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PubMed1995

Analysis of human immunodeficiency virus type 1 integrase mutants.

The human immunodeficiency virus type-1 (HIV-1) integrase protein (IN) mediates the insertion of linear double-stranded viral DNA into the host genome. Mutations in IN can have different effects on the virus life cycle. In this study, Gag-Pol polyprotein processing, Tat synthesis, and viral replication were investigated in integrase-defective HIV-1 mutants. In the absence of IN synthesis, the Gag-Pol polyprotein stability, packaging, and/or processing was reduced. There was limited expression of Tat observed in IN mutants, but no viral replication.

Base Sequence↗
PubMed1999

Isolation and characterization of novel human immunodeficiency virus integrase inhibitors from fungal metabolites.

We have identified a series of novel inhibitors of human immunodeficiency virus type 1 (HIV-1) integrase by randomly screening natural product extracts using an in vitro biochemical assay designed to identify inhibitors of integrase-catalysed strand transfer. Equisetin recovered from the fungus Fusarium heterosporum and a novel enantiomeric homologue of equisetin from Phoma sp. were isolated as inhibitors of HIV-1 integrase in vitro. Two additional analogues, a novel decalin derivative, integric acid, and oteromycin were also discovered to be inhibitors of integrase. Equisetin and related compounds inhibit 3' end-processing and strand transfer as well as disintegration catalysed by either the full-length enzyme or the truncated integrase core domain (amino acids 50-212). These compounds also inhibit strand transfer reactions catalysed by stable complexes assembled in vitro and integration reactions catalysed by pre-integration complexes isolated from HIV-1-infected cells. The compounds described in this report are structurally novel and mechanistically distinct from many previously described inhibitors of HIV-1 integrase. These results demonstrate the utility of using an appropriately configured assay to identify compounds that are effective post-assembly and the potential of isolating novel integrase inhibitors from complex natural product extracts.

Base Sequence↗
PubMedDate not supplied

Stimulation of human flap endonuclease 1 by human immunodeficiency virus type 1 integrase: possible role for flap endonuclease 1 in 5'-end processing of human immunodeficiency virus type 1 integration intermediates.

Human immunodeficiency virus type 1 (HIV-1) DNA integration intermediates consist of viral and host DNA segments separated by a 5-nucleotide gap adjacent to a 5'-AC unpaired dinucleotide. These short-flap (pre-repair) integration intermediates are structurally similar to DNA loci undergoing long-patch base excision repair in mammalian cells. The cellular proteins flap endonuclease 1 (FEN-1), proliferating cell nuclear antigen, replication factor C, DNA ligase I and DNA polymerase delta are required for the repair of this type of DNA lesion. The role of FEN-1 in the base excision repair pathway is to cleave 5'-unpaired flaps in forked structures so that DNA ligase can seal the single-stranded breaks that remain following gap repair. The rate of excision by FEN-1 of 5'-flaps from short- and long-flap oligonucleotide substrates that mimic pre- and post-repair HIV-1 integration intermediates, respectively, and the effect of HIV-1 integrase on these reactions were examined in the present study. Cleavage of 5'-flaps by FEN-1 in pre-repair HIV-1 integration intermediates was relatively inefficient and was further decreased 3-fold by HIV-1 integrase. The rate of removal of 5'-flaps by FEN-1 from post-repair HIV-1 integration intermediates containing relatively long (7-nucleotide) unpaired 5'-tails and short (1-nucleotide) gaps was increased 3-fold relative to that seen with pre-repair substrates and was further stimulated 5- to 10-fold by HIV-1 integrase. Overall, post-repair structures were cleaved 18 times more effectively in the presence of HIV-1 integrase than pre-repair structures. The site of cleavage was 1 or 2 nucleotides 3' of the branch point and was unaffected by HIV-1 integrase. Integrase alone had no detectable activity in removing 5'-flaps from either pre- or post-repair substrates.

DNA Repair↗
PubMed1999

Inhibitors of human immunodeficiency virus integrase.

Integration of the viral DNA into a host cell chromosome is an essential step for HIV replication and maintenance of persistent infection. Two viral factors are essential for integration: the viral DNA termini (the att sites) and IN. Accruing knowledge of the IN structure, catalytic mechanisms, and interactions with other proteins can be used to design strategies to block integration. A large number of inhibitors have been identified that can be used as leads for the development of potent and selective anti-IN drugs with antiviral activity.

Animals↗
PubMed1999

Structure-activity relationships: analogues of the dicaffeoylquinic and dicaffeoyltartaric acids as potent inhibitors of human immunodeficiency virus type 1 integrase and replication.

The dicaffeoylquinic acids (DCQAs) and dicaffeoyltartaric acids (DCTAs) are potent and selective inhibitors of human immunodeficiency virus type 1 (HIV-1) integrase. They also inhibit HIV-1 replication at nontoxic concentrations. Since integrase is an excellent target for anti-HIV therapy, structure-activity relationships were employed to synthesize compounds with: (1) improved potency against HIV-1 integrase, (2) improved anti-HIV effect in tissue culture, and (3) increased selectivity as indicated by low cellular toxicity. Thirty-four analogues of the DCTAs and DCQAs were synthesized and tested for cell toxicity, anti-HIV activity, and inhibition of HIV-1 integrase. Seventeen of the 34 analogues had potent activity against HIV-1 integrase ranging from 0. 07 to >10 microM. Seventeen analogues that were synthesized or purchased had no inhibitory activity against integrase at concentrations of 25 microM. Of the biologically active analogues, 7 of the 17 inhibited HIV replication at nontoxic concentrations. The most potent compounds were D-chicoric acid, meso-chicoric acid, bis(3,4-dihydroxydihydrocinnamoyl)-L-tartaric acid, digalloyl-L-tartaric acid, bis(3,4-dihydroxybenzoyl)-L-tartaric acid, dicaffeoylglyceric acid, and bis(3, 4-dihydroxyphenylacetyl)-L-tartaric acid. Anti-HIV activity of the active compounds in tissue culture ranged from 35 to 0.66 microM. Structure-activity relationships demonstrated that biscatechol moieties were absolutely required for inhibition of integrase, while at least one free carboxyl group was required for anti-HIV activity. These data demonstrate that analogues of the DCTAs and the DCQAs can be synthesized which have improved activity against HIV integrase.

Anti-HIV Agents↗
PubMed1996

Effects of nucleotide analogues on human immunodeficiency virus type 1 integrase.

We extended our previous study with 3'-azido-3'-deoxythymidine nucleotides [Proc. Natl. Acad. Sci. USA 91:5771-5775 (1994)] and examined the effects on human immunodeficiency virus type 1 (HIV-1) integrase of the nucleotides of three nucleoside analogues currently under evaluation in clinical trials: beta-D-2',3'-didehydro-3'-deoxythymidine, beta-D-2'-ara-fluoro-2', 3'-dideoxyadenosine, and beta-L-2',3'-dideoxy-3'-thiacytidine. Beta-D-2',3'-Didehydro-3'-deoxythymidine and beta-D-2'-ara-fluoro-2',3'-dideoxyadenosine nucleotides had IC50 values for strand transfer of 100 and 200 microM, respectively, whereas the corresponding 2',3'-dideoxynucleoside triphosphates, ddT triphosphate and ddA triphosphate, did not inhibit the integrase at 800 and 200 microM, respectively. Beta-L-2',3'-Dideoxy-3'-thiacytidine triphosphate had no effect up to 500 microM. The L-enantiomers of 5-fluoro-2',3'-dideoxycytidine monophosphate and triphosphate had IC50 values of approximately 40 microM, whereas their D-enantiomer isomers showed no inhibition at 200 microM. NAD, pyridoxal phosphate, and coumermycin A1, which exhibit no antiviral activity but are typically used to probe nucleotide binding sites, were also tested. NAD was inactive, and its etheno derivative exhibited activity at 1 mM. In contrast, pyridoxal phosphate (IC50 = 18 microM and coumermycin A1 (IC50 = 5 microM were potent inhibitors. None of the coumermycin monomeric derivatives were active integrase inhibitors. The physiological ribonucleotides ATP and GTP inhibited HIV-1 integrase at or near cellular concentrations, suggesting that they may regulate HIV-1 integrase activity in cells. In general, the active nucleotides tested inhibited binding of HIV-1 integrase to its substrate DNA an inhibited an integrase deletion mutant containing only amino acids 50-212, indicating that nucleotides bind to the enzyme catalytic core. Consisently, the choice of nucleophile in the 3'-processing reaction was blocked to the same extent regardless of the nucleotide used (water, glycerol, or the viral DNA hydroxyl) by the enzyme. These observations suggest new strategies for antiviral drug development that could be based on nucleotide analogues as inhibitors of HIV-1 integrase.

Antiviral Agents↗
PubMed2000

Developing G-quartet oligonucleotides as novel anti-HIV agents: focus on anti-HIV drug design.

Recently, a new class of oligonucleotides, forming G-quartet structures, has been developed as novel anti-HIV agents. Several critical structure-activity relationships between HIV-1 integrase and G-quartet oligonucleotides have been demonstrated. In addition the mechanism of the inhibition of HIV-1 integrase by G-quartet oligonucleotides, such as T30695 and its derivatives, has been explored. This review summarises the preliminary studies of developing G-quartet oligonucleotides as novel anti-HIV agents in several aspects including structure-activity relationship, stability-activity correlation, mechanism of HIV-1 integrase inhibition, substitution of phosphorothioates and targeting HIV-1 integrase in infected cells, which, hopefully, could help for developing a novel, efficient anti-HIV agent.

Animals↗
PubMed2006

Dimerization inhibitors of HIV-1 reverse transcriptase, protease and integrase: a single mode of inhibition for the three HIV enzymes?

The genome of human immunodeficiency virus type 1 (HIV-1) encodes 15 distinct proteins, three of which provide essential enzymatic functions: a reverse transcriptase (RT), an integrase (IN), and a protease (PR). Since these enzymes are all homodimers, pseudohomodimers or multimers, disruption of protein-protein interactions in these retroviral enzymes may constitute an alternative way to achieve HIV-1 inhibition. A growing number of dimerization inhibitors for these enzymes is being reported. This mini review summarizes some approaches that have been followed for the development of compounds that inhibit those three enzymes by interfering with the dimerization interfaces between the enzyme subunits.

Anti-HIV Agents↗
PubMed1999

Analysis of integration activity of human immunodeficiency virus type-1 integrase.

The integration activity of human immunodeficiency virus type-1 (HIV-1) integrase was characterized in vitro by using pre-processed oligonucleotide substrates. The highest level of integration activity was found at pH 6.5 to 7.0, while the endonucleolytic activity was highest at pH 7.4 to 8.0. Although the endonucleolytic and integration reactions are consecutive in retroviral integration, our result indicates that the optimal conditions of the two reactions are quite different. In addition, it is suggested that the endonucleolytic and integration steps can be separated by control of the cellular physiological state in retroviral therapy. Strong integration was detected in the presence of 0.5-10 mM Mn2+ ion, but weak integration at around 10 mM Mg2+ ion. This observation explains that the Mn2+ ion is preferred to the Mg2+ ion as a cofactor in the integration reaction. Although there was no sequence-specificity in the integration site of the target DNA, integration was found to frequently occur at particular regions of the target DNA. Furthermore, the mutant integrases such as Asp116, Ser147, and Glu152, which had been reported previously, were shown to lose integration activity completely, indicating that these residues are critically involved in catalytic action.

Base Sequence↗
PubMed1997

In vitro assays for activities of retroviral integrase.

Integration of retroviral DNA, an essential step during the retroviral life cycle, is mediated by the viral protein integrase. Simple in vitro assays for measuring integrase activities are described, including catalysis (3'-end processing, 3'-end joining, disintegration), juxtaposition of viral DNA ends, DNA binding, and target site selection. The described assays will be useful in elucidating the molecular mechanism of retroviral integration and screening for integrase inhibitors as potential anti-retroviral drugs.

Catalysis↗
PubMed2005

Evaluation of selected South African medicinal plants for inhibitory properties against human immunodeficiency virus type 1 reverse transcriptase and integrase.

Seventeen aqueous and methanol extracts from nine South African medicinal plants, ethnobotanically selected, were screened for inhibitory properties against HIV-1 reverse transcriptase (RT). Isolated compounds were additionally evaluated on HIV-1 integrase (IN). The strongest inhibition against the RNA-dependent-DNA polymerase (RDDP) activity of RT was observed with the methanol extract of the stem-bark of Peltophorum africanum Sond. (Fabaceae) (IC(50) 3.5 microg/ml), while the methanol extract of the roots of Combretum molle R.Br. ex G. Don (Combretaceae) was the most inhibitory on the ribonuclease H (RNase H) activity (IC(50) 9.7 microg/ml). The known compounds bergenin and catechin, and a red coloured gallotannin composed of meta-depside chains of gallic and protocatechuic acids esterified to a 1-O-isobutyroly-beta-D-glucopyranose core, were isolated from the methanol extract of the roots and stem-bark of Peltophorum africanum. The gallotannin inhibited the RDDP and RNase H functions of RT with IC(50) values of 6.0 and 5.0 microM, respectively, and abolished the 3'-end processing activity of IN at 100 microM. Catechin showed no effect on RT but had a moderate activity on HIV-1 IN. Bergenin was inactive on both enzymes. The aqueous and methanol extracts were non-toxic in a HeLaP4 cell line at a concentration of 400 microg/ml.

Anti-HIV Agents↗
PubMed1998

Production of uninfectious human immunodeficiency virus type 1 containing viral protein R fused to a single-chain antibody against viral integrase.

A single-chain antibody (scAb) against human immunodeficiency virus type 1 (HIV-1) integrase was expressed as a fusion protein of scAb and HIV-1 viral protein R (Vpr), together with the HIV-1 genome, in human 293T cells. The expression did not affect virion production much but markedly reduced the infectivity of progeny virions. The fusion protein was found to be incorporated into the virions. The incorporation appears to account for the reduced infectivity.

Cell Line, Transformed↗
PubMed1996

Inhibition of the human immunodeficiency virus type 1 integrase by guanosine quartet structures.

An oligonucleotide (T30177) composed entirely of deoxyguanosine and thymidine has previously been shown to fold upon itself in the presence of potassium into a highly stable four-stranded DNA structure containing two stacked deoxyguanosine quartets (G4s). T30177 also protects host cells from the cytopathic effects of human immunodeficiency virus type 1 (HIV-1). We report that this G4 oligonucleotide is the most potent inhibitor of HIV-1 integrase identified to date, with IC50 values in the nanomolar range. Both the number of quartets formed and the sequence of the loops between the quartets are important for optimal activity. T30177 binds to HIV-1 integrase without being processed and blocks the binding of the normal viral DNA substrate to the enzyme. The normal DNA substrate was not able to compete off T30177 binding to HIV-1 integrase, indicating a tight binding of G4s to the enzyme. Experiments with truncated HIV-1 integrases indicate that the N-terminal region containing a putative zinc finger is required for inhibition by T30177 and that T30177 binds better to full-length or deletion mutant integrases containing the zinc finger region than to a deletion mutant consisting of only the central catalytic domain. The N-terminal region of integrase alone is able to bind efficiently to T30177, but not the linear viral DNA substrate, in the presence of zinc. Hence, G4s represent the first class of compounds that inhibit HIV-1 integrase by interacting with the enzyme N-terminal domain. The greater inhibitory potency of T30177 in buffer containing magnesium versus manganese suggests that divalent metal ion coordination along the phosphodiester backbone may play a role in the inhibitory activity. T30177 inhibited HIV-2 integrase with similar potency as HIV-1 but inhibited feline and simian immunodeficiency virus integrases at higher concentrations, suggesting selectivity can be achieved. We propose that novel AIDS therapies could be based upon guanosine quarters as inhibitors of HIV-1 integrase.

Binding, Competitive↗
PubMed1997

Mode of interaction of G-quartets with the integrase of human immunodeficiency virus type 1.

Oligonucleotides that can form a highly stable intramolecular four-stranded DNA structure containing two stacked guanosine-quartets (G-quartets) have been reported to inhibit the replication of the human immunodeficiency virus type 1 (HIV-1) in cell culture. Two possible mechanisms for the observed antiviral activity have been proposed: interference with virus adsorption to the cell and/or inhibition of HIV-1 integrase. We investigated the molecular interaction of G-quartet-containing oligonucleotides with HIV-1 integrase in comparison with random oligonucleotides and dextran sulfate. The prototypical G-quartet-containing oligonucleotide, T30177 (Zintevir), inhibited the overall integration reaction with an IC50 value of 80 nM. A random oligonucleotide was 10-fold less potent, but dextran sulfate was more potent, with an IC50 value of 7 nM. We developed novel kinetic assays to dissect the overall integration reaction in three steps: the formation of the initial stable complex (ISC), the 3'-processing reaction, and the DNA strand-transfer step. We then analyzed the kinetics of the ISC formation and 3'-processing. The rate constant determined for the conversion of ISC into the cleaved product was 0.08 +/- 0.01 min-1. T30177 did not inhibit 3'-processing or DNA strand transfer, whereas dextran sulfate inhibited DNA strand transfer to some extent. Binding studies using surface plasmon resonance technology revealed that both T30177 and dextran sulfate were capable of preventing the binding of integrase to specific DNA. We propose a model in which the interaction of HIV-1 integrase with G-quartets results in the inhibition of the formation of the ISC between integrase and substrate DNA. Finally, we selected for an HIV-1 strain that was resistant to T30177 in cell culture. DNA sequence analysis revealed mutations in the envelope glycoprotein gp120 but not in the integrase gene. Although gp120 seems to be the main target for the antiviral activity in cell culture of G-quartets, the study of their specific inhibition of HIV-1 integrase may lead to the development of effective integrase inhibitors.

DNA, Viral↗
PubMed2000

Modeling of the inhibition of retroviral integrases by styrylquinoline derivatives.

Styrylquinoline derivatives, known to be potent inhibitors of HIV-1 integrase, have been experimentally tested for their inhibitory effect on the disintegration reaction catalyzed by catalytic cores of HIV-1 and Rous sarcoma virus (RSV) integrases. A modified docking protocol, consisting of coupling a grid search method with full energy minimization, has been specially designed to study the interaction between the inhibitors and the integrases. The inhibitors consist of two moieties that have hydroxyl and/or carboxyl substituents: the first moiety is either benzene, phenol, catechol, resorcinol, or salicycilic acid; the hydroxyl substituents on the second (quinoline) moiety may be in the keto or in the enol forms. Several tautomeric forms of the drugs have been docked to the crystallographic structure of the RSV catalytic core. The computed binding energy of the keto forms correlates best with the measured inhibitory effect. The docking procedure shows that the inhibitors bind closely to the crystallographic catalytic Mg(2+) dication. Additional quantum chemistry computations show that there is no direct correlation between the binding energy of the drugs with the Mg(2+) dication and their in vitro inhibitory effect. The designed method is a leading way for identification of potent integrase inhibitors using in silico experiments.

Algorithms↗
PubMed1998

Cytotoxic T-cell responses to HIV-1 reverse transcriptase, integrase and protease.

OBJECTIVES: To determine immunodominant regions and new epitopes for cytotoxic T cells (CTL) directed against the HIV-1 pol products reverse transcriptase (RT), integrase and protease in a large cohort of patients at different stages of disease. DESIGN AND METHODS: Cross-sectional analysis of 98 patients from the French IMMUNOCO cohort (CD4 counts: 125-1050 x 10(6) cells/l), monitored for CTL recognition of HIV-1 pol products using recombinant vaccinia virus constructs and synthetic peptides. RESULTS: Memory CTL responses against HIV-1 pol products were detected in 78% of all patients whatever the stage of disease. RT was more immunogenic (81%, 30 out of 37 patients) than integrase and protease (51% and 24%, respectively). CTL recognition of RT was more frequent against Pol amino acids 310-460 (61%, 11 out of 18 patients) than against the other three portions (Pol 168-310, Pol 450-600, Pol 590-728) in patients with CD4 counts > 400 x 10(6)/l, whereas in patients at advanced stages no prominent differences were observed. Two new clusters of antigenic regions were found in the NH2 segment: three epitopes between amino-acids Pol 200 and 217 and four epitopes between amino-acids Pol 346 and 387, using five different HLA-restricting elements. A new cluster of three conserved epitopes was found in the COOH segment of RT. CONCLUSIONS: This study shows that memory CTL responses against HIV-1 RT, integrase and protease are detectable in most patients at different stages of disease. The capacity of CTL to recognize simultaneously clusters of epitopes may become important for the immune control to reinforce antiretroviral drug efficiency.

Cohort Studies↗
PubMed2004

Prediction of HIV-1 integrase/viral DNA interactions in the catalytic domain by fast molecular docking.

This study details the separate analyses of binding specificity of HIV-1 integrase (IN) and viral B-DNA forms through ligand-receptor docking studies by means of a fast molecular docking method. The application of solvated electrostatics with the University of Houston Brownian Dynamics Program (UHBD) and configurational sampling by the Daughter of Turnip (DOT) docking program resulted in the computation of energies of more than 113 billion configurations for each ligand-receptor docking study, a procedure considered computationally intractable a few years ago. A specific binding pattern of viral DNA to the IN catalytic domain region has been predicted as a result of these calculations. In a representative docked configuration, we observe the 3'-hydroxyl of the conserved deoxyadenosine to be close to one of the two divalent metal ions that are necessary for catalysis. A superimposition of our energy-minimized docked complex on representative structures from a molecular dynamics (MD) simulation of a crystallographically resolved IN/inhibitor complex revealed an overlap of viral DNA with the inhibitor, indicating that the bound inhibitor might operate by blocking substrate binding. The DOT docking calculation also identified a second, adjacent DNA-binding site, which we believe is the nonspecific host DNA binding site. The binding pattern predicted by DOT complements previous electrostatics, MD simulation, photo-cross-linking, and mutagenesis studies and also provides a further refinement of the IN/viral DNA binding interaction as a basis for new structure-based design efforts.

Catalytic Domain↗