Biomedical subjects

Gilda Tachedjian

Publications and source records attributed to Gilda Tachedjian.

11 recordsLinked to original sources

PubMed2006

Potent nonnucleoside reverse transcriptase inhibitors target HIV-1 Gag-Pol.

Nonnucleoside reverse transcriptase inhibitors (NNRTIs) target HIV-1 reverse transcriptase (RT) by binding to a pocket in RT that is close to, but distinct, from the DNA polymerase active site and prevent the synthesis of viral cDNA. NNRTIs, in particular, those that are potent inhibitors of RT polymerase activity, can also act as chemical enhancers of the enzyme's inter-subunit interactions. However, the consequences of this chemical enhancement effect on HIV-1 replication are not understood. Here, we show that the potent NNRTIs efavirenz, TMC120, and TMC125, but not nevirapine or delavirdine, inhibit the late stages of HIV-1 replication. These potent NNRTIs enhanced the intracellular processing of Gag and Gag-Pol polyproteins, and this was associated with a decrease in viral particle production from HIV-1-transfected cells. The increased polyprotein processing is consistent with premature activation of the HIV-1 protease by NNRTI-enhanced Gag-Pol multimerization through the embedded RT sequence. These findings support the view that Gag-Pol multimerization is an important step in viral assembly and demonstrate that regulation of Gag-Pol/Gag-Pol interactions is a novel target for small molecule inhibitors of HIV-1 production. Furthermore, these drugs can serve as useful probes to further understand processes involved in HIV-1 particle assembly and maturation.

Alkynes↗

PubMed2005

Efavirenz enhances the proteolytic processing of an HIV-1 pol polyprotein precursor and reverse transcriptase homodimer formation.

The non-nucleoside reverse transcriptase inhibitor, efavirenz (EFV), is a potent enhancer of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) p66/p51 heterodimerization. While the mechanism of RT heterodimer formation in HIV-1 infected cells is not completely understood, it has been speculated that Gag-Pol/Gag-Pol and/or RT homodimer interactions may represent important intermediates in the pathway. To elucidate whether EFV impacts on these interactions, we have evaluated the effects of this drug on RT homodimer interactions and HIV-1 Gag-Pol processing. EFV, but not nevirapine, significantly enhanced RT p66/p66 and p51/p51 homodimer interactions and accelerated the proteolytic cleavage of a model HIV-1 Pol polyprotein precursor expressed in bacteria. These data suggest that potent mediators of RT dimerization might interfere with the late-stages of viral replication.

Alkynes↗

PubMed2005

Analysis of the contribution of reverse transcriptase and integrase proteins to retroviral RNA dimer conformation.

All retroviruses contain two copies of genomic RNA that are linked noncovalently. The dimeric RNA of human immunodeficiency virus type 1 (HIV-1) undergoes rearrangement during virion maturation, whereby the dimeric RNA genome assumes a more stable conformation. Previously, we have shown that the packaging of the HIV-1 polymerase (Pol) proteins reverse transcriptase (RT) and integrase (IN) is essential for the generation of the mature RNA dimer conformation. Analysis of HIV-1 mutants that are defective in processing of Pol showed that these mutant virions contained altered dimeric RNA conformation, indicating that the mature RNA dimer conformation in HIV-1 requires the correct proteolytic processing of Pol. The HIV-1 Pol proteins are multimeric in their mature enzymatically active forms; RT forms a heterodimer, and IN appears to form a homotetramer. Using RT and IN multimerization defective mutants, we have found that dimeric RNA from these mutant virions has the same stability and conformation as wild-type RNA dimers, showing that the mature enzymatically active RT and IN proteins are dispensable for the generation of mature RNA dimer conformation. This also indicated that formation of the mature RNA dimer structure occurs prior to RT or IN maturation. We have also investigated the requirement of Pol for RNA dimerization in both Mason-Pfizer monkey virus (M-PMV) and Moloney murine leukemia virus (MoMuLV) and found that in contrast to HIV-1, Pol is dispensable for RNA dimer maturation in M-PMV and MoMuLV, demonstrating that the requirement of Pol in retroviral RNA dimer maturation is not conserved among all retroviruses.

Dimerization↗

PubMed2005

Mutations that abrogate human immunodeficiency virus type 1 reverse transcriptase dimerization affect maturation of the reverse transcriptase heterodimer.

The specific impact of mutations that abrogate human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) dimerization on virus replication is not known, as mutations shown previously to inhibit RT dimerization also impact Gag-Pol stability, resulting in pleiotropic effects on HIV-1 replication. We have previously characterized mutations at codon 401 in the HIV-1 RT tryptophan repeat motif that abrogate RT dimerization in vitro, leading to a loss in polymerase activity. The introduction of the RT dimerization-inhibiting mutations W401L and W401A into HIV-1 resulted in the formation of noninfectious viruses with reduced levels of both virion-associated and intracellular RT activity compared to the wild-type virus and the W401F mutant, which does not inhibit RT dimerization in vitro. Steady-state levels of the p66 and p51 RT subunits in viral lysates of the W401L and W401A mutants were reduced, but no significant decrease in Gag-Pol was observed compared to the wild type. In contrast, there was a decrease in processing of p66 to p51 in cell lysates for the dimerization-defective mutants compared to the wild type. The treatment of transfected cells with indinavir suggested that the HIV-1 protease contributed to the degradation of virion-associated RT subunits. These data demonstrate that mutations near the RT dimer interface that abrogate RT dimerization in vitro result in the production of replication-impaired viruses without detectable effects on Gag-Pol stability or virion incorporation. The inhibition of RT activity is most likely due to a defect in RT maturation, suggesting that RT dimerization represents a valid drug target for chemotherapeutic intervention.

Codon↗

PubMed2005

Antiretroviral compounds: mechanisms underlying failure of HAART to eradicate HIV-1.

During the past decade, combined highly active antiretroviral therapy (HAART) consisting of the nucleoside, non-nucleoside and protease inhibitors has improved the outlook for HIV-infected individuals. However, despite the clinical improvement associated with HAART, current antiviral drug regimens are not able to eradicate HIV due to the persistence of virus in cellular reservoirs (predominantly long-lived memory CD4+ T cells and cells of the macrophage lineage) and anatomical sanctuary sites (brain and possibly testis). Detailed knowledge of viral reservoirs is essential for the effective design of therapeutic eradication strategies such as immunostimulation of virus-persistent reservoirs and better penetration of antiretroviral drugs into sanctuary sites. The recent therapeutic approaches undertaken thus far, including immune activation, intensification protocols combined with HAART, antiretroviral treatment during seroconversion, structured treatment interruptions, activation of latent infection or targeted killing of viral reservoirs have failed to completely eradicate the virus. This review provides an evaluation of the current HAART regimens exploring the reasons for their inability to eradicate HIV from cellular reservoirs and anatomical sanctuary sites. We also provide examples of therapeutic strategies that aim to eradicate the virus, flush out reservoirs and increase antiretroviral drug concentration in these cells and tissue compartments.

Antiretroviral Therapy, Highly Active↗

PubMed2005

The packaging and maturation of the HIV-1 Pol proteins.

The Pol protein of human immunodeficiency virus type 1 (HIV-1) harbours the viral enzymes critical for viral replication; protease (PR), reverse transcriptase (RT), and integrase (IN). PR, RT and IN are not functional in their monomeric forms and must come together as either dimers (PR), heterodimers (RT) or tetramers (IN) to be catalytically active. Our knowledge of the tertiary structures of the functional enzymes is well advanced, and substantial progress has recently been made towards understanding the precise steps leading from Pol protein synthesis through viral assembly to the release of active viral enzymes. This review will summarise our current understanding of how the Pol proteins, which are initially expressed as a Gag-Pol fusion product, are packaged into the assembling virion and discuss the maturation process that results in the release of the viral enzymes in their active forms. Our discussion will focus on the relationship between structure and function for each of the viral enzymes. This review will also provide an overview of the current status of inhibitors against the HIV-1 Pol proteins. Effective inhibitors of PR and RT are well established and we will discuss the next generation inhibitors of these enzymes as well recent investigations that have highlighted the potential of IN and RNase H as antiretroviral targets.

Anti-HIV Agents↗

PubMed2005

Evaluation of a low cost reverse transcriptase assay for plasma HIV-1 viral load monitoring.

We evaluated a low cost manual reverse transcriptase assay (ExaVir Load V.1 and V.2; Cavidi Tech AB) against commercially available HIV RNA assays that quantify viral load to assess its suitability for use in resource-constrained settings. Frozen plasma samples previously tested for RNA by RT-PCR (Roche Diagnostics) and bDNA (Bayer Diagnostics) were retested for RT activity. Text sequence obtained from HIV genotype analysis was submitted to the Stanford HIV Resistance Database V.3.9 and were examined for resistant virus. Detectable RT was present in 98% of samples (V.1; n=127) and in 95% of samples (V.2; n=69) with RNA >10,000 and >1,000 copies/ml respectively. Positive association was found between the log10 RNA copies/ml and log10 RT copies/ml equivalents variables using Pearson's correlation (V.1: r=0.89, n=189; V.2: r=0.89, n=85). The RT activity over time closely followed the trend for RNA levels in samples from 10 HIV seropositive patients with progressive disease. A strong association between RT and RNA was also found with paired samples from 19 patients taken at initiation or change of antiretroviral therapy and again within 2 months. Current (n=40) or no (n=119) exposure to efavirenz therapy had no effect on RT assay performance despite efavirenz binding tightly to the RT enzyme. Samples that demonstrated resistance to the non-nucleoside RT inhibitors (n=112) had a decrease in RT of 0.20 log10 indicating a possible decrease in RT fitness. The RT assay showed good association with current molecular assays, and V.2 is sufficiently sensitive for monitoring HIV viral load in resource-constrained settings.

Alkynes↗

PubMed2004

Virological significance, prevalence and genetic basis of hypersusceptibility to nonnucleoside reverse transcriptase inhibitors.

Nonnucleoside reverse transcriptase inhibitors (NNRTI) are used to treat HIV-infected individuals in combination with nucleoside analogues (NRTI) and protease inhibitors. Long-term treatment with antiretroviral agents results in the emergence of strains with decreased susceptibility (resistance) to the drugs and is one of the major factors in loss of drug efficacy. Conversely, there have been recent reports of HIV strains with increased susceptibility (hypersusceptibility) to NNRTIs. These isolates emerge in patients on long-term antiretroviral therapy particularly in individuals receiving NRTIs. The prevalence of NNRTI hypersusceptibility ranges between 17.5 and 50% in NRTI-treatment experienced compared to 10% in NRTI-naïve patients. There is an inverse correlation between NNRTI hypersusceptibility and phenotypic NRTI resistance and a direct correlation between the number of NRTI resistance mutations present in the HIV reverse transcriptase. Re-sensitisation of phenotypic NNRTI resistance has been reported by NRTI mutations and is not likely to be detected using genotypic resistance assays. Recent studies demonstrate that NNRTI hypersusceptible virus at baseline is likely to predict better virological outcomes in patients on NNRTI-based salvage regimens compared to patients with NNRTI susceptible virus. These studies have implications for the sequence of antiretroviral drug use where patients may benefit from NRTI therapy before the introduction of NNRTIs, however more studies are needed to examine this treatment rationale.

Drug Resistance, Viral↗

PubMed2003

Role of residues in the tryptophan repeat motif for HIV-1 reverse transcriptase dimerization.

The tryptophan repeat motif of the human immunodeficiency virus type-1 (HIV-1) reverse transcriptase (RT) is comprised of a cluster of six tryptophan residues at codons 398, 401, 402, 406, 410 and 414 that are highly conserved amongst primate lentiviral RTs. To determine the contributions of each of these residues for HIV-1 RT dimerization, we introduced changes into cloned DNA and tested the mutant subunits for their capacity to mediate heterodimerization in the yeast two-hybrid system. Changes of residue 401 to either leucine or alanine (but not phenylalanine) and residue 414 to leucine resulted in major reductions in beta-galactosidase activity produced from the reporter gene as compared to yeast expressing wild-type p66 bait and p51 prey fusions. Subunit selective mutagenesis revealed that the effect of these mutations was mediated mainly through the p66 subunit. Introduction of tryptophan mutants into the bacterial expression vector pRT6H/NB-PROT showed that RTs containing W401A or W401L substitutions (but not W401F) and W414L were defective for dimerization in vitro. Consistent with their dimerization defect, the W401A, W401L and W414L mutants were devoid of RT activity. Using the yeast two-hybrid system, we identified several second-site suppressors in p66 that restored interaction of the p66W401A bait to the p51W401A prey. The suppressors (T409I, D110G, V372A and I393M) also restored heterodimerization of bacterially expressed W401A subunits. When introduced into the W401A mutant, T409I was able to restore RT activity to 50% of the wild-type level. Examination of the RT structures revealed that K331 in p51 makes multiple hydrogen bond contacts with residues in the p66 loop spanned by W401 and W414. Consistent with this observation, the K331A RT mutant was dimerization-defective. We conclude that mutations at codons 401 and 414 in p66 impair dimerization by altering the proper positioning of structural elements in between these residues that make important contacts with p51.

Binding Sites↗

PubMed2003

The effect of NNRTIs on HIV reverse transcriptase dimerization.

Non-nucleoside reverse transcriptase inhibitors (NNRTIs) are chemically diverse compounds that allosterically inhibit HIV-1 reverse transcriptase (RT). NNRTIs are highly effective in the treatment of HIV-infected individuals when used in combination with other anti-HIV drugs. Recent studies have demonstrated that various NNRTIs can enhance, destabilize or have no effect on HIV-1 RT dimerization, suggesting that these drugs have different modes of binding to the HIV-1 RT. The strong enhancement of RT dimerization by many conventional NNRTIs such as efavirenz may contribute in part to the inhibitory activity of these drugs, while subunit destabilization by unconventional NNRTIs is thought to interfere with the RT heterodimerization-induced conformational changes that are essential for enzyme function. These studies suggest that modulation of subunit interaction by NNRTIs represents a previously unrecognized property of these RT inhibitors that could be exploited to produce more potent inhibitors of HIV-1 RT.

Anti-HIV Agents↗

PubMed2002

Modulation of the oligomeric structures of HIV-1 retroviral enzymes by synthetic peptides and small molecules.

The efficacy of antiretroviral agents approved for the treatment of HIV-1 infection is limited by the virus's ability to develop resistance. As such there is an urgent need for new ways of thinking about anti-HIV drug development, and accordingly novel viral and cellular targets critical to HIV-1 replication need to be explored and exploited. The retroviral RNA genome encodes for three enzymes essential for viral replication: HIV-1 protease (PR), HIV-1 reverse transcriptase (RT) and HIV-1 integrase (IN). The enzymatic functioning of each of these enzymes is entirely dependent on their oligomeric structures, suggesting that inhibition of subunit-subunit assembly or modulation of their quaternary structures provide alternative targets for HIV-1 inhibition. This review discusses the recent advances in the design and/or identification of synthetic peptides and small molecules that specifically target the subunit-subunit interfaces of these retroviral enzymes, resulting in the inactivation of their enzymatic functioning.

Anti-HIV Agents↗