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PubMed2004

QSAR study on some anti-HIV HEPT analogues using physicochemical and topological parameters.

A Quantitative Structure-Activity Relationship (QSAR) study has been carried out using topological indices, physicochemical and indicator parameters on a series of HEPT analogues for their HIV reverse transcriptase inhibitory activity. Correlation analysis and multiple linear regression (MLR) method were used to find out the best QSAR model. The results clearly explained that decreased hydrophobicity of substituents at R(1) and R(2) positions are favorable for the activity and presence of di-substitution at phenyl ring as well as i-Pr at R(1) position have detrimental effect but presence of OH group at R(2) position increases the activity. The atoms numbered as 1, 2, 3, 4, 5, 6 and 11 may constitute pharmacophore moiety of the HEPT analogues for their anti-HIV activity. Leave one out (LOO-) cross validation method was used to judge the predictive power of final equations. From the study one can propose that atom or fragmental level descriptors are more useful to interpret drug-receptor interactions in these analogues. The potentiality of ETSA index to recognize these atoms (Pharmacophoric atoms) was established.

Anti-HIV Agents↗
PubMed2004

PCR amplification of DNA containing non-standard base pairs by variants of reverse transcriptase from Human Immunodeficiency Virus-1.

As the next step towards generating a synthetic biology from artificial genetic information systems, we have examined variants of HIV reverse transcriptase (RT) for their ability to synthesize duplex DNA incorporating the non-standard base pair between 2,4-diaminopyrimidine (pyDAD), a pyrimidine presenting a hydrogen bond 'donor-acceptor-donor' pattern to the complementary base, and xanthine (puADA), a purine presenting a hydrogen bond 'acceptor-donor-acceptor' pattern. This base pair fits the Watson-Crick geometry, but is joined by a pattern of hydrogen bond donor and acceptor groups different from those joining the GC and AT pairs. A variant of HIV-RT where Tyr 188 is replaced by Leu, has emerged from experiments where HIV was challenged to grow in the presence of drugs targeted against the RT, such as L-697639, TIBO and nevirapine. These drugs bind at a site near, but not in, the active site. This variant accepts the pyDAD-puADA base pair significantly better than wild type HIV-RT, and we used this as a starting point. A second mutation, E478Q, was introduced into the Y188L variant, in the event that the residual nuclease activity observed is due to the RT, and not a contaminant. The doubly mutated RT incorporated the non-standard pair with sufficient fidelity that the variant could be used to amplify oligonucleotides containing pyDAD and puADA through several rounds of a polymerase chain reaction (PCR) without losing the non-standard base pair. This is the first time where DNA containing non-standard base pairs with alternative hydrogen bonding patterns has been amplified by a full PCR. This work also illustrates a research strategy that combines in clinico pre-evolution of proteins followed by rational design to obtain an enzyme that meets a particular technological specification.

Amino Acid Substitution↗
PubMed1992

Fidelity of the RNA-dependent DNA synthesis exhibited by the reverse transcriptases of human immunodeficiency virus types 1 and 2 and of murine leukemia virus: mispair extension frequencies.

Human immunodeficiency viruses type 1 and 2 (HIV-1 and HIV-2, respectively) exhibit extensive genetic variations. It was postulated that much of this genetic variability stems from the low fidelity of the reverse transcription step. Both HIV reverse transcriptases (RTs) were shown to be particularly error-prone during the in vitro DNA-dependent DNA synthesis relative to other retroviral RTs. Extension of mismatched 3'-termini of the primer DNA was shown to be a major determinant in the infidelity of HIV RTs. However, reverse transcriptases generally exhibit dual template specificities. Therefore, we determined in the current study the fidelity of RNA-dependent DNA synthesis catalyzed in vitro by the RTs of HIV-1 and HIV-2 in comparison with that of murine leukemia virus (MLV) RT. Consequently, we examined the ability of these enzymes to extend preformed 3'-terminal A.A, A.C, and A.G mispairs by quantitating the amount and length of extended primers in a primer extension assay using ribosomal RNA as a template. The results demonstrate that the three RTs studied exhibited efficient extensions from 3'-terminal mispairs with a specificity of A.C greater than A.A greater than A.G. Nevertheless, the HIV RTs are qualitatively as well as quantitatively more error-prone than MLV RT. The mispair extension efficiency appears to be affected mainly by the increase of apparent Km values, rather than by the change in Vmax values.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed2004

Phenotypic susceptibility to nonnucleoside inhibitors of virion-associated reverse transcriptase from different HIV types and groups.

OBJECTIVES: To evaluate a phenotype assay based on plasma reverse transcriptase (RT) to assess HIV susceptibility to nonnucleoside RT inhibitors (NNRTIs). To compare RT-based phenotype with recombinant virus assay (RVA) phenotype- and genotype-based analysis. To assess group O and HIV-2 susceptibility to NNRTIs in correlation with genotype polymorphisms. METHODS: RT activity was quantified and its susceptibility to efavirenz, nevirapine, and delavirdine measured as drug concentration resulting in 50% inhibition. RT phenotype was compared with genotype analysis. Eighteen plasma samples from 14 group M- and culture supernatants from 4 group M-, 9 group O-, and 7 HIV-2-infected patients were investigated. RT-based and RVA-based phenotypes were compared for identical plasma from 9 group M-infected patients. RESULTS: RT-based and RVA-based phenotypes were in complete agreement. RT-based phenotype- and genotype-predicted susceptibility were concordant for all but 1 group M samples. One plasma showed susceptibility to 3 NNRTIs by phenotypes, despite the presence of 101E and 106I/V residues. The HIV-2 RTs were totally resistant to the NNRTIs tested. Among HIV-1 group O, 6 were totally resistant to NNRTIs independently of the presence of the 181C mutation and 3 were susceptible to some NNRTIs. CONCLUSION: Plasma RT-based phenotype could be useful as a simple alternative for monitoring resistance to NNRTIs. This assay is suitable for highly divergent strains. It would be particularly useful for large epidemiologic survey of the natural HIV polymorphism and the potential impact in emergence of drug resistance, particularly to nevirapine, widely used to prevent mother-to-child transmission.

Anti-HIV Agents↗
PubMed1994

Mechanism of enzyme inhibition mediated by anti-reverse transcriptase antibodies from HIV type 1-infected individuals.

We have examined the mechanisms of reverse transcriptase (RT) inhibition mediated by anti-RT antibodies, isolated by affinity chromatography, from four HIV-1-positive individuals. In kinetics assays, anti-RT immunoglobulin (Ig) obtained from three of the sera mediated a noncompetitive type of inhibition against template primer; two of these three also mediated noncompetitive inhibition with respect to deoxyribonucleoside triphosphate. Such inhibition did not require that the Ig be preincubated with RT prior to the addition of reaction components. In contrast, a more complicated pattern of inhibition was exhibited by anti-RT Ig from the fourth serum. Preincubation of this Ig with enzyme markedly enhanced the inhibition. The results demonstrate that the specificities of RT-inhibiting antibodies vary among HIV-1-infected individuals, but that one prevalent mechanism of inhibition involves interactions with epitopes outside of the enzyme active site.

Antibody Specificity↗
PubMed2001

Molecular anatomy and pathophysiologic implications of drug resistance in hepatitis B virus infection.

Synthesis of the hepatitis B virus (HBV) DNA genome occurs within the viral nucleocapsid in a mechanistically ordered fashion. The nucleocapsid contains small pores that permit influx of nucleotide triphosphates and metabolites of nucleoside analogues such as lamivudine for DNA synthesis. Lamivudine is a potent inhibitor of HBV and human immunodeficiency virus (HIV) reverse transcriptases, but substitutions of isoleucine or valine for methionine within the tyrosine-methionine-aspartate-aspartate (YMDD) motif are associated with virologic and clinical resistance to lamivudine therapy. Under lamivudine selection pressure, the high viral production rate and the low fidelity viral polymerase contribute to frequent development of the YMDD mutants. However, the pattern and dynamics of emergence of the mutant viruses over the wild-type virus are determined by multiple factors including replication efficiency, host immune response, and availability of replication space. Structural modeling of HIV reverse transcriptase has permitted key insights into the molecular basis of lamivudine resistance of HBV based on evolutionary relatedness of HIV and HBV. The side groups of isoleucine and valine of the YMDD mutants sterically prevent lamivudine from appropriately configuring into the nucleotide binding site of the reverse transcriptase. Aminotransferase flares are associated with lamivudine therapy and may signify clinical resistance with emergence of YMDD mutants. They may also herald the recovery phase with seroconversion and viral clearance. Reconstitution of the endogenous anti-HBV immune response may be equally important in the control of viral replication by lamivudine and other nucleoside analogues.

Drug Resistance, Microbial↗
PubMed2003

Predictors of the virological response to a change in the antiretroviral treatment regimen in HIV-1-infected patients enrolled in a randomized trial comparing genotyping, phenotyping and standard of care (Narval trial, ANRS 088).

OBJECTIVE: To identify predictors of the virological response to antiretroviral therapy in patients in whom initial therapy has failed. METHODS: The Narval trial was designed to compare phenotyping, genotyping and standard of care for the choice of antiretroviral therapy in patients in whom a protease inhibitor (PI)-containing regimen had failed. Virological success was defined as viral load below 200 copies/ml at week 12. Baseline variables including demographic, clinical and biological characteristics, HIV reverse transcriptase and protease mutations, the randomization arm, the drugs prescribed, as well as adherence to treatment and plasma concentrations of PIs and non-nucleoside reverse transcriptase inhibitors (NNRTIs) at week 12 were tested in the model. Variables that were significantly associated with virological success in univariate analysis were included in a logistic regression model. RESULTS: Five-hundred-and-forty-one patients were randomized. Virological success at week 12 was obtained in 200 patients. In multivariate analysis, the following factors were significantly associated with virological success: prescription of efavirenz to NNRTI-naive patients (OR=4.37; 95% CI: 2.76-6.90), randomization to the genotyping arm (OR=2.13, 1.20-3.79), prescription of lamivudine (OR=1.69, 1.01-2.83) and prescription of abacavir to abacavir-naive patients (OR=1.66, 1.02-2.72). Factors significantly associated with virological failure were prescription of nelfinavir (OR=0.30, 0.13-0.68), a high baseline viral load (OR=0.37, 0.28-0.50), the presence of at least five PI mutations (OR=0.42, 0.26-0.66), the presence of at least three thymidine analogue mutations (OR=0.61, 0.39-0.97) and at least 30 months of prior PI exposure (OR=0.64, 0.41-0.99). CONCLUSIONS: These results confirm that among heavily pretreated patients, prescription of efavirenz to NNRTI-naive patients is associated with a good virological response, while a high baseline viral load, a large number of PI mutations and nelfinavir prescription at baseline are associated with a poor virological response. Genotyping was found to be beneficial, while this was not the case for phenotyping. This work was presented at the XI International HIV Drug Resistance Workshop, Sevilla, Spain, July 3-6 2002 (Abstract N(o)133); and at the XIV International Conference on AIDS, Barcelona, Spain, July 7-11 2002 (Abstract N(o)ThOrB138).

Adult↗
PubMed1988

Factors determining the activity of 2',3'-dideoxynucleosides in suppressing human immunodeficiency virus in vitro.

Mitsuya and Broder [Proc. Natl. Acad. Sci. USA 83:1911-1915 (1986)] demonstrated that every purine (adenosine, guanosine, and inosine) and pyrimidine (cytidine and thymidine) nucleoside containing the 2',3'-dideoxyribose configuration, when evaluated against human immunodeficiency virus (HIV) in vitro, significantly suppressed both the infectivity and the cytopathic effect of the virus, with 2',3'-dideoxycytidine (ddCyd) being the most potent of the series (total antiviral protection at 0.5-1.0 microM). We have compared three factors likely to be of significance in determining the pharmacological activity of these compounds, i.e., (i) their abilities to influence pool sizes of physiological deoxynucleoside-5'-triphosphates, (ii) their capacity to generate the corresponding 2',3'-dideoxynucleoside-5'-triphosphates, and (iii) the effectiveness of these nucleoside-5'-triphosphates as inhibitors of HIV reverse transcriptase. In MOLT-4 cells (a human T cell line), ddCyd was the compound most efficiently converted to its 5'-triphosphate, whereas 2',3'-dideoxyguanosine and 2',3'-dideoxythymidine were the compounds least efficiently converted, generating levels of their corresponding 5'-triphosphates less than 0.1% of that seen with ddCyd when these nucleosides were compared on an equimolar basis (5 microM). The 3'-azido analogue of 2',3'-dideoxythymidine fell intermediate between these two extremes. As inhibitors of HIV reverse transcriptase, however, all the 5'-triphosphates, with the exception of 2',3'-dideoxyinosine-5'-triphosphate, fell within a narrow range of activity (Ki, 0.10-0.26 microM), affinities some 40-60 fold greater than those of the corresponding physiological 2'-deoxynucleoside-5'-triphosphates. Significant alterations in pool sizes of physiological 2'-deoxynucleoside-5'-triphosphates were not observed at pharmacologically effective drug levels. The relative ability of 2',3'-dideoxynucleosides to generate 5'-triphosphates intracellularly thus correlates much more closely than do the other two factors examined, in capacity to block HIV replication. These studies support the conclusion that, for purposes of design of new compounds of this general class, factors influencing efficiency of nucleotide formation and degradation (e.g., membrane transport mechanisms, affinities for nucleoside kinases and for nucleotide kinases and phosphatases) may be of equal or even greater importance than differences in the relative abilities of the resultant 2',3'-dideoxynucleoside-5'-triphosphates to inhibit the viral reverse transcriptase.

Antiviral Agents↗
PubMed1994

Human immunodeficiency virus 1 (HIV-1)-specific reverse transcriptase (RT) inhibitors may suppress the replication of specific drug-resistant (E138K)RT HIV-1 mutants or select for highly resistant (Y181C-->C181I)RT HIV-1 mutants.

Mutant HIV-1 that expresses a Glu138-->Lys substitution in its RT [(E138K)RT] is resistant to the HIV-1-specific RT inhibitor 2',5'-bis-O-(tert-butyldimethylsilyl)-3'-spiro-5"-(4"-amino-1",2"- oxathiole-2",2"-dioxide)pyrimidine (TSAO). However, cell cultures infected with this mutant were completely protected against virus-mediated destruction by micromolar concentrations of the HIV-1-specific RT inhibitors tetrahydroimidazo[4,5,1-jk][1,4]benzodiazepin-2(1H)-one and -thione (TIBO), nevirapine, and bis(heteroaryl)piperazine (BHAP). In contrast, cells infected with a virus mutant that expresses a Tyr181-->Cys substitution in its RT [(Y181C)RT] were not protected by nevirapine and TIBO and were only temporarily protected by BHAP. HIV-1 mutant that emerged under the latter conditions contained a Cys181-->Ile substitution in their RT [(LC181I)RT]. This mutant proved highly resistant to all HIV-1-specific RT inhibitors tested, except for several 1-(2-hydroxyethoxymethyl)-6-(phenylthio)thymine (HEPT) derivatives. When recombinant (C181I)RT was evaluated for susceptibility to the HIV-1-specific RT inhibitors, it was resistant to all inhibitors except the HEPT compounds. Since a (Y181F)RT HIV mutant strain was isolated from cells infected with (Y181C)RT HIV-1 and treated with BHAP, we postulate that the Ile codon was derived from a Cys-->Phe transversion mutation (TGT-->TTT), followed by a Phe-->Ile transversion mutation (TTT-->ATT).

Amino Acid Sequence↗
PubMed2003

Heterogeneous clearance rates of long-lived lymphocytes infected with HIV: intrinsic stability predicts lifelong persistence.

Viral replication and latently infected cellular reservoirs persist in HIV-infected patients achieving undetectable plasma virus levels with potent antiretroviral therapy. We exploited a predictable drug resistance mutation in the HIV reverse transcriptase to label and track cells infected during defined intervals of treatment and to identify cells replenished by ongoing replication. Decay rates of subsets of latently HIV-infected cells paradoxically decreased with time since establishment, reflecting heterogeneous lymphocyte activation and clearance. Residual low-level replication can replenish cellular reservoirs; however, it does not account for prolonged clearance rates in patients without detectable viremia. In patients receiving potent antiretroviral therapy, the latent pool has a heterogeneous and dynamic composition that comprises a progressively increasing proportion of stable lymphocytes. Eradication will not be achieved with complete inhibition of viral replication alone.

Antiretroviral Therapy, Highly Active↗
PubMed2003

A conformationally locked analogue of the anti-HIV agent stavudine. An important correlation between pseudorotation and maximum amplitude.

The synthesis and biological evaluation of a bicyclo[3.1.0]hexene nucleoside designed as a conformational mimic of the anti-HIV agent stavudine (1, D4T) is described. The unsaturated methanocarbocyclic pseudosugar of N-MCD4T (2) was constructed from an iodo-substituted precursor by a DBU-catalyzed olefination reaction. Mitsunobu coupling with N(3)-benzoylthymine afforded the desired target after deprotection. Both D4T and N-MCD4T are in the North (N) hemisphere of the pseudorotational cycle but 70 degrees away from a perfect N (P = 0 degrees ) conformation toward the East and West hemispheres, respectively. Despite this large difference, the double bond reduces the puckering amplitude (nu(max)) of N-MCD4T to 6.81 degrees, and the superposition of both structures showed a RMS deviation of only 0.039 A. The combined structural analysis of P and nu(max) shows that while the value of P may differ substantially, the low nu(max) resolves the differences and becomes the dominant pseudorotational parameter. N-MCD4T is active against HIV-1 and HIV-2 in CEM, MT-2, and MT-4 cells, and while it is somewhat less potent than D4T, it also appears to be less toxic. The triphosphate (N-MCD4TTP) inhibits HIV reverse transcriptase with a 10-fold higher IC(50) than D4TTP. By virtue of its carbocyclic nature, N-MCD4T (2) is a more robust molecule stable to conditions that would cleave D4T.

Animals↗
PubMed2006

Virologic and enzymatic studies revealing the mechanism of K65R- and Q151M-associated HIV-1 drug resistance towards emtricitabine and lamivudine.

Emtricitabine (FTC) and lamivudine (3TC) are deoxycytidine analogues with potent and selective inhibition of human immunodeficiency virus (HIV) and hepatitis B virus (HBV) replication. The K65R mutation in the HIV reverse transcriptase (RT) confers reduced susceptibility to 3TC, ddC, ddI, abacavir, and tenofovir in vitro. The Q151M mutation confers reduced susceptibility to many of the approved anti-HIV nucleoside analogues with the exception of 3TC and tenofovir. The double mutation K65R/Q151M has been shown to be more resistant to many NRTIs than either of the single mutations alone. In this study, we measured the antiviral activity of FTC and 3TC against HIV-1 containing K65R, Q151M, and K65R/Q151M mutations. We also studied the steady-state kinetic properties for the inhibition of dCTP incorporation by FTC 5'-triphosphate (TP) and 3TC-TP In addition, we measured the incorporation of dCTP, FTC-TP, and 3TC-TP into a random sequence DNA/DNA primer/template by the HIV-1 RTs using pre-steady-state kinetic analysis. Finally, we studied the incorporation of these deoxycytidine analogues into a HIV-1 genomic DNA/DNA primer/template by K65R HIV-1 RT to address certain concerns associated with DNA sequence specificity. Overall, this study demonstrated that K65R and K65R/Q151M related drug resistance to FTC and 3TC was mainly due to a significant decrease in the rate of incorporation. There was little to no effect on the binding affinities of the mutant HIV-1 RTs for the deoxycytidine analogues. The Q151M mutation remained sensitive to both FTC and 3TC in both cell culture and enzymatic assays. At a molecular level, FTC-TP was incorporated at least as efficiently as 3TC-TP for all of the HIV-1 RT and primer/templates tested.

Amino Acid Substitution↗
PubMed1999

Synthesis and evaluation of analogs of Efavirenz (SUSTIVA) as HIV-1 reverse transcriptase inhibitors.

Efavirenz (SUSTIVA) is a potent non-nucleoside reverse transcriptase inhibitor. Due to the observation of breakthrough mutations of the reverse transcriptase enzyme during Efavirenz therapy, we sought to develop an optimized second generation series. To that end, SAR of the substituents on the aromatic ring was undertaken and the results are summarized here. The 5,6-difluoro (4f) and the 6-methoxy (4m) substituted benzoxazinones were determined to be equipotent, and as a result such substitution patterns will be incorporated in second generation scaffolds.

Alkynes↗
PubMed2003

Modest decreases in NNRTI susceptibility do not influence virological outcome in patients receiving initial NNRTI-containing triple therapy.

OBJECTIVE: To assess the prevalence of modest (< 10-fold) decreases in baseline non-nucleoside reverse transcriptase inhibitor (NNRTI) susceptibility and their impact on virological response to NNRTI-containing triple therapy in drug-naive individuals. METHODS: Baseline HIV resistance phenotype, genotype and response to therapy were examined retrospectively for all antiretroviral-naive individuals initiating therapy with two nucleoside analogues and an NNRTI in British Columbia, Canada, between 05/1997 and 08/1999 (n = 279), followed until July 31 2001. Time to viral suppression (first of at least two consecutive plasma viral loads < 400 copies HIV RNA copies/ml) and viral rebound (to > or = 400 copies/ml after first pVL < 400 copies HIV RNA copies/ml), were estimated by Kaplan-Meier methods. Multivariate analyses were performed using Cox proportional hazards regression. RESULTS: Nevirapine was the most commonly prescribed NNRTI (96%). Four- to 10-fold decreased susceptibility to NNRTIs was observed in > 30% of untreated individuals at baseline, an observation strongly driven by decreased susceptibility to delavirdine (22.4%). A > 10-fold decrease in susceptibility to any NNRTI was observed only rarely (< 2%). There was no association between four- and 10-fold decreased baseline susceptibility to NNRTIs and virological outcome (P > 0.05). In multivariate analyses, the strongest predictors of poor virological response to NNRTI-based therapy were baseline plasma viral load and the proportion of time on therapy in the first year of follow-up. There was no relationship between the presence of previously reported mutations associated with decreased NNRTI susceptibility (at codons 135 and 283 in HIV reverse transcriptase) and virological response. CONCLUSIONS: These data suggest that the clinically significant level of resistance to NNRTIs, particularly nevirapine, in drug-naive individuals is likely greater than four- to 10-fold.

Adult↗
PubMed1994

Didanosine resistance in HIV-infected patients switched from zidovudine to didanosine monotherapy.

OBJECTIVE: To determine the frequency and pattern of development of specific drug resistance mutations for human immunodeficiency virus (HIV) reverse transcriptase in patients switched from zidovudine to didanosine therapy and to examine the relation of the didanosine resistance mutation at codon 74 of the HIV reverse-transcriptase gene to CD4+ T-cell changes and virus burden. DESIGN: Retrospective analysis of all patients enrolled at Stanford University in protocols where patients were switched from zidovudine to didanosine monotherapy. SETTING: A university hospital. PATIENTS: 64 patients infected with HIV who were switched from zidovudine to didanosine monotherapy. Patients had the acquired immunodeficiency syndrome (AIDS), AIDS-related complex, or were asymptomatic (mean [+/- SD] starting CD4+ T-cell count of 129 +/- 88 cells/mm3). MEASUREMENTS: Serial serum specimens were tested for the didanosine resistance mutation at codon 74 of the HIV reverse-transcriptase gene and for a zidovudine resistance mutation at codon 215 using selective polymerase chain reactions (PCR). Serum HIV RNA levels were determined by quantitative PCR. CD4+ T-cell counts were determined at serial time points. RESULTS: By 24 weeks of didanosine therapy, the proportion of patients with the didanosine resistance mutation at codon 74 increased from 0% to 56% (36 of 64). In contrast, the proportion of patients with the zidovudine resistance mutation at codon 215 decreased from 84% at the start to 59% after 24 weeks of didanosine therapy (a 25% decrease, 95% lower CI, 15%; P < 0.0001). Patients who developed the codon 74 mutation had a greater decrease in CD4+ T cells after the development of the mutation than did patients without the mutation (P < 0.001). In addition, after 24 weeks of didanosine, patients who developed the codon 74 mutation had a greater serum HIV RNA burden than patients who remained wild type (did not have the mutation) at codon 74 (225,000 compared with 82,400 HIV RNA copies/mL serum; P = 0.01). CONCLUSIONS: Among patients infected with HIV who had advanced disease and were switched from zidovudine to didanosine therapy, more than one half developed the didanosine resistance mutation at codon 74 by 24 weeks of didanosine therapy. Patients who developed the codon 74 mutation had a greater decline in CD4+ T cells after the development of the mutation and had a greater serum virus burden than did patients without the codon 74 mutation.

CD4-Positive T-Lymphocytes↗
PubMed2004

Synthesis of 5-alkynylated d4T analogues as potential HIV-1 reverse transcriptase inhibitors.

A series of 2',3'-didehydro-2',3'-dideoxynucleosides substituted with an alkynylhydroxy- (6, 7, 12 and 13) and alkynylamino- (20) groups at the C-5 position were synthesized. All these five target modified nucleosides were tested for anti-human immunodeficiency virus type 1 activity in CEM-SS and MT-4 cells and unfortunately displayed no improvement in antiviral activity.

Alkynes↗
PubMed2003

Viable Mycobacterium avium is required for the majority of human immunodeficiency virus-induced upregulation in monocytoid cells.

The Mycobacterium avium complex (MAC), an intracellular pathogen of cells of the macrophage lineage, often clinically coexists with human immunodeficiency virus type 1 (HIV). It was shown previously that coinfection of the monocytoid cell line U937 with HIV and MAC results in the enhancement of HIV replication. To determine whether MAC-mediated HIV upregulation is due to the exposure of intact organisms to HIV-infected cells or if actual infection with viable organisms is required for the effect, U937 cells were coinfected simultaneously with HIV and live or heat-killed MAC. Live MAC (infection) consistently increased HIV reverse transcriptase (RT) activity by more than 3-fold. Heat-killed MAC, however, failed to enhance RT activity significantly. Further investigation showed that infection of U38 cells [a U937-derived cell line containing regions of the HIV-1 long terminal repeat (LTR) linked to chloramphenicol acetyl transferase (CAT)] with live or heat-killed MAC resulted in a similar enhancement of HIV LTR-CAT transcription. In addition, transient transfection of U937 cells with a full-length wild-type HIV LTR-CAT construct revealed that heat-killed MAC stimulated LTR-mediated CAT activity to levels comparable to those of viable MAC. Finally, both live and heat-killed MAC mediated similar enhancement of NF-kappa B DNA-binding activity. Taken together, these observations confirm previous findings that MAC-induced NF-kappa B-dependent LTR-CAT activity is not a major factor in upregulating HIV expression in a coinfection model. It also indicates that MAC infection plays a significant role in the enhancement of HIV replication and suggests that viable MAC either contains or induces the production of an as-yet-unidentified factor(s) that mediates the enhancement of HIV replication.

Electrophoretic Mobility Shift Assay↗