucleotides (ODNs) carrying a phosphorothioate (PS) modification are strong inhibitors of HIV-1 RT. The affinity of PS-ODNs for the enzyme was monitored by gel mobility shift electrophoresis. Experiments with HIV-1-infected human cells (MT-2 cells) were performed with the latter ODNs. A PS-ODN corresponding to the 3' end of tRNALys3 (acceptor stem [AS]) was able to inhibit HIV-1 replication. No effect of the other modified ODNs was observed in infected cells. The analysis of HIV-1 RNase H activity in a cell-free system strongly suggests that the inhibitory effect of the PS-AS may be mediated via both a sense and an antisense mechanism.

Adsorption↗
PubMed2001

Mutations at amino acid positions 63, 189, and 396 of human immunodeficiency virus type 1 reverse transcriptase (RT) partially restore the DNA polymerase activity of a Trp229Tyr mutant RT.

Trp229 is part of the nonnucleoside reverse transcriptase inhibitor (NNRTI)-binding pocket of HIV-1 reverse transcriptase (RT). It is also an important constituent of the so-called "primer grip." Using a recombinant virus assay, we tried to obtain recombinant virus containing a Trp229Phe or a Trp229Tyr mutation in its RT. Previous studies already established the very low DNA polymerase activities of both the Trp229Phe and the Trp229Tyr mutant RT enzymes. We were able to obtain a Trp229Tyr but not a Trp229Phe mutant virus. However, in addition to the Trp229Tyr mutation this mutant virus also contained an Ile63Met, a Val189Ile, and a Glu396Gly mutation in its RT. When we evaluated the quadruple mutant virus for sensitivity/resistance against a variety of NNRTIs, no significant difference with the sensitivity/resistance profile of the single Trp229Tyr mutant RT enzyme could be observed. We found that the three additional mutations partly restored the low RNA- and DNA-dependent DNA polymerase activities of the Trp229Tyr mutant enzyme. Kinetic analysis revealed that both template/primer binding and dNTP incorporation are affected by the Trp229Tyr mutation. Our findings demonstrate that a mutation at position 229 is unlikely to occur under NNRTI drug pressure due to the poor catalytic activity of the singly mutated RT and the favorable drug sensitivity profile of the mutated enzyme/viruses in both the absence and the presence of the compensatory mutations. Therefore, amino acid position 229 may be regarded as an excellent amino acid target within the NNRTI pocket for rational drug design.

Amino Acid Substitution↗