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PubMed1994

Biophysical and enzymatic properties of the catalytic domain of HIV-1 integrase.

A deletion derivative of the integrase protein from human immunodeficiency virus type-1 (HIV-1) consisting of the central core domain (amino acids 50-212) has been characterized biophysically and biochemically. This deletion mutant is of particular interest for structural studies as it can carry out the disintegration reaction suggesting the presence of an active site and, under certain conditions, is more soluble than full-length integrase. The circular dichroism and fluorescence of the deletion mutant and the 288-residue full-length integrase were similar, indicating that the core residues maintain similar overall conformations in both proteins. The deletion mutant is approximately 10% more alpha-helical than the full-length protein. Analytical centrifugation demonstrated that both proteins undergo monomer-dimer association although the truncated protein showed slightly less tendency to dimerize; the dissociation constants were 2.5 x 10(-5) M for the full-length protein and 8.0 x 10(-5) M for the truncated protein. The disintegration activity of both proteins was also compared. Although a higher concentration of the truncation mutant was required for optimal activity, the mutant did not have altered pH or Mn2+ requirements relative to the full-length protein. The combined biophysical and enzymatic studies suggest that this truncated form of HIV-1 integrase is likely to be useful for structural studies.

Amino Acid Sequence↗
PubMedDate not supplied

HIV-1 integration: an interplay between HIV-1 integrase, cellular and viral proteins.

To achieve a productive infection, the reverse transcribed cDNA of the human immunodeficiency virus type 1 (HIV-1) has to be inserted in the host cell genome. The main protein required to accomplish this reaction is the virally encoded integrase. In vitro, the recombinant integrase is capable of catalyzing the two subsequent reactions of the integration process, namely the 3' processing followed by the strand transfer, without other viral and/or cellular proteins. However, a number of studies indicate that the in vivo integration process also involves cellular proteins, assisting the virus to integrate in the cellular genome. These cellular proteins can play a role during different steps of the integration process, including nuclear import, integrase catalysis, integration site selection and DNA gap repair. In this review we summarize the candidate cellular proteins involved in the HIV-1 integration process identified so far and discuss their potential roles during HIV-1 replication.

Apoptosis↗
PubMed1998

Application of electrospray ionization mass spectrometry for studying human immunodeficiency virus protein complexes.

Mass spectrometry (MS) with electrospray ionization (ESI) has shown utility for studying noncovalent protein complexes, as it offers advantages in sensitivity, speed, and mass accuracy. The stoichiometry of the binding partners can be easily deduced from the molecular weight measurement. In many examples of protein complexes, the gas phase-based measurement is consistent with the expected solution phase binding characteristics. This quality suggests the utility of ESI-MS for investigating solution phase molecular interactions. Complexes composed of proteins from the human immunodeficiency virus (HIV) have been studied using ESI-MS. Multiply charged protein dimers from HIV integrase catalytic core (F185K) and HIV protease have been observed. Furthermore, the ternary complex between HIV protease dimer and inhibitor pepstatin A was studied as a function of solution pH. Zinc binding to zinc finger-containing nucleocapsid protein (NCp7) and the NCp7-psi RNA 1:1 stoichiometry complex was also studied by ESI-MS. No protein-RNA complex was observed in the absence of zinc, consistent with the role of the zinc finger motifs for RNA binding.

Amino Acid Sequence↗
PubMed2005

Enantioselective total synthesis of a novel polyketide natural product (+)-integrasone, an HIV-1 integrase inhibitor.

Enantioselective total synthesis of the recently isolated, novel polyketide natural product (+)-integrasone has been accomplished from the readily available Diels-Alder adduct of cyclopentadiene and p-benzoquinone. An enzymatically desymmetrized epoxyquinone building block has been elaborated through a series of regio- , chemo- and stereocontrolled steps to the final bicyclic framework of the natural product.

Bridged Bicyclo Compounds, Heterocyclic↗
PubMed1999

The mobility of an HIV-1 integrase active site loop is correlated with catalytic activity.

Replication of HIV-1 requires the covalent integration of the viral cDNA into the host chromosomal DNA directed by the virus-encoded integrase protein. Here we explore the importance of a protein surface loop near the integrase active site using protein engineering and X-ray crystallography. We have redetermined the structure of the integrase catalytic domain (residues 50-212) using an independent phase set at 1.7 A resolution. The structure extends helix alpha4 on its N-terminal side (residues 149-154), thus defining the position of the three conserved active site residues. Evident in this and in previous structures is a conformationally flexible loop composed of residues 141-148. To probe the role of flexibility in this loop, we replaced Gly 140 and Gly 149, residues that appear to act as conformational hinges, with Ala residues. X-ray structures of the catalytic domain mutants G149A and G140A/G149A show further rigidity of alpha4 and the adjoining loop. Activity assays in vitro revealed that these mutants are impaired in catalysis. The DNA binding affinity, however, is minimally affected by these mutants as assayed by UV cross-linking. We propose that the conformational flexibility of this active site loop is important for a postbinding catalytic step.

Alanine↗
PubMedDate not supplied

Linear indices of the "molecular pseudograph's atom adjacency matrix": definition, significance-interpretation, and application to QSAR analysis of flavone derivatives as HIV-1 integrase inhibitors.

This report describes a new set of molecular descriptors of relevance to QSAR/QSPR studies and drug design, atom linear indices fk(xi). These atomic level chemical descriptors are based on the calculation of linear maps on Rn[fk(xi): Rn--> Rn] in canonical basis. In this context, the kth power of the molecular pseudograph's atom adjacency matrix [Mk(G)] denotes the matrix of fk(xi) with respect to the canonical basis. In addition, a local-fragment (atom-type) formalism was developed. The kth atom-type linear indices are calculated by summing the kth atom linear indices of all atoms of the same atom type in the molecules. Moreover, total (whole-molecule) linear indices are also proposed. This descriptor is a linear functional (linear form) on Rn. That is, the kth total linear indices is a linear map from Rn to the scalar R[ fk(x): Rn --> R]. Thus, the kth total linear indices are calculated by summing the atom linear indices of all atoms in the molecule. The features of the kth total and local linear indices are illustrated by examples of various types of molecular structures, including chain-lengthening, branching, heteroatoms-content, and multiple bonds. Additionally, the linear independence of the local linear indices to other 0D, 1D, 2D, and 3D molecular descriptors is demonstrated by using principal component analysis for 42 very heterogeneous molecules. Much redundancy and overlapping was found among total linear indices and most of the other structural indices presently in use in the QSPR/QSAR practice. On the contrary, the information carried by atom-type linear indices was strikingly different from that codified in most of the 229 0D-3D molecular descriptors used in this study. It is concluded that the local linear indices are an independent indices containing important structural information to be used in QSPR/QSAR and drug design studies. In this sense, atom, atom-type, and total linear indices were used for the prediction of pIC50 values for the cleavage process of a set of flavone derivatives inhibitors of HIV-1 integrase. Quantitative models found are significant from a statistical point of view (R of 0.965, 0.902, and 0.927, respectively) and permit a clear interpretation of the studied properties in terms of the structural features of molecules. A LOO cross-validation procedure revealed that the regression models had a fairly good predictability (q2 of 0.679, 0.543, and 0.721, respectively). The comparison with other approaches reveals good behavior of the method proposed. The approach described in this paper appears to be an excellent alternative or guides for discovery and optimization of new lead compounds.

Computer Simulation↗
PubMed2006

A series of 5-aminosubstituted 4-fluorobenzyl-8-hydroxy-[1,6]naphthyridine-7-carboxamide HIV-1 integrase inhibitors.

A series of 5-amino derivatives of 8-hydroxy[1,6]-naphthyridine-7-carboxamide exhibiting sub-micromolar potency against replication of HIV-1 in cell culture was identified. One of these analogs, compound 12, displayed excellent pharmacokinetic properties when dosed orally in rats and in monkeys. This compound was demonstrated to be efficacious against replication of simian-human immunodeficiency virus (SHIV) 89.6P in infected rhesus macaques.

Amination↗
PubMed2006

HIV-1 integrase is capable of targeting DNA to the nucleus via an importin alpha/beta-dependent mechanism.

In addition to its well-documented role in integration of the viral genome, the HIV-1 enzyme IN (integrase) is thought to be involved in the preceding step of importing the viral cDNA into the nucleus. The ability of HIV to transport its cDNA through an intact nuclear envelope allows HIV-1 to infect non-dividing cells, which is thought to be crucial for the persistent nature of HIV/AIDS. Despite this, the mechanism utilized by HIV-1 to import its cDNA into the nucleus, and the viral proteins involved, remains ill-defined. In the present study we utilize in vitro techniques to assess the nuclear import properties of the IN protein, and show that IN interacts with members of the Imp (Importin) family of nuclear transport proteins with high affinity and exhibits rapid nuclear accumulation within an in vitro assay, indicating that IN possesses potent nucleophilic potential. IN nuclear import appears to be dependent on the Imp alpha/beta heterodimer and Ran GTP (Ran in its GTP-bound state), but does not require ATP. Importantly, we show that IN is capable of binding DNA and facilitating its import into the nucleus of semi-intact cells via a process that involves basic residues within amino acids 186-188 of IN. These results confirm IN as an efficient mediator of DNA nuclear import in vitro and imply the potential for IN to fulfil such a role in vivo. These results may not only aid in highlighting potential therapeutic targets for impeding the progression of HIV/AIDS, but may also be relevant for non-viral gene delivery.

Animals↗
PubMed1993

Characterization of a DNA binding domain in the C-terminus of HIV-1 integrase by deletion mutagenesis.

The integrase (IN) protein of human immunodeficiency virus type 1 (HIV-1) catalyzes site-specific cleavage of 2 bases from the viral long terminal repeat (LTR) sequence yet it binds DNA with little DNA sequence specificity. We have previously demonstrated that the C-terminal half of IN (amino acids 154-288) possesses a DNA binding domain. In order to further characterize this region, a series of clones expressing truncated forms of IN as N-terminal fusion proteins in E.coli were constructed and analyzed by Southwestern blotting. Proteins containing amino acids 1-263, 1-248 and 170-288 retained the ability to bind DNA, whereas a protein containing amino acids 1-180 showed no detectable DNA binding. This defines a DNA binding domain contained within amino acids 180-248. This region contains an arrangement of 9 lysine and arginine residues each separated by 2-4 amino acids (KxxxKxxxKxxxxRxxxRxxRxxxxKxxxKxxxK), spanning amino acids 211-244, which is conserved in all HIV-1 isolates. A clone expressing full-length IN with a C-terminal fusion of 16 amino acids was able to bind DNA comparably to a cloned protein with a free C-terminus, and an IN-specific monoclonal antibody which recognizes an epitope contained within amino acids 264-279 was unable to block DNA binding, supporting the evidence that a region necessary for binding lies upstream of amino acid 264.

Amino Acid Sequence↗
PubMed1994

Binding and stimulation of HIV-1 integrase by a human homolog of yeast transcription factor SNF5.

Upon entry into a host cell, retroviruses direct the reverse transcription of the viral RNA genome and the establishment of an integrated proviral DNA. The retroviral integrase protein (IN) is responsible for the insertion of the viral DNA into host chromosomal targets. The two-hybrid system was used to identify a human gene product that binds tightly to the human immunodeficiency virus-type 1 (HIV-1) integrase in vitro and stimulates its DNA-joining activity. The sequence of the gene suggests that the protein is a human homolog of yeast SNF5, a transcriptional activator required for high-level expression of many genes. The gene, termed INI1 (for integrase interactor 1), may encode a nuclear factor that promotes integration and targets incoming viral DNA to active genes.

Amino Acid Sequence↗
PubMed2006

Identification of the LEDGF/p75 binding site in HIV-1 integrase.

Lens epithelium-derived growth factor (LEDGF)/p75 is an important cellular co-factor for human immunodeficiency virus (HIV) replication. We originally identified LEDGF/p75 as a binding partner of integrase (IN) in human cells. The interaction has been mapped to the integrase-binding domain (IBD) of LEDGF/p75 located in the C-terminal part. We have subsequently shown that IN carrying the Q168A mutation remains enzymatically active but is impaired for interaction with LEDGF/p75. To map the integrase/LEDGF interface in more detail, we have now identified and characterized two regions within the enzyme involved in the interaction with LEDGF/p75. The first region centers around residues W131 and W132 while the second extends from I161 up to E170. For the different IN mutants the interaction with LEDGF/p75 and the enzymatic activities were determined. IN(W131A), IN(I161A), IN(R166A), IN(Q168A) and IN(E170A) are impaired for interaction with LEDGF/p75, but retain 3' processing and strand transfer activities. Due to impaired integration, an HIV-1 strain containing the W131A mutation in IN displays reduced replication capacity, whereas virus carrying IN(Q168A) is replication defective. Comparison of the wild-type IN-LEDGF/p75 co-crystal structure with that of the modelled structure of the IN(Q168A) and IN(W131A) mutant integrases corroborated our experimental data.

Amino Acid Sequence↗
PubMed2003

Design and synthesis of 8-hydroxy-[1,6]naphthyridines as novel inhibitors of HIV-1 integrase in vitro and in infected cells.

Naphthyridine 7 inhibits the strand transfer of the integration process catalyzed by integrase with an IC50 of 10 nM and inhibits 95% of the spread of HIV-1 infection in cell culture at 0.39 microM. It does not exhibit cytotoxicity in cell culture at < or =12.5 microM and shows a good pharmacokinetic profile when dosed orally to rats. The antiviral activity of 7 and its effect on integration were confirmed using viruses with specific integrase mutations.

Administration, Oral↗
PubMed2000

A new class of HIV-1 integrase inhibitors: the 3,3,3', 3'-tetramethyl-1,1'-spirobi(indan)-5,5',6,6'-tetrol family.

Integration is a required step in HIV replication, but as yet no inhibitors of the integration step have been developed for clinical use. Many inhibitors have been identified that are active against purified viral-encoded integrase protein; of these, many contain a catechol moiety. Though this substructure contributes potency in inhibitors, it is associated with toxicity and so the utility of catechol-containing inhibitors has been questioned. We have synthesized and tested a systematic series of derivatives of a catechol-containing inhibitor (1) with the goal of identifying catechol isosteres that support inhibition. We find that different patterns of substitution on the aromatic ring suffice for inhibition when Mn(2+) is used as a cofactor. Importantly, the efficiency is different when Mg(2+), the more likely in vivo cofactor, is used. These data emphasize the importance of assays with Mg(2+) and offer new catechol isosteres for use in integrase inhibitors.

Antiviral Agents↗
PubMed2005

Structural basis for the recognition between HIV-1 integrase and transcriptional coactivator p75.

Integrase (IN) is an essential retroviral enzyme, and human transcriptional coactivator p75, which is also referred to as lens epithelium-derived growth factor (LEDGF), is the dominant cellular binding partner of HIV-1 IN. Here, we report the crystal structure of the dimeric catalytic core domain of HIV-1 IN complexed to the IN-binding domain of LEDGF. Previously identified LEDGF hotspot residues anchor the protein to both monomers at the IN dimer interface. The principal structural features of IN that are recognized by the host factor are the backbone conformation of residues 168-171 from one monomer and a hydrophobic patch that is primarily comprised of alpha-helices 1 and 3 of the second IN monomer. Inspection of diverse retroviral primary and secondary sequence elements helps to explain the apparent lentiviral tropism of the LEDGF-IN interaction. Because the lethal phenotypes of HIV-1 mutant viruses unable to interact with LEDGF indicate that IN function is highly sensitive to perturbations of the structure around the LEDGF-binding site, we propose that small molecule inhibitors of the protein-protein interaction might similarly disrupt HIV-1 replication.

Adaptor Proteins, Signal Transducing↗
PubMed2002

Mapping the epitope of an inhibitory monoclonal antibody to the C-terminal DNA-binding domain of HIV-1 integrase.

Integrase (IN) catalyzes the insertion of retroviral DNA into chromosomal DNA of a host cell and is one of three virus-encoded enzymes that are required for replication. A library of monoclonal antibodies against human immunodeficiency virus type 1 (HIV-1) IN was raised and characterized in our laboratory. Among them, monoclonal antibody (mAb) 33 and mAb32 compete for binding to the C-terminal domain of the HIV-1 IN protein. Here, we show that mAb33 is a strong inhibitor of IN catalytic activity, whereas mAb32 is only weakly inhibitory. Furthermore, as the Fab fragment of mAb32 had no effect on IN activity, inhibition by this mAb may result solely from its bivalency. In contrast, Fab33 did inhibit IN catalytic activity, although bivalent binding by mAb33 may enhance the inhibition. Interaction with Fab33 also prevented DNA binding to the isolated C-terminal domain of IN. Results from size-exclusion chromatography, gel electrophoresis, and matrix-assisted laser desorption ionization time-of-flight mass spectrometric analyses revealed that multiple Fab33 small middle dotIN C-terminal domain complexes exist in solution. Studies using heteronuclear NMR showed a steep decrease in (1)H-(15)N cross-peak intensity for 8 residues in the isolated C-terminal domain upon binding of Fab33, indicating that these residues become immobilized in the complex. Among them, Ala(239) and Ile(251) are buried in the interior of the domain, whereas the remaining residues (Phe(223), Arg(224), Tyr(226), Lys(244), Ile(267), and Ile(268)) form a contiguous, solvent-accessible patch on the surface of the protein likely including the epitope of Fab33. Molecular modeling of Fab33 followed by computer-assisted docking with the IN C-terminal domain suggested a structure for the antibody-antigen complex that is consistent with our experimental data and suggested a potential target for anti-AIDS drug design.

Alanine↗
PubMed2006

HIV-1 integrase inhibitors: 2003-2004 update.

The integration of viral cDNA into the host genome is an essential step in the HIV-1-life cycle and is mediated by the virally encoded enzyme, integrase (IN). Inhibition of this process provides an attractive strategy for antiviral drug design. The discovery of beta-diketo acid inhibitors played a major role in validating IN as a legitimate antiretroviral drug target. Over a decade of research, a plethora of IN inhibitors have been discovered and some showed antiviral activity consistent with their effect on IN. To date, at least two compounds have been tested in human but none are close to the FDA approval. In this review, we provide a comprehensive report of all small-molecule IN inhibitors discovered during the years 2003 and 2004. Compilation of such data will prove beneficial in developing QSAR, virtual screening, pharmacophore hypothesis generation, and validation.

Anti-Retroviral Agents↗
PubMed2000

Mapping epitopes of monoclonal antibodies against HIV-1 integrase with limited proteolysis and matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

Monoclonal antibodies (mAbs) have been used extensively in the biochemical analysis of proteins. Molecular identification of a specific epitope can enhance our understanding of the relationship between the structure and function of a protein. We recently developed a protein footprint technique for mapping mAb epitopes that employs limited proteolysis followed by peptide analysis with matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Here we describe the rational for the technique and illustrate its use in mapping the epitopes of two mAbs that bind to the C-terminal domain of human immunodeficiency virus type-1 integrase. The results provide a plausible explanation for the fact that one mAb inhibits enzyme activity while the second does not.

Amino Acid Sequence↗