SEARCH · Search PubMed

Results for “Integrase”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

SourceResearch connectionFrom yearThrough year

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 127 records · Page 7Linked to original sources

PubMed2004

CD8+ T lymphocyte responses target functionally important regions of Protease and Integrase in HIV-1 infected subjects.

BACKGROUND: CD8+ T cell responses are known to be important to the control of HIV-1 infection. While responses to reverse transcriptase and most structural and accessory proteins have been extensively studied, CD8 T cell responses specifically directed to the HIV-1 enzymes Protease and Integrase have not been well characterized, and few epitopes have been described in detail. METHODS: We assessed comprehensively the CD8 T cell responses to synthetic peptides spanning Protease and Integrase in 56 HIV-1 infected subjects with acute, chronic, or controlled infection using IFN-gamma-Elispot assays and intracellular cytokine staining. Fine-characterization of novel CTL epitopes was performed on peptide-specific CTL lines in Elispot and 51Chromium-release assays. RESULTS: Thirteen (23%) and 38 (68%) of the 56 subjects had detectable responses to Protease and Integrase, respectively, and together these targeted most regions within both proteins. Sequence variability analysis confirmed that responses cluster largely around conserved regions of Integrase, but responses against a large, highly conserved region of the N-terminal DNA-binding domain of Integrase were not readily detected. CD8 T cell responses targeted regions of Protease that contain known Protease inhibitor mutation residues, but strong Protease-specific CD8 T cell responses were rare. Fine-mapping of targeted epitopes allowed the identification of three novel, HLA class I-restricted, frequently-targeted optimal epitopes. There were no significant correlations between CD8 T cell responses to Protease and Integrase and clinical disease category in the study subjects, nor was there a correlation with viral load. CONCLUSIONS: These findings confirm that CD8 T cell responses directed against HIV-1 include potentially important functional regions of Protease and Integrase, and that pharmacologic targeting of these enzymes will place them under both drug and immune selection pressure.

Journal Article↗

PubMed2000

Mapping target site selection for the non-specific nuclease activities of retroviral integrase.

To identify the parts of retroviral integrase that interact with its DNA substrates, we compared the patterns of target site usage by chimeric enzymes and protein fragments in assays that reveal integrase's non-specific nuclease activities. The central region of 12 chimeric proteins between the human immunodeficiency virus type 1 and visna virus integrases was found to be responsible for selecting non-viral target DNA sites when small alcohols provide the attacking nucleophilic OH group during non-specific alcoholysis assays. Testing deletion derivatives of the integrase protein in this assay, which has similarities to the DNA joining reaction that occurs during retroviral integration, defined a smaller central domain that is sufficient for activity. Thus, this core domain likely contains both the host DNA site and the nucleophile site. Surprisingly, the region of integrase responsible for selecting non-viral target DNA sites when the viral DNA end is the attacking nucleophile could not similarly be mapped with the standard oligonucleotide joining assay. We therefore tested the proteins in a more sensitive assay that displays preferred sites of viral DNA insertion in a plasmid DNA target. All 12 chimeras yielded novel patterns compared with the wild-type enzymes in this assay, although local insertion patterns indicated that the central domain plays an important role in target site selection. Together, these data suggest that other protein regions must be involved when the attacking nucleophilic group is provided by viral DNA. Because specific recognition of viral DNA ends was previously mapped to the central domain, two different regions of integrase must interact with retroviral DNA.

Alcohols↗

PubMed2002

Disruption of HIV-1 integrase-DNA complexes by short 6-oxocytosine-containing oligonucleotides.

We recently found that oligonucleotides containing the 6-oxocytosine heterocyclic base are efficient inhibitors of the HIV-1 integrase in vitro [Brodin, P., et al. (2001) Nucleosides Nucleotides Nucleic Acids 20, 481-486]. In this report, we demonstrate that the inhibition arises from a noncompetitive mechanism in which the modified oligonucleotide attacks the integrase-DNA complex, leading to its active disruption. This conclusion is based on the following results. First, despite the fact that the respective affinities of a 6-oxocytosine-containing oligonucleotide and of its nonmodified counterpart for integrase were identical, only the modified compound inhibited the enzyme activities. Second, DNA binding and UV cross-linking assays indicated that the 6-oxocytosine-containing oligonucleotide prevented the formation of a stable integrase-DNA complex. Third, the kinetics of the dissociation of the integrase-DNA complex were dramatically accelerated in the presence of the modified ODN, whereas the nonmodified counterpart did not influence the dissociation. This mechanism was supported by the ability of the 6-oxocytosine-containing oligonucleotide to inhibit the strand transfer activity of HIV-1 preintegration complexes in vitro. Disruption of integrase-DNA complexes by 6-oxocytosine-containing oligonucleotides constitutes an original mechanism of integration inhibition, therefore suggesting a strategy for searching for inhibitors of the HIV-1 preintegration complexes.

Binding, Competitive↗

PubMed1998

Styrylquinoline derivatives: a new class of potent HIV-1 integrase inhibitors that block HIV-1 replication in CEM cells.

On the basis of the fact that several polynucleotidyl transferases, related to HIV integrase, contain in their active site two divalent metal cations, separated by ca. 4 A, new potential HIV integrase inhibitors were designed, in which a quinoline substructure is linked to an aryl nucleus possessing various hydroxy substitution patterns, by means of an ethylenic spacer. Although the most active compounds contain the catechol structure, this group is not essential for the activity, since compound 21 that lacks such a moiety is a potent drug, implicating the presence of a different pharmacophore. The most promising styrylquinolines thus synthesized inhibit HIV-1 integrase in vitro at micromolar or submicromolar concentrations and block HIV replication in CEM cells, with no significant cellular toxicity in a 5-day period assay. These inhibitors are active against integrase core domain-mediated disintegration, suggesting that fragment 50-212 is their actual target. These new styrylquinolines may provide lead compounds for the development of novel antiretroviral agents for AIDS therapeutics, based upon inhibition of HIV integrase. They might also be used in the elucidation of the mechanism of inhibition of this enzyme; e.g., they could serve as candidates for cocrystallization studies with HIV integrase.

Anti-HIV Agents↗

PubMed2004

A naphthyridine carboxamide provides evidence for discordant resistance between mechanistically identical inhibitors of HIV-1 integrase.

The increasing incidence of resistance to current HIV-1 therapy underscores the need to develop antiretroviral agents with new mechanisms of action. Integrase, one of three viral enzymes essential for HIV-1 replication, presents an important yet unexploited opportunity for drug development. We describe here the identification and characterization of L-870,810, a small-molecule inhibitor of HIV-1 integrase with potent antiviral activity in cell culture and good pharmacokinetic properties. L-870,810 is an inhibitor with an 8-hydroxy-(1,6)-naphthyridine-7-carboxamide pharmacophore. The compound inhibits HIV-1 integrase-mediated strand transfer, and its antiviral activity in vitro is a direct consequence of this ascribed effect on integration. L-870,810 is mechanistically identical to previously described inhibitors from the diketo acid series; however, viruses selected for resistance to L-870,810 contain mutations (integrase residues 72, 121, and 125) that uniquely confer resistance to the naphthyridine. Conversely, mutations associated with resistance to the diketo acid do not engender naphthyridine resistance. Importantly, the mutations associated with resistance to each of these inhibitors map to distinct regions within the integrase active site. Therefore, we propose a model of the two inhibitors that is consistent with this observation and suggests specific interactions with discrete binding sites for each ligand. These studies provide a structural basis and rationale for developing integrase inhibitors with the potential for unique and nonoverlapping resistance profiles.

Animals↗

PubMed1998

Dicaffeoylquinic and dicaffeoyltartaric acids are selective inhibitors of human immunodeficiency virus type 1 integrase.

Current pharmacological agents for human immunodeficiency virus (HIV) infection include drugs targeted against HIV reverse transcriptase and HIV protease. An understudied therapeutic target is HIV integrase, an essential enzyme that mediates integration of the HIV genome into the host chromosome. The dicaffeoylquinic acids (DCQAs) and the dicaffeoyltartaric acids (DCTAs) have potent activity against HIV integrase in vitro and prevent HIV replication in tissue culture. However, their specificity against HIV integrase in cell culture has been questioned. Thus, the ability of the DCQAs and DCTAs to inhibit binding of HIV type 1 (HIV-1) gp120 to CD4 and their activities against HIV-1 reverse transcriptase and HIV RNase H were studied. The DCQAs and DCTAs inhibited HIV-1 integrase at concentrations between 150 and 840 nM. They inhibited HIV replication at concentrations between 2 and 12 microM. Their activity against reverse transcriptase ranged from 7 microM to greater than 100 microM. Concentrations that inhibited gp120 binding to CD4 exceeded 80 microM. None of the compounds blocked HIV-1 RNase H by 50% at concentrations exceeding 80 microM. Furthermore, when the effects of the DCTAs on reverse transcription in acutely infected cells were measured, they were found to have no activity. Therefore, the DCQAs and DCTAs exhibit > 10- to > 100-fold specificity for HIV integrase, and their activity against integrase in biochemical assays is consistent with their observed anti-HIV activity in tissue culture. Thus, the DCQAs and DCTAs are a potentially important class of HIV inhibitors that act at a site distinct from that of current HIV therapeutic agents.

Acquired Immunodeficiency Syndrome↗

PubMed2006

Dihydroxythiophenes are novel potent inhibitors of human immunodeficiency virus integrase with a diketo acid-like pharmacophore.

We have identified dihydroxythiophenes (DHT) as a novel series of human immunodeficiency virus type 1 (HIV-1) integrase inhibitors with broad antiviral activities against different HIV isolates in vitro. DHT were discovered in a biochemical integrase high-throughput screen searching for inhibitors of the strand transfer reaction of HIV-1 integrase. DHT are selective inhibitors of integrase that do not interfere with virus entry, as shown by the inhibition of a vesicular stomatitis virus G-pseudotyped retroviral system. Moreover, in quantitative real-time PCR experiments, no effect on the synthesis of viral cDNA could be detected but rather an increase in the accumulation of 2-long-terminal-repeat cycles was detected. This suggests that the integration of viral cDNA is blocked. Molecular modeling and the structure activity relationship of DHT demonstrate that our compound fits into a two-metal-binding motif that has been suggested as the essential pharmacophore for diketo acid (DKA)-like strand transfer inhibitors (Grobler et al., Proc. Natl. Acad. Sci. USA 99:6661-6666, 2002.). This notion is supported by the profiling of DHT on retroviral vectors carrying published resistance mutations for DKA-like inhibitors where DHT showed partial cross-resistance. This suggests that DHT bind to a common site in the catalytic center of integrase, albeit with an altered binding mode. Taken together, our findings indicate that DHT are novel selective strand transfer inhibitors of integrase with a pharmacophore homologous to DKA-like inhibitors.

Amino Acid Motifs↗

PubMed2003

Development of resistance against diketo derivatives of human immunodeficiency virus type 1 by progressive accumulation of integrase mutations.

The diketo acid L-708,906 has been reported to be a selective inhibitor of the strand transfer step of the human immunodeficiency virus type 1 (HIV-1) integration process (D. Hazuda, P. Felock, M. Witmer, A. Wolfe, K. Stillmock, J. A. Grobler, A. Espeseth, L. Gabryelski, W. Schleif, C. Blau, and M. D. Miller, Science 287:646-650, 2000). We have now studied the development of antiviral resistance to L-708,906 by growing HIV-1 strains in the presence of increasing concentrations of the compound. The mutations T66I, L74M, and S230R emerged successively in the integrase gene. The virus with three mutations (T66I L74M S230R) was 10-fold less susceptible to L-708,906, while displaying the sensitivity of the wild-type virus to inhibitors of the RT or PRO or viral entry process. Chimeric HIV-1 strains containing the mutant integrase genes displayed the same resistance profile as the in vitro-selected strains, corroborating the impact of the reported mutations on the resistance phenotype. Phenotypic cross-resistance to S-1360, a diketo analogue in clinical trials, was observed for all strains. Interestingly, the diketo acid-resistant strain remained fully sensitive to V-165, a novel integrase inhibitor (C. Pannecouque, W. Pluymers, B. Van Maele, V. Tetz, P. Cherepanov, E. De Clercq, M. Witvrouw, and Z. Debyser, Curr. Biol. 12:1169-1177, 2002). Antiviral resistance was also studied at the level of recombinant integrase. Single mutations did not appear to impair specific enzymatic activity. However, 3' processing and strand transfer activities of the recombinant integrases with two (T66I L74M) and three (T66I L74M S230R) mutations were notably lower than those of the wild-type integrase. Although the virus with three mutations was resistant to inhibition by diketo acids, the sensitivity of the corresponding enzyme to L-708,906 or S-1360 was reduced only two- to threefold. As to the replication kinetics of the selected strains, the replication fitness for all strains was lower than that of the wild-type HIV-1 strain.

Acetoacetates↗

PubMed2004

Specificity of interaction of INI1/hSNF5 with retroviral integrases and its functional significance.

Integrase interactor 1 (INI1)/hSNF5 is a host factor that directly interacts with human immunodeficiency virus type 1 (HIV-1) integrase and is incorporated into HIV-1 virions. Here, we show that while INI1/hSNF5 is completely absent from purified microvesicular fractions, it is specifically incorporated into HIV-1 virions with an integrase-to-INI1/hSNF5 stoichiometry of approximately 2:1 (molar ratio). In addition, we show that INI1/hSNF5 is not incorporated into related primate lentiviral and murine retroviral particles despite the abundance of the protein in producer cells. We have found that the specificity in incorporation of INI1/hSNF5 into HIV-1 virions is directly correlated with its ability to exclusively interact with HIV-1 integrase but not with other retroviral integrases. This specificity is also reflected in our finding that the transdominant mutant S6, harboring the minimal integrase interaction domain of INI1/hSNF5, blocks HIV-1 particle production but not that of the other retroviruses in 293T cells. Taken together, these results suggest that INI1/hNSF5 is a host factor restricted for HIV-1 and that S6 acts as a highly specific and potent inhibitor of HIV-1 replication.

Binding Sites↗

PubMed2002

Microarray compound screening (microARCS) to identify inhibitors of HIV integrase.

A novel high-throughput strand transfer assay has been developed, using Microarray Compound Screening (microARCS) technology, to identify inhibitors of human immunodeficiency virus (HIV) integrase. This technology utilizes agarose matrices to introduce a majority of the reagents throughout the assay. Integration of biotinylated donor DNA with fluorescein isothiocyanate (FITC)-labeled target DNA occurs on a SAM membrane in the presence of integrase. An anti-FITC antibody conjugated to alkaline phosphatase (AP) was used to do an enzyme-linked immunosorbent assay with the SAM. An agarose gel containing AttoPhos, a substrate of AP, was used for detection of the integrase reactions on the SAM. For detection, the AttoPhos gel was separated from the SAM after incubation and then the gel was imaged using an Eagle Eye II closed-circuit device camera system. Potential integrase inhibitors appear as dark spots on the gel image. A library of approximately 250,000 compounds was screened using this HIV integrase strand transfer assay in microARCS format. Compounds from different structural classes were identified in this assay as novel integrase inhibitors.

HIV Integrase↗

PubMed1997

Characterization of feline immunodeficiency virus integrase and analysis of functional domains.

The wild-type and mutant derivatives of the integrase protein of feline immunodeficiency virus (FIV) were cloned and expressed in Escherichia coli. The purified proteins were examined using various model DNA substrates for their catalytic activities: 3'-end processing, 3'-end joining, and disintegration. The reactions required the presence of either Mn2+ or Mg2+ as a divalent cation. The N-terminal and C-subterminal domains (residues 1-52 and 189-235, respectively) were necessary for 3'-end processing and joining reactions but not for disintegration. Substitution of asparagine for the highly conserved aspartic acid at position 118 resulted in a complete loss of all three activities, confirming that the catalytic domain resides in the central core region (residues 53-188) of the protein. Deletion of the C-terminus (residues 236-281) resulted in a FIV integrase mutant that had efficient 3'-end processing and disintegration activities but weak 3'-end joining activity, a finding that has not been reported previously with other retroviral integrases. The result suggests that the C-terminus is the primary binding site for target DNA. Attachment of a histidine-tag at the N-terminus of the wild-type and deletion derivatives increased the binding affinity to the DNA substrate, resulting in altered levels of catalytic activities and selection of integration sites. Similar to other retroviral integrases, certain pairs of mutant derivatives of FIV integrase could complement each other to restitute 3'-end processing and joining activities, suggesting that formation of functional multimers is a general feature of proteins in the integrase family.

Animals↗

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↗

PubMed2000

Core-binding specificity of bacteriophage integrases.

The site-specific recombination systems of bacteriophages lambda and HK022 share the same mechanism and their integrase proteins show strong homology. Nevertheless the integrase protein of each phage can only catalyze recombination between its own att sites. Previous work has shown that the specificity determinants in the att sites are located within the sequences that bind the integrase to the core of att. DNA fragments that carry attL and attR sites of each phage were challenged with each of the two integrases and the DNA-protein complexes were examined by the gel-retardation technique. The results show that each integrase can form higher-order DNA-protein complexes only with its cognate att sites, suggesting that differences in the mode of binding to the core are responsible for the specificity difference between the two integrases.

Bacterial Proteins↗

PubMed1995

Inhibition of human immunodeficiency virus type-1 integrase by curcumin.

Curcumin (diferuloylmethane) is the yellow pigment in turmeric (Curcuma longa L.) that is widely used as a spice, food coloring (curry) and preservative. Curcumin exhibits a variety of pharmacological effects including antitumor, anti-inflammatory, and anti-infectious activities and is currently in clinical trials for AIDS patients. The effects of curcumin have been determined on purified human immunodeficiency virus type 1 (HIV-1) integrase. Curcumin has an inhibitory concentration50 (IC50) for strand transfer of 40 microM. Inhibition of an integrase deletion mutant containing only amino acids 50-212 suggests that curcumin interacts with the integrase catalytic core. Two structural analogs, methyl cinnamate and chlorogenic acid, were inactive. Energy minimization studies suggest that the anti-integrase activity of curcumin could be due to an intramolecular stacking of two phenyl rings that brings the hydroxyl groups into close proximity. The present data suggest that HIV-1 integrase inhibition may contribute to the antiviral activity of curcumin. These observations suggest new strategies for antiviral drug development that could be based upon curcumin as a lead compound for the development of inhibitors of HIV-1 integrase.

Antiviral Agents↗

PubMed2002

Site-specific recombination in eukaryotic cells mediated by mutant lambda integrases: implications for synaptic complex formation and the reactivity of episomal DNA segments.

Mutant lambda integrases catalyze site-specific DNA recombination in the absence of accessory factors IHF, XIS, and negative DNA supercoiling. Here we investigate the effects that a human cellular environment exerts on these reactions in order to (i) gain further insights into mechanistic aspects of recombination in eukaryotic cells and (ii) to further develop the Int system for biotechnological applications. First, we compared intra- and intermolecular integrative as well as excisive recombination pathways on episomal substrates after co-transfection with recombinase expression vectors. Our results demonstrate that, within 24 hours after transfection, intermolecular recombination by mutant integrase is at least as efficient as intramolecular recombination. Second, a significant intermolecular recombination activity was observed between two copies of a recombination site containing only the 21 bp comprising core-type DNA sequence. This basic activity was stimulated several-fold when arm-type DNA sequences were present in addition to core sites. Therefore, one recombination pathway in human cells involves mutant integrases bound solely at core sites, which is reminiscent of the Flp/FRT and Cre/loxP pathways. The stimulatory effect of arm-type sequences could be explained by an increase in integrase concentration in the vicinity of core sites. We show, in addition, that an N-terminal truncated mutant integrase exhibited only a very weak recombinogenic activity in a eukaryotic background. This result strengthens a functional role for the N-terminal domain in recombination in addition to its arm-type DNA-binding activity. Finally, we demonstrate that low level integrative recombination by wild-type integrase is stimulated when purified integration host factor is co-transfected. This corroborates our previous conclusion that sufficient amounts of eukaryotic protein co-factors, which could functionally replace IHF, are not present in human cells. It also provides a potential means to control site-specific recombination in eukaryotic cells.

Attachment Sites, Microbiological↗

PubMed2001

Inhibition of human immunodeficiency virus type 1 reverse transcriptase, protease and integrase by bovine milk proteins.

Different proteins have been isolated from bovine milk including lactoferrin, lactoperoxidase, glycolactin, angiogenin-1, lactogenin, alpha-lactalbumin, lactoglobulin and casein. These proteins have been assayed for inhibitory activity against human immunodeficiency virus type 1 (HIV-1) reverse transcriptase, protease and integrase, enzymes crucial to the HIV-1 life cycle. It was found that different milk proteins inhibited the three aforementioned HIV enzymes to different extents. Lactoferrin strongly inhibited HIV-1 reverse transcriptase but only slightly inhibited HIV-1 protease and integrase. On the other hand, alpha-lactalbumin, beta-lactoglobulin and casein inhibited HIV-1 protease and integrase to an appreciable extent but did not inhibit HIV-1 reverse transcriptase. Glycolactin and angiogenin-1 suppressed the activity of HIV-1 reverse transcriptase by a moderate extent but more powerfully inhibited HIV-1 protease and integrase. In comparison with the other milk proteins glycolactin was a strong inhibitor of HIV-1 protease and integrase and a moderate inhibitor of HIV-1 reverse transcriptase. Lactogenin was a strong inhibitor of HIV-1 integrase, a moderate inhibitor of HIV-1 reverse transcriptase and a weak inhibitor of HIV-1 protease.

Animals↗

PubMed2002

Isolation of two highly potent and non-toxic inhibitors of human immunodeficiency virus type 1 (HIV-1) integrase from Salvia miltiorrhiza.

Water soluble extracts of the herbal plant, Salvia miltiorrhiza (Danshen) exhibited potent effect against HIV-1 integrase activity in vitro and viral replication in vivo. We have developed an extensive purification scheme to isolate effective, non-toxic inhibitors against human immunodeficiency virus type 1 (HIV-1) using the 3'-processing activity of integrase as a purification guide and assay. Two water soluble compounds, M(5)22 and M(5)32, have been discovered by isolating them from S. miltiorrhiza roots in purities of >99.5% as shown by NMR spectral analysis with yields of 0.018 and 0.038%, respectively. Structural determination revealed that M(5)22 is lithospermic acid and M(5)32 is lithospermic acid B. These two structurally related compounds are potent anti-HIV inhibitors and showed no cytotoxicity to H9 cells at high concentrations (CC(100)>297 microM for M(5)22 and >223 microM for M(5)32). The IC50 for inhibition of 3'-processing by HIV-1 integrase was found to be 0.83 microM for M(5)22 and 0.48 microM for M(5)32. In addition, M(5)22 and M(5)32 inhibited HIV-1 integrase catalytic activities of 3'-joining to the target DNA with IC50 of 0.48 microM for M(5)22 and 0.37 microM for M(5)32. Furthermore, kinetic and mechanistic studies suggested that drug binding to HIV-1 integrase and inhibition of enzymatic activity occur at a fast rate. Both M(5)22 and M(5)32 do not prevent HIV entry in H9 cells. They also show no inhibition of reverse transcriptase activity in infected cells. The levels of intracellular strong stop and full-length viral DNA remained unchanged following drug treatment. However, both inhibitors strongly suppressed the acute HIV-1 infection of H9 cells with IC50 values of 2 and 6.9 microM for M(5)22 and M(5)32, respectively. Thus these two selective integrase inhibitors hold promise as a novel class of therapeutic drugs for AIDS based on their high potencies and absence of cytotoxicity.

Benzofurans↗

PubMed1997

The solution structure of the amino-terminal HHCC domain of HIV-2 integrase: a three-helix bundle stabilized by zinc.

BACKGROUND: Integrase mediates a crucial step in the life cycle of the human immunodeficiency virus (HIV). The enzyme cleaves the viral DNA ends in a sequence-dependent manner and couples the newly generated hydroxyl groups to phosphates in the target DNA. Three domains have been identified in HIV integrase: an amino-terminal domain, a central catalytic core and a carboxy-terminal DNA-binding domain. The amino-terminal region is the only domain with unknown structure thus far. This domain, which is known to bind zinc, contains a HHCC motif that is conserved in retroviral integrases. Although the exact function of this domain is unknown, it is required for cleavage and integration. RESULTS: The three-dimensional structure of the amino-terminal domain of HIV-2 integrase has been determined using two-dimensional and three-dimensional nuclear magnetic resonance data. We obtained 20 final structures, calculated using 693 nuclear Overhauser effects, which display a backbone root-mean square deviation versus the average of 0.25 A for the well defined region. The structure consists of three alpha helices and a helical turn. The zinc is coordinated with His 12 via the N epsilon 2 atom, with His16 via the N delta 1 atom and with the sulfur atoms of Cys40 and Cys43. The alpha helices form a three-helix bundle that is stabilized by this zinc-binding unit. The helical arrangement is similar to that found in the DNA-binding domains of the trp repressor, the prd paired domain and Tc3A transposase. CONCLUSION: The amino-terminal domain of HIV-2 integrase has a remarkable hybrid structure combining features of a three-helix bundle fold with a zinc-binding HHCC motif. This structure shows no similarity with any of the known zinc-finger structures. The strictly conserved residues of the HHCC motif of retroviral integrases are involved in metal coordination, whereas many other well conserved hydrophobic residues are part of the protein core.

Amino Acid Sequence↗