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At least 703 records · Page 39Linked to original sources
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↗
PubMed2004

Circular dichroism and fluorescence of a tyrosine side-chain residue monitors the concentration-dependent equilibrium between U-shaped and coiled-coil conformations of a peptide derived from the catalytic core of HIV-1 integrase.

The peptide denoted K159 (30 residues) derives from the catalytic core (CC) sequence of HIV-1 integrase (IN, residues 147-175). In the crystal structure of CC, the corresponding segment belongs to the alpha4 helix (residues 148-168, including residues Glu 152, Lys 156 and Lys 159, crucial for enzyme activity and DNA recognition), a loop (residues 169-171) and a part of the alpha5 helix (171-175), involved in enzyme dimerization. We used the fluorescence and the circular dichroism (CD) properties in the near-UV of the aromatic side chain of a tyrosine residue added at the C-terminal end of K159 in order to analyze the behavior of the concentrated and diluted peptide in aqueous trifluoroethanol (TFE), in an attempt to connect the information obtainable at high (NMR), medium (CD) and low (fluorescence) concentrations of the peptide. Altogether, the C-terminal tyrosine residue provided indirect information on the global conformation of K159 and on the local orientation and environment of the residue. The propensity of TFE to stabilize alpha-helical conformations in peptides was confirmed in CD and fluorescence experiments at relatively high (20-160 microM) and low (2-16 microM) concentrations, respectively. At relatively high concentration, stabilization of the peptide into alpha-helical conformation favored its auto-association likely in parallel coiled-coil dimers, as pointed out in our previous work [Eur. J. Biochem. 253 (1998) 236]. This was further confirmed by ANS (1-anilinonaphtalene-8-sulfonic acid) analysis and fluorescence temperature coefficient measurement. With diluted K159, a Stern-Volmer analysis with positively and negatively charged quenchers indicated that, when the intermolecular interactions were absent, the tyrosine was in a positively charged environment, as if the peptide folded into a U-shaped conformation similar to that present in the crystal structure of the enzyme.

Catalytic Domain↗
PubMed2003

Analysis of the full-length integrase-DNA complex by a modified approach for DNA docking.

A model of the full-length HIV-1 integrase dimer was constructed assembling the experimentally determined structures of the single domains. Subsequently, the three-domain protein-viral DNA complex was generated for the first time through an automated docking algorithm, obtained modifying the ESCHER program, a well-known method for protein-protein docking. A detailed study of the contacts established with DNA by the enzyme revealed that the predicted model reproduced the results of mutagenesis and cross-linking experiments, confirming the validity of our docking approach in predicting the base specificity in the DNA-protein interaction.

Base Sequence↗
PubMed2005

Evaluation of the activity of HIV-1 integrase over-expressed in eukaryotic cells.

Since the integration of viral DNA in the host genome is an essential step in the replication cycle of HIV-1, an active search for inhibitors of the integration step is ongoing. Our laboratory has been working on the development of a cellular integration system. Such a system would be helpful in the study of the HIV-1 integration process and, eventually, could be used in the search for new inhibitors that selectively interfere with HIV integration. We have previously selected stable cell lines (293T-INS) that constitutively express high levels of HIV-1 integrase (IN) from a synthetic gene [FASEB J. 14 (2000) 1389]. We have now constructed linear DNA substrates containing the terminal HIV LTR sequences (so called 'mini-HIV') and EGFP as reporter gene to evaluate whether IN can improve the integration of transfected linear DNA. After electroporation of this mini-HIV we observed a 2- to 3-fold increase in EGFP expression in IN expressing cell lines relative to control cells. The increase in EGFP expression was still evident after passaging of the cells. The effect was observed with linear DNA but not with circular DNA, thus excluding an effect on DNA uptake. The increase was the highest in the 293T-INS(D64V) cell line due to an increase in the amount of total mini-HIV DNA and 2-LTR circles as quantified by Q-PCR. Our data suggest that IN over-expressed in our cell lines interacts with the incoming DNA, protects it from nuclease degradation but does not catalyze the integration as such.

Aspartic Acid↗
PubMed2004

Synthesis of novel thiazolothiazepine based HIV-1 integrase inhibitors.

Thiazolothiazepines are among the smallest and most constrained inhibitors of human immunodeficiency virus type-1 integrase (HIV-1 IN) inhibitors (J. Med. Chem. 1999, 42, 3334). Previously, we identified two thiazolothiazepines lead IN inhibitors with antiviral activity in cell-based assays. Structural optimization of these molecules necessitated the design of easily synthesizable analogs. In order to design similar molecules with least number of substituent, herein we report the synthesis of 10 novel analogs. One of the new compounds (1) exhibited similar potency as the reference compounds, confirming that a thiazepinedione fused to a naphthalene ring system is the best combination for the molecule to accommodate into the IN active site. Thus, the replacement of sulfur in the thiazole ring with an oxygen does not seem considerably affect potency. On the other hand, the introduction of an extra methyl group at position 1 of the polycyclic system or the shift from a thiazepine to an oxazepine skeleton decreased potency. In order to understand their mode of interactions with IN active site, we docked all the compounds onto the previously reported X-ray crystal structure of IN. We observed that compounds 7-9 occupied an area close to D64 and Mg(2+) and surrounded by amino acid residues K159, K156, N155, E152, D116, H67, and T66. The oxygen atom of the oxazolo ring of 7 and 8 could chelate Mg(2+). These results indicate that the new analogs potentially interact with the highly conserved residues important for IN catalytic activities.

Anti-HIV Agents↗
PubMed2006

Design, synthesis, and biological evaluation of chicoric acid analogs as inhibitors of HIV-1 integrase.

A series of analogs of the potent HIV-1 integrase (HIV IN) inhibitor chicoric acid (CA) was designed with the intention of ameliorating some of the parent natural product's undesirable properties, in particular its toxicity, instability, and poor membrane permeability. More than 70 analogs were synthesized and assayed for three types of activity: (1) the ability to inhibit 3'-end processing and strand transfer reactions using recombinant HIV IN in vitro, (2) toxicity against the CD4+ lymphoblastoid cell line, MT2, and (3) anti-HIV activity against HIV(LAI). CA analogs lacking one of the carboxyl groups of CA and with 3,4,5-trihydroxycinnamoyl sidechains in place of the caffeoyl group of CA exhibited the most potent inhibition of HIV replication and end-processing activity. Galloyl-substituted derivatives also displayed very potent in vitro and in vivo activities, in most cases exceeding the inhibitory effects of CA itself. Conversely, analogous monocarboxy caffeoyl analogs exhibited only modest inhibition, while the corresponding 3,4-dihydroxybenzoyl-substituted compounds were devoid of activity.

Anti-HIV Agents↗
PubMed2006

Structure-activity relationship study of flavone compounds with anti-HIV-1 integrase activity: a density functional theory study.

Human immunodeficiency virus type-1 integrase (HIV-1 IN) is an essential enzyme for effective viral replication. Flavone compounds have been very much studied due to their activity during the inhibition process of HIV-1 IN. In this study, we employed density functional theory (DFT) using the B3LYP hybrid functional to calculate a set of molecular properties for 32 flavonoid compounds with anti-HIV-1 IN activity. The stepwise discriminant analysis (SDA), principal component analysis (PCA) and hierarchical cluster analysis (HCA) methods were employed to reduce dimensionality and investigate possible relationship between the calculated properties and the anti-HIV-1 IN activity. These analyses showed that the molecular hydrophobicity (ClogP), charge on atom 11 and electrophilic index (omega) are responsible for the separation between anti-HIV-1 IN active and inactive compounds.

Cluster Analysis↗
PubMed2004

6-aryl-2,4-dioxo-5-hexenoic acids, novel integrase inhibitors active against HIV-1 multiplication in cell-based assays.

A series of 6-aryl-2,4-dioxo-5-hexenoic acids, were synthesized and tested against HIV-1 in cell-based assays and against recombinant HIV-1 integrase (rIN) in enzyme assays. Compound 8a showed potent antiretroviral activity (EC(50)=1.5 microM) and significant inhibition against rIN (strand transfer: IC(50)=7.9 microM; 3'-processing: IC(50)=7.0 microM). A preliminary molecular modeling study was carried out to compare the spatial conformation of 8a with those of L-731988 (4) and 5CITEP (7) in the IN core.

Anti-HIV Agents↗
PubMed2004

Enzymology of a carbonyl reduction clearance pathway for the HIV integrase inhibitor, S-1360: role of human liver cytosolic aldo-keto reductases.

S-1360, a 1,3-diketone derivative, was the first HIV integrase inhibitor to enter human trials. Clinical data suggested involvement of non-cytochrome P450 clearance pathways, including reduction and glucuronidation. Reduction of S-1360 generates a key metabolite in humans, designated HP1, and constitutes a major clearance pathway. For characterization of subcellular location and cofactor dependence of HP1 formation, [(14)C]-S-1360 was incubated with commercially available pooled human liver fractions, including microsomes, cytosol, and mitochondria, followed by HPLC analysis with radiochemical detection. Incubations were performed in the presence and absence of the cofactors NADH or NADPH. Results showed that the enzyme system responsible for generation of HP1 in vitro is cytosolic and NADPH-dependent, implicating aldo-keto reductases (AKRs) and/or short-chain dehydrogenases/reductases (SDRs). A validated LC/MS/MS method was developed for investigating the reduction of S-1360 in detail. The reduction reaction exhibited sigmoidal kinetics with a K(m,app) of 2 microM and a Hill coefficient of 2. The ratio of V(max)/K(m) was approximately 1 ml/(min mg cytosolic protein). The S-1360 kinetic data were consistent with positive cooperativity and a single enzyme system. The relative contributions of AKRs and SDRs were examined through the use of chemical inhibitors. For these experiments, non-radiolabeled S-1360 was incubated with pooled human liver cytosol and NADPH in the presence of inhibitors, followed by quantitation of HP1 by LC/MS/MS. Quercetin and menadione produced approximately 30% inhibition at a concentration of 100 microM. Enzymes sensitive to these inhibitors include the carbonyl reductases (CRs), a subset of the SDR enzyme family predominantly located in the cytosol. Flufenamic acid and phenolphthalein were the most potent inhibitors, with > 67% inhibition at a concentration of 20 microM, implicating the AKR enzyme family. The cofactor dependence, subcellular location, and chemical inhibitor results implicated the aldo-keto reductase family of enzymes as the most likely pathway for generation of the major metabolite HP1 from S-1360.

Alcohol Oxidoreductases↗
PubMed2005

Lambda integrase: armed for recombination.

Bacteriophage lambda moves its viral genome into and out of the bacterial chromosome using site-specific recombination. Crystal structures of reaction intermediates in this recombination pathway provide exciting new snapshots of full length lambda integrase interacting with both core and regulatory DNA elements.

Bacteriophage lambda↗
PubMed2006

Three-dimensional quantitative structure: activity relationship studies on diverse structural classes of HIV-1 integrase inhibitors using CoMFA and CoMSIA.

Comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA), three-dimensional quantitative structure-activity relationship (3D-QSAR) techniques, were applied to a set of 89 HIV-1 integrase (IN) inhibitors (training set=61, test set=28), belonging to 11 structurally different classes. The biological data for 3' processing mechanism were used. For CoMFA calculations, three different fitting methods for alignment process were investigated. The best CoMFA model yielded the cross-validated r(2) r(2)(cv) =0.698 and the non-cross-validated r(2) (r(2))=0.947. The derived model indicated the importance of steric (60.8%) as well as electrostatic (39.2%) contributions. For CoMSIA calculations, different combinations of the fields were tested. The best CoMSIA model gave r(2)(cv) =0.724 and r(2)=0.864. This model showed that steric (30.3%), hydrogen bond donor (43.4%) and hydrogen bond acceptor (26.3%) properties played major roles in HIV-1 IN inhibition. The mapping of hydrogen bond interaction fields with the HIV-1 IN active site gave details on hydrogen bond forming between ligands and enzyme. These obtained results agree well with the experimental observations that there should be hydrogen bond interactions between ligands and Glu152, Lys156 and Lys159 residues. The results not only lead to a better understanding of structural requirements of HIV-1 IN inhibitors but also can help in the design of new IN inhibitors.

HIV Integrase Inhibitors↗
PubMed2005

Design, synthesis and biological evaluation of heteroaryl diketohexenoic and diketobutanoic acids as HIV-1 integrase inhibitors endowed with antiretroviral activity.

Highly active anti-retroviral therapy (HAART) using reverse transcriptase (RT) and protease (PR) inhibitors and, more recently, inhibitors of the fusion is currently the best clinical approach in combating acquired immunodeficiency syndrome (AIDS), caused by infection from human immunodeficiency virus type 1 (HIV-1). However, this therapy does not completely eradicate the virus, so that resistant strains easily emerge. The above problem calls urgently for research on inhibitors of further viral targets such as integrase (IN), the third enzyme produced by HIV. Recently, our research group was engaged in studies on conformationally restrained cinnamoyl compounds related to curcumin as anti-IN agents. Compounds containing both a 3,4,5-trihydroxyphenyl group and a carboxylic acid function were potent IN inhibitors active against viral replication. More recently, a promising new class of inhibitors synthesized by Merck Company has emerged, which contain aryldiketoacid (ADK) functionality. The ADKs selectively inhibited the stand transfer (ST) step of integration and were proven to be effective IN inhibitors in vivo. Our interest in the field of IN inhibitors led us to design pyrrole and indole derivatives containing both a cinnamoyl moiety and a diketoacid group. A number of the cited derivatives were proven potent IN inhibitors, which selectively inhibited the ST step at submicromolar concentrations and were effective against virus replication in HIV-1 infected cells.

Anti-Retroviral Agents↗
PubMed2005

Determination of an investigational HIV integrase inhibitor in human plasma using high performance liquid chromatography with tandem mass spectrometric detection.

An HPLC-MS/MS assay for the determination of an HIV integrase inhibitor, 5-(1,1-dioxido-1,2-thiazinan-2-yl)-N-(4-fluorobenzyl)-8-hydroxy-1,6-naphthyridine-7-carboxamide (I) in human plasma has been developed and validated. Compound I and a stable isotope labeled internal standard (II) were isolated from 0.5 mL plasma samples by solid phase extraction using an Ansys SPEC C-8 96-well plate. Extracts were separated on a Hypersil BDS C-18 HPLC column (3.0 mmx50 mm, 3 microm) with a mobile phase consisting of 25 mM ammonium formate pH 3.0:acetonitrile (60:40) vol%/vol% pumped at 0.5 mL/min. A Sciex API 365 mass spectrometer equipped with an atmospheric pressure chemical ionization source was operated in selected reaction monitoring (SRM) mode with the precursor-to-product ion transitions m/z 431-->109 (I) and m/z 437-->115 (II) used for quantitation. The assay was validated over the concentration range of 10-5000 ng/mL and was found to have acceptable accuracy, precision, linearity, and selectivity. The mean extraction recovery from spiked plasma samples was 69%. The intra-day accuracy of the assay was within 4% of nominal and intra-day precision was better than 4% C.V. Following a 200 mg dose of the compound administered to human subjects, concentrations of I ranged from 21.1 to 1500 ng/mL in plasma samples collected up to 12 h after dosing. Inter-day accuracy and precision results for quality control samples run over a 3-month period alongside clinical samples showed mean accuracies of within 6% of nominal and precision better than 3.5% C.V.

Chromatography, High Pressure Liquid↗
PubMed2004

A synthetic peptide bearing the HIV-1 integrase 161-173 amino acid residues mediates active nuclear import and binding to importin alpha: characterization of a functional nuclear localization signal.

In spite of recent efforts to elucidate the nuclear import pathway of the human immunodeficiency virus type 1 (HIV-1) integrase protein (IN), its exact route as well as the domains that mediate its import are still unknown. Here, we show that a synthetic peptide bearing the amino acid residues 161-173 of the HIV-1 IN is able to mediate active import of covalently attached bovine serum albumin molecules into nuclei of permeabilized cells and therefore was designated as nuclear localization signal-IN (NLS(IN)). A peptide bearing residues 161-173 in the reversed order showed low karyophilic properties. Active nuclear import was demonstrated by using fluorescence microscopy and a quantitative ELISA-based assay system. Nuclear import was blocked by addition of the NLS(IN) peptide, as well as by a peptide bearing the NLS of the simian virus 40 T-antigen (NLS-SV40). The NLS(IN) peptide partially inhibited nuclear import mediated by the full-length recombinant HIV-1 IN protein, indicating that the sequence of the NLS(IN) is involved in mediating nuclear import of the IN protein. The NLS(IN) as well as the full-length IN protein interacted specifically with importin alpha, binding of which was blocked by the NLS(IN) peptide itself as well as by the NLS-SV40.

Active Transport, Cell Nucleus↗
PubMed2004

The efficiency of mispaired ligations by lambda integrase is extremely sensitive to context.

The integrase protein (Int) of phage lambda is a well-studied representative of the tyrosine recombinase family, whose defining features are two sequential pairs of DNA cleavage/ligation reactions that proceed via a 3' phosphotyrosine covalent intermediate to first form and then resolve a Holliday junction recombination intermediate. We devised an assay that takes advantage of DNA hairpin formation at one Int target site to trap Int cleavages at a different target site, and thereby reveal iterative cycles of cleavage and ligation that would otherwise be undetected. Using this assay and others to compare wild-type Int and a mutant (R169D) defective in forming proper dimer/tetramer interfaces, we found that the efficiency of "bottom-strand" DNA cleavage by wild-type Int, but not R169D, is very sensitive to the base-pair at the "top-strand" cleavage site, seven base-pairs away. We show that this is related to the finding that hairpin formation involving ligation of a mispaired base is much faster for R169D than for wild-type Int, but only in the context of a multimeric complex. During resolution of Holliday junction recombination intermediates, wild-type Int, but not R169D, is very sensitive to homology at the sites of ligation. A long-sought insight from these results is that during Holliday junction resolution the tetrameric Int complex remains intact until after ligation of the product helices has been completed. This contrasts with models in which the second pair of DNA cleavages is a trigger for dissolution of the recombination complex.

Bacteriophage lambda↗
PubMed2006

Single-particle image reconstruction of a tetramer of HIV integrase bound to DNA.

The HIV integrase enzyme (IN) catalyzes the initial DNA breaking and joining reactions that integrate viral DNA in the host chromosome. Structures for individual IN domains have been determined by X-ray crystallography and NMR spectroscopy, but the structure of the complete IN-DNA complex has remained elusive. Homogeneous complexes of IN tetramers were assembled on DNA three-way junction substrates designed to resemble integration intermediates. Electron microscopy and single-particle image analysis of these complexes yielded a three-dimensional reconstruction at approximately 27 A resolution. The map of the IN-DNA complex displays four lobes of density approximately 50 A in diameter. Three of the lobes form a roughly triangular base with a central channel approximately 20 A in diameter. The fourth lobe is centered between two lobes and extends approximately 40 A above the base. We propose that the central channel tethers the target DNA, and two of the lobes may bind the ends of the viral DNA. The asymmetry of the complex is a feature not incorporated in previous structural models and potentially provides the first view of an asymmetric reaction intermediate.

Base Sequence↗
PubMed2004

Rapid, high-throughput purification of HIV-1 integrase using microtiter plate technology.

The human immunodeficiency virus type-1 (HIV-1) integrase (IN) catalyzes the insertion of the retroviral genome into the chromosome of an infected host cell. HIV-1 IN was expressed as a N-terminal hexa-histidine fusion in Escherichia coli. A high-throughput purification strategy was developed using denaturing methods for the initial protein extraction, followed by a one-step nickel-chelating chromatography purification and step-wise refolding. IN was routinely greater than 90% pure with yields exceeding 14 microg of purified IN per ml of E. coli culture. In vitro 3' processing and strand transfer assays showed the enzyme preparations to be highly active. The specific activity of the purified IN was 2.65 pmol/h/microg IN, which is very similar to the activity of IN routinely produced by large-scale column chromatographic methods. This high-throughput platform should be of general utility to those interested in defining the structure-function relationship of proteins and enzymes.

Cell Culture Techniques↗