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PubMed2005

Anti-human immunodeficiency virus type 1 activity of the nonnucleoside reverse transcriptase inhibitor GW678248 in combination with other antiretrovirals against clinical isolate viruses and in vitro selection for resistance.

GW678248, a novel nonnucleoside reverse transcriptase inhibitor, has been evaluated for anti-human immunodeficiency virus activity in a variety of in vitro assays against laboratory strains and clinical isolates. When GW678248 was tested in combination with approved drugs in the nucleoside and nucleotide reverse transcriptase inhibitor classes or the protease inhibitor class, the antiviral activities were either synergistic or additive. When GW678248 was tested in combination with approved drugs in the nonnucleoside reverse transcriptase inhibitor class, the antiviral activities were either additive or slightly antagonistic. Clinical isolates from antiretroviral drug-experienced patients were selected for evaluation of sensitivity to GW678248 in a recombinant virus assay. Efavirenz (EFV) and nevirapine (NVP) had > or = 10-fold increases in their 50% inhibitory concentrations (IC50s) for 85% and 98% of the 55 selected isolates, respectively, whereas GW678248 had a > or = 10-fold increase in the IC50 for only 17% of these isolates. Thus, 81 to 83% of the EFV- and/or NVP-resistant viruses from this data set were susceptible to GW678248. Virus populations resistant to GW678248 were selected by in vitro dose-escalating serial passage. Resistant progeny viruses recovered after eight passages had amino acid substitutions V106I, E138K, and P236L in the reverse transcriptase-coding region in one passage series and amino acid substitutions K102E, V106A, and P236L in a second passage series.

Acquired Immunodeficiency Syndrome↗
PubMed1992

Domain structure of the human immunodeficiency virus reverse transcriptase.

The spatial arrangement of subunits p51 and p66 of the HIV-1 reverse transcriptase and the position of the RNase H containing domain, p15, have been determined by means of neutron small-angle scattering. The reverse transcriptase (p66/p51) is a flat molecule, which can be approximated by an ellipsoid with the half axes of 5.2 nm, 4.8 nm and 1.4 nm. The two subunits p51 and p66 having a centre-to-centre distance of 3.3 +/- 0.3 nm are attached at their flat sides, slightly shifted sideways. The p15 domain is located at the long axis of the ellipsoidal reverse transcriptase having a distance of 5.0 +/- 0.5 nm to the centre of the p51d domain, which is part of the p66 subunit, and a distance of 5.3 +/- 1.2 nm to the centre of the neighbouring p51s subunit.

Chromatography, Affinity↗
PubMed2005

Anti-HIV-1 activities in extracts from some medicinal plants as assessed in an in vitro biochemical HIV-1 reverse transcriptase assay.

An in vitro HIV-1 reverse transcriptase (RT) assay was used for screening of anti-HIV activity of extracts obtained from some Kenyan medicinal plants. The assay utilises [3H]-methyl thymidine triphosphate (dTTP) as the enzyme substrate and polyadenylic acid.oligodeoxythymidylic acid [poly(rA).p(dT)(12-18)] as the template-primer dimmer. This assay was optimised and standardised with respect to the various experimental parameters in a microtiter plate methodology. The assay was then applied to test for potential antiviral activities of several Kenyan medicinal plant extracts and the concentrations producing 50% inhibition (IC50) of the HIV-1 RT were determined. This assay is described in this report and results obtained with some of the extracts are presented.

Anti-HIV Agents↗
PubMed1997

HIV-1 drug susceptibilities and reverse transcriptase mutations in patients receiving combination therapy with didanosine and delavirdine.

Previous studies have shown that the human immunodeficiency virus type 1 (HIV-1) reverse transcriptase mutation Y181C, which confers high-level resistance to nonnucleoside reverse transcriptase inhibitors (NNRTIs), develops rarely during therapy with NNRTIs plus zidovudine. To determine whether didanosine (ddI) is also effective in preventing the emergence of Y181C, we analyzed delavirdine (DLV) susceptibilties and reverse transcriptase sequences of isolates obtained from patients enrolled in a pharmacokinetic study of DLV and ddI. Nine NNRTI-naive patients were evaluated. Seven received DLV/ddI and two received DLV/ddI/zidovudine. Median durations of prior zidovudine and ddI were 26 and 15 months, respectively. Isolates from eight of nine patients had a mutation(s) associated with nucleoside resistance at entry. After treatment with DLV and ddI alone, isolates from five of seven patients developed Y181C, four in combination with K103N. Thus, in this group of nucleoside-experienced patients, combination therapy with DLV/ddI did not prevent the emergence of Y181C.

Adult↗
PubMed2002

A rapid phenotypic assay for detecting multiple nucleoside analogue reverse transcriptase inhibitor-resistant HIV-1 in plasma.

Zidovudine and other nucleoside analogue reverse transcriptase inhibitors (NRTIs), like zalcitabine and didanosine used for treatment of individuals infected with HIV-1, can select for viruses with Q151M and other associated mutations (for example, A62V, S68G, V751, F77L, F116Y) in the reverse transcriptase (RT) enzyme. These mutations confer resistance to multiple nucleoside analogues, and thereby compromise the efficacy of this class of drugs. Presently available phenotypic assays for detection of multiple nucleoside analogue resistant (MNR) HIV-1 require testing for each NRTI individually. Here we report an enzymatic RT assay that uses resistance to zidovudine triphosphate (zidovudine-TP) as a diagnostic biochemical marker of MNR HIV-1. This assay exploits the different biochemical mechanisms for zidovudine-resistance conferred by either Q151 M or T215Y/F mutations and the inability of conventional RT assays to detect T215Y/F-associated zidovudine resistance. The assay detects RT activity directly in plasma by using Amp-RT, an ultra-sensitive PCR-based RT assay. We show that enzymatic resistance to zidovudine-TP is specific to MNR RT and is distinguishable from both wild-type (WT) and RT containing classical zidovudine-resistant mutations (D67N, K70R, T215Y/F, K219Q). Compared to WT, MNR HIV-1 RT had 5- to 36-fold increases in the concentration of drug required to inhibit 50% (IC50) of RT activity, depending on the presence of Q151 M alone or with additional MNR mutations. A screening assay utilizing 1 microM zidovudine-TP was developed and validated on 14 reference isolates, 37 plasma specimens, and seven patient-derived viruses. Twenty-three specimens were found to have reduced susceptibility to zidovudine-TP, and all had Q151 M. In contrast, 21 specimens were sensitive to zidovudine-TP, of which 12 had WT genotypes, four had T215Y/F, and five had T69S-insertions along with T215Y/F mutations. This RT-based phenotypic assay provides a specific and rapid tool for the direct identification and monitoring of Q151M-associated MNR HIV-1 in plasma.

Adenosine Monophosphate↗
PubMed2005

Antiviral activity of GW678248, a novel benzophenone nonnucleoside reverse transcriptase inhibitor.

The compound GW678248 is a novel benzophenone nonnucleoside reverse transcriptase inhibitor (NNRTI). Preclinical assessment of GW678248 indicates that this compound potently inhibits wild-type (WT) and mutant human immunodeficiency virus type 1 (HIV-1) reverse transcriptase in biochemical assays, with 50% inhibitory concentrations (IC(50)s) between 0.8 and 6.8 nM. In HeLa CD4 MAGI cell culture virus replication assays, GW678248 has an IC(50) of < or =21 nM against HIV-1 isogenic strains with single or double mutations known to be associated with NNRTI resistance, including L100I, K101E, K103N, V106A/I/M, V108I, E138K, Y181C, Y188C, Y188L, G190A/E, P225H, and P236L and various combinations. An IC(50) of 86 nM was obtained with a mutant virus having V106I, E138K, and P236L mutations that resulted from serial passage of WT virus in the presence of GW678248. The presence of 45 mg/ml human serum albumin plus 1 mg/ml alpha-1 acid glycoprotein increased the IC(50) approximately sevenfold. Cytotoxicity studies with GW678248 indicate that the 50% cytotoxicity concentration is greater than the level of compound solubility and provides a selectivity index of >2,500-fold for WT, Y181C, or K103N HIV-1. This compound exhibits excellent preclinical antiviral properties and, as a prodrug designated GW695634, is being developed as a new generation of NNRTI for the treatment of HIV-1 in combination with other antiretroviral agents.

Anti-HIV Agents↗
PubMed1993

Biochemical analysis of human immunodeficiency virus-1 reverse transcriptase containing a mutation at position lysine 263.

Site-directed mutagenesis has been used to assess the importance of lysine 263 in substrate binding of human immunodeficiency virus-1 (HIV-1) reverse transcriptase. Previous studies have indicated that lysine 263 functions in the binding of 2'-deoxynucleoside 5'-triphosphate (dNTP) substrates (Basu, A., Tirumalai, R. S., and Modak, M. J. (1989) J. Biol. Chem. 264, 8746-8752). We studied this interaction directly by using site-specific mutagenesis to change lysine 263 to a serine. Highly purified mutant enzyme K263S bound natural dNTP substrates and primed polynucleic acid substrates with equal affinity when compared to the wild type reverse transcriptase. No difference was observed in the binding of 3'-azido-2',3'-dideoxythymidine 5'-triphosphate to the mutant reverse transcriptase on the basis of Km and Ki determinations. The serine substitution had no effect on RNase H activity. These results indicate that lysine 263 is not essential in the binding of substrates to HIV-1 reverse transcriptase.

Base Sequence↗
PubMed1991

Reconstitution in vitro of RNase H activity by using purified N-terminal and C-terminal domains of human immunodeficiency virus type 1 reverse transcriptase.

Two constituent protein domains of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase were expressed separately and purified to homogeneity. The N-terminal domain (p51) behaves as a monomeric protein exhibiting salt-sensitive DNA polymerase activity. The C-terminal domain (p15) on its own has no detectable RNase H activity. However, the combination of both isolated p51 and p15 in vitro leads to reconstitution of RNase H activity on a defined substrate. These results demonstrate that domains of HIV-1 reverse transcriptase are functionally interdependent to a much higher degree than in the case of reverse transcriptase from Moloney murine leukemia virus.

Base Sequence↗
PubMed1997

The role of genotypic heterogeneity in wild type virus populations on the selection of nonnucleoside reverse transcriptase inhibitor-resistant viruses.

Virus populations were selected in cell culture using two widely used protocols in order to evaluate the role of selection methodology on the genotype and phenotype of nonnucleoside reverse transcriptase inhibitor resistant viruses. Selection was performed by serial passage of virus in the presence of gradually increasing concentrations of antiviral compound or passage in the presence of a constant high concentration of compound. Using the CEM-SS cell line, the IIIB strain of HIV-1, and identical nonnucleoside reverse transcriptase inhibitors, resistant viruses were obtained and their phenotypic and genotypic properties were defined. Resistant virus populations containing the Y181C amino acid change in the reverse transcriptase were predominantly selected with each of the tested compounds. Several of the compounds selected secondary amino acid changes using both methods. A comparison of the resistant viruses selected in our laboratory using each of the two protocols with viruses reported by a second laboratory employing one of the two methods suggests that genotypic differences in the selected virus isolates may most likely result from the variation in the genetic composition of the respective wild type virus pools, rather than the specific selection methodology employed. These results imply that HIV may select a wide variety of amino acid changes to avoid the inhibitory effects of the nonnucleoside reverse transcriptase inhibitors and the selection of compounds for clinical use in combination with agents possessing non-overlapping resistance phenotypes will require evaluation of the agents against virus isolates possessing each of the mutations known to confer drug resistance.

Anti-HIV Agents↗
PubMed1993

Active site labeling of HIV-1 reverse transcriptase.

The human immunodeficiency virus-1 reverse transcriptase (HIV-1 RT) heterodimer (M(r) = 66,000 and M(r) = 51,000) has been photoaffinity labeled using 4-thiodeoxyuridine triphosphate (S4-dUTP) as a probe. A nascent polymerization complex was assembled from a single-stranded DNA template, a 12-mer DNA primer, and the necessary dNTPs (one of which was alpha-32P-labeled) to extend the primer to produce the n-1 product. The photoaffinity probe was then uniquely added at the 3'-terminal position of the extended primer bound at the catalytic site and photolyzed. The larger subunit (p66) was exclusively derivatized. The unique radioactive peptide resulting from proteolysis was isolated and identified by amino acid sequencing.

Acquired Immunodeficiency Syndrome↗
PubMed2005

Polymorphism and drug-selected mutations in the reverse transcriptase gene of HIV-2 from patients living in southeastern France.

Few data are available about the susceptibility and the genotypic resistance pattern of human immunodeficiency virus type 2 (HIV-2) to nucleoside reverse transcriptase inhibitors (NRTIs). The HIV-2 reverse transcriptase (RT) gene from 25 HIV-2-infected patients followed-up in Marseilles and the surrounding area was analyzed. The aims of this study were to characterize the polymorphism of HIV-2 RT in the absence of drug, to determine whether it naturally harbors codons associated with drug-resistance in HIV-1, and to identify mutations emerging under NRTI-selective pressure. Fourteen patients had never undergone antiretroviral therapy and 11 received NRTI. Seventy sequences were analyzed. In untreated patients, 12 spots of high natural polymorphism (at positions 10, 11, 20, 43, 104, 121, 135, 162, 176, 180, 200, and 227) were observed; 4 of them were specific of HIV-2 (10, 176, 180, 227). Moreover, results showed four positions that could be associated with natural resistance to NRTI (75I, 118I, 219E, and perhaps 215S), in addition to those described previously for non-nucleoside reverse transcriptase inhibitors (NNRTIs) (181I, 188L, 190A). In HIV-2-infected patients receiving NRTI-containing therapies, specific genotypic patterns were observed with a high frequency of mutation Q151M (in 45% of patients) often associated with 70R, 115F, 214L, and/or 223R, which might compose an HIV-2 multi-NRTI resistance complex. Four newly or rarely described NRTI-selected mutations were observed: I5V, K35R, F214L, and K223R. As in HIV-1, substitution M184V was found in 3TC-treated patients. In conclusion, these findings highlight the need for specific guidelines for determining genotypic resistance and treatment of HIV-2.

Adult↗
PubMed1990

Selective action of 3'-azido-3'-deoxythymidine 5'-triphosphate on viral reverse transcriptases and human DNA polymerases.

The action of 3'-azido-3'-deoxythymidine 5'-triphosphate (N3dTTP) on DNA strand elongation catalyzed by human immunodeficiency virus type 1 reverse transcriptase was evaluated in comparison with human DNA polymerase alpha and proliferating cell nuclear antigen-independent DNA polymerase delta. Sequencing gel analysis demonstrated that the human immunodeficiency virus 1 reverse transcriptase preferentially incorporated N3dTTP into the T sites of the growing DNA strands and caused chain termination in a dose-dependent manner. This effect was observed even when the N3dTTP concentration was 0.3 microM, 100-fold less than dTTP. Studies with reverse transcriptases from avian myeloblastosis virus and Moloney murine leukemia virus showed that N3dTTP was also efficiently incorporated into DNA by these enzymes and terminated DNA strand elongation. In contrast, human DNA polymerases alpha and delta did not incorporate detectable amounts of N3dTTP into the DNA and were not inhibited by 300 microM N3dTTP. The selective incorporation of the chain-terminating nucleotide by the viral reverse transcriptases appears to be a molecular basis for the positive therapeutic index of 3'-azido-3'-deoxythymidine.

Antiviral Agents↗
PubMed1989

Azidothymidine triphosphate is an inhibitor of both human immunodeficiency virus type 1 reverse transcriptase and DNA polymerase gamma.

The reverse transcriptase from human immunodeficiency virus type 1 was purified from the virus to near homogeneity. The enzyme was shown to possess both RNA-dependent and DNA-dependent DNA-synthesizing activity. Activated DNA as a heteropolymeric substrate was used as efficiently as was the homopolymeric substrate poly(rA)-oligo(dT). The Michaelis-Menten constants were determined for each of the four nucleotides needed to elongate a natural template primer. Azidothymidine triphosphate, a well-known inhibitor of the enzyme, inhibited the enzyme competitively with respect to dTTP and noncompetitively with respect to the other nucleotides. Azidothymidine triphosphate acted as an efficient inhibitor of cellular DNA polymerase gamma, whereas other enzymes of eucaryotic DNA metabolism, namely, DNA polymerase alpha-primase and DNA polymerase beta, were not inhibited. This finding may explain why some acquired immunodeficiency syndrome patients suffer side effects during azidothymidine therapy.

DNA Polymerase III↗
PubMed1991

Characterization of an HIV-1 isolate displaying an apparent absence of virion-associated reverse transcriptase activity.

In characterizing a group of independent human immunodeficiency virus (HIV-1) isolates, we noted that certain isolates had anomolously low levels of virion-associated reverse transcriptase activity. In an attempt to understand the basis of this phenomenon, we examined in detail one such isolate, HIV-1G. We found correctly processed forms of the viral reverse transcriptase in virions as well as processed forms of other viral proteins, suggesting that viral proteins are both expressed and properly processed. We have detected a nuclease activity associated with the outer face of the HIV-1G envelope. This nuclease degrades the DNA product generated during the reverse transcription assay. The nuclease activity is more sensitive to mild protein denaturation than is the viral reverse transcriptase, and it is stimulated by the presence of Ca2+. The amount of virion-associated nuclease activity relative to reverse transcriptase activity varies between virus isolates and can vary also for one isolate during virus spread through a culture. The origin of the nuclease activity is unknown but is presumed to be cellular. The variability in amount of nuclease activity may reflect variability in the interaction of the virus with different cellular components during maturation.

Calcium↗
PubMed2004

Detection and quantitation of akabane and aino viruses by multiplex real-time reverse-transcriptase PCR.

A multiplex, quantitive reverse-transcriptase real-time PCR, using MGB TaqMan chemistry, for detecting akabane virus (AKAV) and aino virus (AINV) is described. Each specific probe was labeled with a different fluorescent dye--VIC for detecting AKAV and 6-carboxy-fluorescein (FAM) for detecting AINV. All available sequences of viral S RNA were aligned and primers and probes were designed so that AKAV primers and probes would recognize all AKAVs but not AINV, and vice versa. The parameters for multiplex reactions enabled the detection of both viruses in one tube reaction with similar efficiency. To quantitate the viruses, cDNA amplicons containing the real-time amplicon were prepared using forward primers carrying the T7 promoter sequences. The cDNAs were used directly as templates for run-off transcription and 10-fold dilutions of the products served as standards to quantitate unknown viral samples. Using this system had shown that it could detect approximately 3-30 copies of viral S genome.

Animals↗
PubMed2002

Quantitative structure-activity relationship study of 2-arylsulfonyl-6-substituted benzonitriles as non-nucleoside reverse transcriptase inhibitors of HIV-1.

The reverse transcriptase inhibition of HIV-1, the most common form of HIV, by non-nucleoside 2-arylsulfonyl-6-substituted benzonitriles is analysed through Fujita-Ban and Hansch approaches. The analyses have helped to ascertain the role of different substituents in explaining the observed inhibitory actions of these compounds. From both approaches it appeared that SO2 instead of SO or S at X; and NH2 instead of F at Y (see Figure 1) are advantageous to improving the activity of a compound against HIV-1. This in turn leads to the suggestion that the 2-arylsulfonyl-6-aminobenzonitrile scaffold is the only appropriate structural entity that may further result into potential compounds. Further, the compounds having a OMe substituent at the orthoposition, the bulkier substituents at meta-positions and "no" substituent at para-position of 2-arylsulfonyl moiety are beneficial in raising the activity. The two quantitative structure-activity relationship (QSAR) analyses, differing in parametric approach, therefore, provided the grounds for rationalizing the substituent selection in designing more potent compounds of the series.

Anti-HIV Agents↗
PubMed2003

Toxicity of non-nucleoside analogue reverse transcriptase inhibitors.

The non-nucleoside reverse transcriptase inhibitors (NNRTI) nevirapine (NVP), efavirenz (EFV), and delaviridine (DLV) are increasingly being used to treat HIV infection. Studies have shown excellent tolerance and efficacy and less development of virological resistance with HIV regimens that include NNRTIs. Nevertheless, abnormalities in liver enzymes are common in patients with HIV infection, and there are multiple etiologies for these abnormalities, including drug toxicity, viral hepatitis, opportunistic infections, and substance abuse. In particular, highly active antiretroviral therapy (HAART) can result in hepatotoxicity through a variety of mechanisms, such as mitochondrial toxicity, lipodystrophy syndrome, and steatohepatitis. The NNRTIs have been most frequently implicated in hypersensitivity reactions. NVP-containing HAART regimens may be more hepatotoxic than are those with EFV and DLV, at least for the first 6 weeks, although the data are still contradictory. Coinfection with hepatitis C and B viruses appears to significantly increase the risk of toxicity, and therefore all patients should be screened for viral hepatitis prior to commencing HAART. Close monitoring of transaminases is suggested in all patients commencing HAART, especially those with preexisting liver disease and coinfection with viral hepatitis.

Alkynes↗