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PubMed2005

Alternative approach to blood screening using the ExaVir reverse transcriptase activity assay.

408 non-selected samples were obtained from healthy, adult individuals donating blood at the Ethiopian Red Cross Society-National Blood Transfusion Service. All samples were screened for HIV using the Vironostika Ag/Ab test, the Amplicor DNA PCR and examined for the presence of HIV reverse transcriptase (RT) using the ExaVir Load test (version 2). A panel of supplementary tests was used to evaluate the HIV status of the discordant samples and to confirm positivity. One aim was to assess an RT based test for screening for HIV in comparison with other more conventional tests. An HIV-prevalence of 3.4 % (14/408) was found. The Vironostika Ag/Ab test produced 391 negative, and according to the supplementary testing, 14 true- and three false- positive test results. The corresponding figures for the Amplicor DNA PCR test was 384 negative, 14 true- and two extra probably false -positive samples. In addition, the DNA PCR generated eight indeterminate results. The colorimetric version of the ExaVir load test exhibited 100 % specificity and detected RT in 13 of the true positive samples, but failed to detect one sample containing 200 HIV RNA copies /mL. This sample was detectable in the fluorimetric version of the test. The detection of RT activity in addition to the currently used markers would seem to have a potential for use in blood screening.

Blood Donors↗
PubMed2004

AZT binds RNA at multiple sites.

Azidothymidine (AZT) is a widely used inhibitor of type I human immunodeficiency virus (HIV) reverse transcriptase that act as a DNA chain terminator. Studies have shown primer unblocking and rescue of DNA synthesis AZT-resistant HIV-1 reverse transcriptase on DNA and RNA templates. Our recent study showed AZT bindings to the G-C, A-T base pairs and the backbone phosphate group of DNA duplex resulting in partial DNA conformational changes. This study was designed to examine the interaction of AZT with RNA in aqueous solution at physiological condition, using different drug/RNA (phosphate) molar ratios of 1/800 to 1/2 and constant RNA concentration of 1.25 or 12.5 mM (phosphate). Capillary electrophoresis, FTIR, and UV-visible difference spectroscopic methods and molecular modeling were used to determine the drug binding sites, binding constants, and the effects of AZT complexation on RNA conformation. Structural analysis showed that AZT binds RNA through G-C and A-U bases with two binding constants of K1=7.3 x 10(5) M(-1) and K2=1.90 x 10(5) M(-1). The drug distributions were 54% with G-C, 36% A-U, and 10% with the backbone phosphate group. RNA remains in A-family structure and drug sugar pucker in the C2'-endo/anti conformation in the AZT-RNA complexes. Molecular modeling studies show hydrogen bondings between RNA and AZT donor groups.

Electrophoresis, Capillary↗
PubMed1992

Interference with HIV-1 reverse transcriptase-catalyzed DNA chain elongation by the 5'-triphosphate of the carbocyclic analog of 2'-deoxyguanosine.

In an effort to better understand features in nucleotide analogs that result in the inhibition of HIV-1 reverse transcriptase, we have evaluated this enzyme with the 5'-triphosphate of the carbocyclic analog of 2'-deoxyguanosine (CdG-TP). CdG-TP was a reasonably potent competitive inhibitor of the incorporation of dGTP into DNA by HIV-1 reverse transcriptase using either a RNA or DNA template (Ki, 1 microM). CdG-TP was a good substrate for HIV-1 reverse transcriptase on both templates, but the DNA chain was poorly extended beyond the incorporation of CdG. These results indicate that substitution of ribose with a cyclopentane ring in nucleotides is not well tolerated by HIV-1 reverse transcriptase.

Antiviral Agents↗
PubMed1997

Efficacy, pharmacokinetics, and in vivo antiviral activity of UC781, a highly potent, orally bioavailable nonnucleoside reverse transcriptase inhibitor of HIV type 1.

A series of compounds related to oxathiin carboxanilide has been identified as nonnucleoside reverse transcriptase inhibitors (NNRTIs) of HIV-1, and structure-activity relationships have been described (Buckheit RW, et al.: Antimicrob Agents Chemother 1995;39:2718-2727). Three new analogs (UC040, UC82, and UC781) inhibited laboratory and clinical isolates of HIV-1, including isolates representative of the various clades of HIV-1 found worldwide, in both established and fresh human cells. Virus isolates with the amino acid changes L100I, K103N, V106I, and Y181C in the reverse transcriptase were partially resistant to these compounds. However, UC781 inhibited these virus isolates at low nontoxic concentrations, presenting a broad in vitro therapeutic index. As with other NNRTIs, each of the compounds synergistically interacted with AZT to inhibit HIV-1 replication. UC781 possesses a favorable pharmacokinetic profile in mice with a high level of oral bioavailability. Plasma concentrations reached maximum levels within 2 to 4 hr of oral administration and remained in excess of those required for in vitro anti-HIV activity for at least 24 hr after a single oral dose. When evaluated in a murine hollow fiber implant model of HIV infection, UC781 dosed orally or parenterally was able to suppress HIV replication completely in this model system, providing evidence of the in vivo efficacy of the compound.

Administration, Oral↗
PubMed2004

In vitro characterization of a simian immunodeficiency virus-human immunodeficiency virus (HIV) chimera expressing HIV type 1 reverse transcriptase to study antiviral resistance in pigtail macaques.

Antiviral resistance is a significant obstacle in the treatment of human immunodeficiency virus type 1 (HIV-1)-infected individuals. Because nonnucleoside reverse transcriptase inhibitors (NNRTIs) specifically target HIV-1 reverse transcriptase (RT) and do not effectively inhibit simian immunodeficiency virus (SIV) RT, the development of animal models to study the evolution of antiviral resistance has been problematic. To facilitate in vivo studies of NNRTI resistance, we examined whether a SIV that causes immunopathogenesis in pigtail macaques could be made sensitive to NNRTIs. Two simian-human immunodeficiency viruses (SHIVs) were derived from the genetic background of SIV(mne): SIV-RT-YY contains RT substitutions intended to confer NNRTI susceptibility (V181Y and L188Y), and RT-SHIV(mne) contains the entire HIV-1 RT coding region. Both mutant viruses grew to high titers in vitro but had reduced fitness relative to wild-type SIV(mne). Although the HIV-1 RT was properly processed into p66 and p51 subunits in RT-SHIV(mne) particles, the RT-SHIV(mne) virions had lower levels of RT per viral genomic RNA than HIV-1. Correspondingly, there was decreased RT activity in RT-SHIV(mne) and SIV-RT-YY particles. HIV-1 and RT-SHIV(mne) were similarly susceptible to the NNRTIs efavirenz, nevirapine, and UC781. However, SIV-RT-YY was less sensitive to NNRTIs than HIV-1 or RT-SHIV(mne). Classical NNRTI resistance mutations were selected in RT-SHIV(mne) after in vitro drug treatment and were monitored in a sensitive allele-specific real-time RT-PCR assay. Collectively, these results indicate that RT-SHIV(mne) may be a useful model in macaques for the preclinical evaluation of NNRTIs and for studies of the development of drug resistance in vivo.

Amino Acid Sequence↗
PubMed2005

Effect of the Q207D mutation in HIV type 1 reverse transcriptase on zidovudine susceptibility and replicative fitness.

Although present as polymorphisms in HIV-1 sequences from untreated patients, mutations at reverse transcriptase (RT) codon 207 are found at higher frequency in samples from zidovudine-treated patients. Introduction of the Q207D mutation into the RT of a zidovudine (ZDV)-resistant isolate by site-directed mutagenesis increased ZDV resistance 2.7-fold but had no effect on ZDV susceptibility when introduced into wild-type RT. Zidovudine-resistant recombinant HIV-1 with and without 207D showed comparable fitness in growth competition assays in the absence of ZDV, but this mutation enhanced the fitness of ZDV-resistant recombinants in the presence of drug. These results suggest that when present with other thymidine analogue resistance mutations, 207D serves as a resistance mutation that improves the fitness of ZDV-resistant HIV-1. Analyzing viral fitness can provide important insights into the role of polymorphisms in drug resistance.

Drug Resistance, Viral↗
PubMed2003

Alovudine Medivir.

Medivir is developing alovudine, an anti-HIV compound for the potential treatment of drug-resistant HIV infection. A phase IIa trial involving patients with multiresistant HIV was initiated in October 2001 and by July 2002, the trial had been completed.

Clinical Trials, Phase I as Topic↗
PubMed2003

New antiretroviral drugs.

Despite the availability of 16 antiretroviral drugs approved for the treatment of HIV infection, current combination regimens present challenges. Newer antiretroviral drugs are needed to improve convenience, reduce toxicity and, of particular importance, to provide antiretroviral activity against viral strains resistant to the currently available antiretroviral agents. Candidate drugs with novel properties are in development in the two currently available drug classes: HIV reverse transcriptase inhibitors (nucleoside analogs, non-nucleoside analogs and nucleotide analogs) and HIV protease inhibitors (PI). Investigational nucleoside analog reverse transcriptase inhibitors (nRTI) include emtricitabine (FTC) and amdoxovir (DAPD), and investigational non-nucleoside reverse transcriptase inhibitors (NNRTI) include DPC 083 and TMC 125. New protease inhibitors under investigation include atazanavir (BMS-232 632), tipranavir, and TMC 114. In addition, newer agents with novel mechanisms of action such as HIV entry inhibitors (that inhibit the three steps of HIV entry: CD4 attachment, chemokine receptor binding and membrane fusion) and HIV integrase inhibitors are under investigation. Investigational entry inhibitors include PRO 542 (a CD4 attachment inhibitor), Schering C (a chemokine receptor inhibitor), enfuvirtide (T-20) and T-1249, inhibitors of membrane fusion. Investigational HIV integrase inhibitors include S-1360. Continued progress in the treatment of HIV disease will result from the development of new antiretroviral drugs.

Anti-HIV Agents↗
PubMed2002

Trends in the design of nucleoside analogues as anti-HIV drugs.

HIV infection remains incurable although several anti-HIV drugs have been identified and developed. Among these the nucleoside analogues were and remain in the forefront of anti-HIV chemotherapeutic regimens. Most of these nucleoside analogues are modified mainly in the sugar moiety. In general, they lack a free hydroxy group at the 3'-position. Consequently, they cannot participate in the formation of a 3',5'-phosphodiester linkage, which renders the nucleoside 5'-triphosphates of such nucleoside analogues effective anti-HIV agents. Preventing the formation of 3',5'-phosphodiester linkages leads to inhibition of the viral DNA strand elongation and ultimately chain termination. The phosphorylation of nucleoside analogues is a key factor in their efficacy as anti-HIV agents. Efficient phosphorylation depends largely on the structure of the nucleoside. Therefore, modification of the structure of nucleoside analogues are sought to enhance their phosphorylation and ultimately, effective inhibition of the HIV reverse transcriptase. This review is concerned with the trends in the design of nucleoside analogues as anti-HIV agents.

Anti-HIV Agents↗
PubMed1995

Human immunodeficiency virus reverse transcriptase substitutes for DNA polymerase I in Escherichia coli.

We present evidence that human immunodeficiency virus (HIV) reverse transcriptase (RT) can substitute for DNA polymerase I in bacteria. Expression of HIV RT enables an Escherichia coli mutant, polA12 recA718, containing a temperature-sensitive mutation in DNA polymerase I, to grow at a nonpermissive temperature. The plasmid pBR322 contains a DNA polymerase I-dependent origin of replication. Expression of HIV RT enables the same E. coli mutant to maintain this plasmid at a nonpermissive temperature. Furthermore, expression of HIV RT in this mutant renders it sensitive to 3'-azido-3'-deoxythymidine, a commonly used anti-AIDS drug that targets HIV RT. These combined findings on the genetic complementation of DNA polymerase I by HIV RT provide a bacterial assay to screen for drugs directed against HIV RT. Genetic complementation provides a method for positive selection of large numbers of functional HIV RT mutants for studies on structure-function relationships.

DNA Polymerase I↗
PubMed2005

Mechanism of action and resistant profile of anti-HIV-1 coumarin derivatives.

Dicamphanoyl khellactone (DCK) is a coumarin derivative that can potently inhibit HIV-1 replication. DCK does not inhibit RNA-dependent DNA synthesis. However, an HIV reverse transcriptase (RT) inhibitor-resistant strain, HIV-1/RTMDR1, is resistant to DCK. Thus, it is possible that HIV-1 RT is the target of DCK. To test this possibility, DCK-resistant viruses were selected in the presence of DCK. Our results indicate that a single amino acid mutation, E138K in HIV-1 RT, is sufficient to confer DCK resistance. Interestingly, a DCK derivative, 3'R,4'R-Di-O-(-)-camphanoyl-2-ethyl-2',2'-dimethyldihydropyrano[2,3-f]chromone (DCP8), is effective against HIV-1/RTMDR1. However, the DCK-escape virus carrying the E138K mutation remains resistant to DCP8. Since DCK did not inhibit the RNA-dependent DNA polymerase activity of HIV-1 RT when using poly-rA or poly-rC as template, we evaluated the effect of DCK on the DNA-dependent DNA polymerase activity of HIV-1 RT. Our results indicate that DCK can inhibit the DNA-dependent DNA polymerase activity of HIV-1 RT. In conclusion, DCK is a unique HIV-1 RT inhibitor that inhibits the DNA-dependent DNA polymerase activity. In contrast, DCK did not significantly affect the RNA-dependent DNA polymerase activity when poly-rA or poly-rC was used as templates. An E138K mutation in the non-nucleoside RT inhibitors (NNRTIs) binding pocket of HIV-1 RT confers resistance to DCK and its chromone derivative, DCP8.

Animals↗
PubMedDate not supplied

[Study of reverse transcriptase selectivity. Substrate properties of new 2',3'-unsaturated acyclic nucleotide analogs].

A new series of nucleotide analogs, (Z)-pyrophosphoryl (phosphonyloxymethyl) but-2-enyl derivatives of pyrimidines and purines, was synthesized. Their substrate and inhibitory properties toward some DNA polymerases and reverse transcriptases were evaluated. They were shown to be selective inhibitors of HIV reverse transcriptase. The structure-substrate properties relationships for nucleotide analogs were discussed.

Base Sequence↗
PubMed1999

Michellamine alkaloids inhibit protein kinase C.

Michellamines A, B, and C have shown antiviral activity against HIV-1 and HIV-2 in cell culture. They act in a complex manner by at least two reported antiviral mechanisms, inhibition of HIV reverse transcriptase and inhibition of HIV-induced cellular fusion. On the basis of their structural similarity to other protein kinase C (PKC) inhibitors, we have investigated another possible mechanism-inhibition of PKC. The michellamines were found to inhibit rat brain PKC with IC50 values in the 15-35 microM range. Michellamine B was a noncompetitive PKC inhibitor with respect to ATP with a Ki value of 4-6 microM, whereas mixed-type inhibition was observed when the peptide concentration was varied. Michellamine B inhibited the kinase domain of PKC similarly. These results indicate that the michellamines bind to the PKC kinase domain and not its regulatory domain. Molecular modeling showed that all three michellamines can bind in the active site cleft of the PKC kinase domain, to block both the ATP and the peptide substrate subsites.

Adenosine Triphosphate↗