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PubMed2004

Spectroscopic studies of diketoacids-metal interactions. A probing tool for the pharmacophoric intermetallic distance in the HIV-1 integrase active site.

The interactions with divalent cations of 4-phenyl-4-oxo-2-hydroxybuten-2-oic acid (benzoylpyruvic acid (BPA)), the pharmacophore of HIV-1 integrase inhibitors, were investigated using spectroscopic tools. In the absence of the enzyme, a 2:2 metal-ligand complex was characterized with an intermetallic distance of 4-6 A. Molecular modeling allowed us to propose a compatible structure for the metal-ligand complex. BPA does not inhibit the reactions catalyzed by HIV-1 IN, emphasizing the importance of the aromatic ring substitution in the antiviral activity.

Cations, Divalent↗
PubMed1992

Localization of DNA binding activity of HIV-1 integrase to the C-terminal half of the protein.

Human immunodeficiency virus type 1 (HIV-1) integrase (IN) is the viral protein required for integration of the HIV-1 genome into host cell DNA. A series of clones expressing portions of IN as lambda cII fusion proteins has been constructed in an Escherichia coli expression system; a Southwestern procedure was used to examine binding of the expressed proteins to DNA oligonucleotides. Proteins expressed by clone pHIP106, encoding the entire IN protein but no other pol sequence, and pKNA101, which expresses an IN fusion protein containing 23 amino acids of HIV-1 reverse transcriptase at its amino terminus, exhibited similar levels of oligonucleotide binding. Little DNA sequence specificity was associated with binding activity and there was a preference for Mn2+ over Mg2+ and Ca2+. Interestingly, the protein expressed by an N-terminal clone containing nucleotides coding for IN amino acids 1-141 (including a conserved His-Cys box) was unable to bind oligonucleotide, whereas the protein expressed by a C-terminal clone containing nucleotides coding for amino acids 142-288 exhibited binding equivalent to that of full-length IN. The C-terminal protein was unreactive with a MAb to the lambda cII leader peptide and with an antipeptide serum directed against amino acids 141-158. These results are consistent with the previously reported internal initiation of IN protein synthesis in E. coli at met 154, and indicate that the C-terminal clone does not express IN amino acids 142-153. These amino acids represent part of a conserved region termed D(35)E, containing amino acids 116-152, which has been implicated in IN DNA binding.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMedDate not supplied

Bayesian model based clustering analysis: application to a molecular dynamics trajectory of the HIV-1 integrase catalytic core.

This work describes the application of a Bayesian method for clustering protein conformations sampled during a molecular dynamics simulation of the HIV-1 integrase catalytic core. A clustering analysis is carried out under the assumption of normal distribution without fixing the number of clusters in advance. Some performance measures, such as posterior probability and class cross entropy, are used to determine the most probable set of clusters. The Bayesian clustering method results in meaningful groups identifying transitions between conformational ensembles. The dihedral angles involved in such transitions are also examined in detail. The conformations in high dimensional space are projected into 3D space employing a multidimensional scaling technique to provide a visual inspection.

Algorithms↗
PubMed2002

QSAR of HIV-1 integrase inhibitors by genetic function approximation method.

Quantitative structure--activity relationship (QSAR) paradigm, using genetic function approximation (GFA) technique was used to examine the correlations between the calculated physicochemical descriptors and the in vitro activities (3'-processing and 3'-strand transfer inhibition) of a series of human immunodeficiency virus type 1 (HIV-1) integrase inhibitors. Depending on the chemical structure, all molecules were divided into two classes---catechols and noncatechols. Eighty-one molecules were used in the present study and they were divided into training set and test set. The training set in each class consisted of 35 molecules and QSAR models were generated separately for both catechols and noncatechols. Equations were evaluated using internal as well as external test set predictions. Models generated for catechols show that electronic, shape related, and thermodynamic parameters are important whereas for noncatechols, spatial, structural, and thermodynamic properties play an important role for the activity.

Anti-HIV Agents↗
PubMed1998

Solution structure of the His12 --> Cys mutant of the N-terminal zinc binding domain of HIV-1 integrase complexed to cadmium.

The solution structure of His12 --> Cys mutant of the N-terminal zinc binding domain (residues 1-55; IN(1-55)) of HIV-1 integrase complexed to cadmium has been solved by multidimensional heteronuclear NMR spectroscopy. The overall structure is very similar to that of the wild-type N-terminal domain complexed to zinc. In contrast to the wild-type domain, however, which exists in two interconverting conformational states arising from different modes of coordination of the two histidine side chains to the metal, the cadmium complex of the His12 --> Cys mutant exists in only a single form at low pH. The conformation of the polypeptide chain encompassing residues 10-18 is intermediate between the two forms of the wild-type complex.

Binding Sites↗
PubMed2006

Sequence-based design and discovery of peptide inhibitors of HIV-1 integrase: insight into the binding mode of the enzyme.

Integration of viral DNA into the host chromosome is an essential step in the HIV life cycle. This process is mediated by integrase (IN), a 32 kDa viral enzyme that has no mammalian counterpart, rendering it an attractive target for antiviral drug design. Herein, we present a novel approach toward elucidating "hot spots" of protein-protein or protein-nucleic acid interactions of IN through the design of peptides that encompass conserved amino acids and residues known to be important for enzymatic activity. We designed small peptides (7-17 residues) containing at least one amino acid residue that is important for IN catalytic activities (3'-processing and strand transfer) or viral replication. All these peptides were synthesized on solid phase by fluorenylmethoxycarbonyl (Fmoc) chemistry and evaluated for their inhibition of IN catalytic activities. Such specific sites of interest (i.e., protein-DNA or protein-drug interactions) could potentially be used as drug targets. This novel "sequence walk" strategy across the entire 288 residues of IN has allowed the identification of two peptides NL-6 and NL-9 with 50% inhibitory concentration (IC50) values of 2.7 and 56 microM for strand transfer activity, respectively. Amino acid substitution analysis on these peptides revealed essential residues for activity, and the rational truncation of NL-6 produced a novel hexapeptide (peptide NL6-5) with inhibitory potency equal to that of the parent dodecapeptide (peptide NL-6). More significantly, the retroinverso analogue of NL-6 (peptide RDNL-6) in which the direction of the sequence is reversed and the chirality of each amino acid residue is inverted displayed improved inhibitory potency against 3'-processing of HIV-1 IN by 6-fold relative to the parent NL-6, serving as a metabolically stable derivative for further in vitro and in vivo analyses.

Amino Acid Sequence↗
PubMed2002

Flavanone and flavonol glycosides from the leaves of Thevetia peruviana and their HIV-1 reverse transcriptase and HIV-1 integrase inhibitory activities.

Two new flavanone glucosides, (2R)- and (2S)-5-O-beta-D-glucopyranosyl-7,4'-dihydroxy-3',5'-dimethoxyflavanone[pervianoside I (3), peruvianoside II(4)] and a new flavonol glycoside, quercetin 3-O-[beta-D-glucopyranosyl-(1-->2)-[alpha-L-rhamnopyranosyl-(1-->6)]-beta-D-galactopyranoside] (peruvianoside III, 13) were isolated from the leaves of Thevetia peruviana Schum., together with nine known flavonol glycosides and two known iridoid glucosides. The structures of all compounds were determined on the basis of chemical and spectroscopic methods. Their inhibitory effects against HIV-1 reverse transcriptase and HIV-1 integrase were also investigated.

Flavonoids↗
PubMed1995

The DNA-binding domain of HIV-1 integrase has an SH3-like fold.

We have determined the solution structure of the DNA-binding domain of HIV-1 integrase by nuclear magnetic resonance spectroscopy. In solution, this carboxyterminal region of integrase forms a homodimer, consisting of two structures that closely resemble Src-homology 3 (SH3) domains. Lys 264, previously identified by mutagenesis studies to be important for DNA binding of the integrase, as well as several adjacent basic amino acids are solvent exposed. The identification of an SH3-like domain in integrase provides a new potential target for drug design.

Amino Acid Sequence↗
PubMed2002

X-ray absorption spectroscopic studies of zinc in the N-terminal domain of HIV-2 integrase and model compounds.

X-ray absorption spectroscopy (XAS), including extended X-ray absorption fine structure (EXAFS) and X-ray absorption near-edge structure (XANES) analysis, has been carried out at the Zn K edge of the N-terminal part of the integrase protein of the human immunodeficiency virus, type 2 (HIV-2), and of some zinc coordination compounds. In the presence of excess beta-mercaptoethanol, which was present in the NMR structure elucidation of the protein [Eijkelenboom et al. (1997), Curr. Biol. 7, 739-746; (2000), J. Biomol. NMR, 18, 119-28], the protein spectrum was nearly identical to that recorded in its absence. Comparison of the XANES of the protein with that of model compounds and literature data permits the conclusion that the Zn ion is four-coordinated. The major shell of the EXAFS provides evidence for a mixed (N or O as well as S) coordination sphere, while the minor shells indicate imidazole coordination. Our approach to the analysis of the EXAFS, including quantification of the imidazole by multiple scattering simulations with EXCURV92, was validated on the model compounds. An important result is that with multiple scattering simulations using restraints on the parameters of the imidazole rings the number of imidazoles and their orientation could be determined. The integrase spectra can be fitted with two sulfur ligands at 2.26 A (Debye-Waller-type factor 0.009 A(2)) and two imidazole ligands with the N atoms at 1.99 A (Debye-Waller-type factor 0.005 A(2)). The XAS-derived geometry is fully consistent with that found in the NMR structure determination and, allowing for the Volume contraction due to the temperature difference between the experiments, justifies the restraints applied in the structure calculation (Zn-S and Zn-N distances of 2.3 A and 2.0 A, respectively).

Absorptiometry, Photon↗
PubMed2002

HIV-1 integrase interaction with U3 and U5 terminal sequences in vitro defined using substrates with random sequences.

Successful integration of viral genome into a host chromosome depends on interaction between viral integrase and its recognition sequences. We have used a reconstituted concerted human immunodeficiency virus, type 1 (HIV-1), integration system to analyze the role of integrase (IN) recognition sequences in formation of the IN-viral DNA complex capable of concerted integration. HIV-1 integrase was presented with substrates that contained all 4 bases at 8 mismatched positions that define the inverted repeat relationship between U3 and U5 long terminal repeats (LTR) termini and at positions 17-19, which are conserved in the termini. Evidence presented indicates that positions 17-20 of the IN recognition sequences are needed for a concerted DNA integration mechanism. All 4 bases were found at each randomized position in sequenced concerted DNA integrants, although in some instances there were preferences for specific bases. These results indicate that integrase tolerates a significant amount of plasticity as to what constitutes an IN recognition sequence. By having several positions randomized, the concerted integrants were examined for statistically significant relationships between selections of bases at different positions. The results of this analysis show not only relationships between different positions within the same LTR end but also between different positions belonging to opposite DNA termini.

Base Sequence↗
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↗
PubMed2006

Antiviral activity, pharmacokinetics, and dose response of the HIV-1 integrase inhibitor GS-9137 (JTK-303) in treatment-naive and treatment-experienced patients.

BACKGROUND: GS-9137 is a potent low-nanomolar strand transfer inhibitor of HIV-1 integrase. METHODS: The antiviral activity, tolerability, pharmacokinetics, and pharmacodynamics of GS-9137 were evaluated in a randomized, double-blind, placebo-controlled monotherapy study in 40 HIV-1- infected patients not receiving antiretroviral therapy with an HIV-1 RNA between 10,000 and 300,000 copies/mL and a CD4 count of 200 cells/microL or greater. GS-9137 or matching placebo was administered with food for 10 days at 5 dosage regimens (200, 400, or 800 mg BID, 800 mg QD, or 50 mg+100 mg ritonavir QD; 6 active, 2 placebo per dose level). The primary end point was the maximum reduction from baseline in log10 HIV-1 RNA. RESULTS: Forty patients were enrolled, with a mean baseline viral load of 4.75 log10 copies/mL and a CD4 count of 442 cells/microL. Each GS-9137 dosing regimen exhibited significant, exposure-dependent (mean reductions, -0.98 to -1.99 log10 copies/mL) antiviral activity compared with placebo (P<0.01). Twice-daily administrations of GS-9137 at doses of 400 or 800 mg or once-daily dosing of 50 mg with ritonavir demonstrated mean reductions from baseline in HIV-1 RNA of 1.91 log10 copies/mL or greater, with all patients exhibiting 1 log10 or greater and 50% having 2 log10 or greater reductions. No patient developed evidence of integrase resistance. GS-9137 showed an adverse event profile similar to placebo, and there were no study drug discontinuations. CONCLUSIONS: GS-9137 demonstrated substantial short-term antiviral activity and was well tolerated as monotherapy, thus warranting further study.

Acquired Immunodeficiency Syndrome↗
PubMed2001

Structure-based HIV-1 integrase inhibitor design: a future perspective.

The pol gene of HIV-1 encodes for three essential enzymes, protease (PR), reverse transcriptase (RT) and integrase (IN). More than 16 drugs, targeting two of these enzymes, PR and RT have been approved by the FDA. At present, there are no clinically useful agents that inhibit the third enzyme, IN. Combination chemotherapy consisting of PR and RT inhibitors has shown remarkable success in the clinic and has benefited many patients. It is thought that a combination of drugs targeting all three enzymes should further incapacitate the virus. Discovery of highly selective PR inhibitors owe their success to the recent development in structure-guided drug design. During the past several years a plethora of structures of HIV-1 PR in complex with an inhibitor have been solved by x-ray crystallography. This incredible wealth of information provided opportunities for the discovery of second and third generation inhibitors. Due to the inherent nature of IN and insufficient structural information, structure-based inhibitor design selective for IN has not kept pace. However, because of recent developments in the field such information could soon become available. In this review, emphasis is placed on inhibitors with identified or proposed drug binding sites on IN.

Amino Acid Sequence↗
PubMed1995

Incorporation of a fluorescent guanosine analog into oligonucleotides and its application to a real time assay for the HIV-1 integrase 3'-processing reaction.

We have synthesized a highly fluorescent (quantum yield 0.88) guanosine analog, (3-methyl-8-(2-deoxy-beta-D-ribofuranosyl) isoxanthopterin (3-Mi) in a dimethoxytrityl, phosphoramidite protected form, which can be site-specifically inserted into oligonucleotides through a 3',5'-phosphodiester linkage using an automated DNA synthesizer. Fluorescence is partially quenched within an oligonucleotide and the degree of quench is a function of the fluorophore's proximity to purines and its position in the oligonucleotide. As an example of the potential utility of this class of fluorophores, we developed a continuous assay for HIV-1 integrase 3'-processing reaction by incorporating 3-MI at the cleavage site in a double-stranded oligonucleotide identical to the U5 terminal sequence of the HIV genome. Integrase cleaves the 3'-terminal dinucleotide containing the fluorophore, resulting in an increase in fluorescence which can be monitored on a spectrofluorometer. Substitution of the fluorophore for guanosine at the cleavage site does not inhibit integrase activity. This assay is specific for the 3'-processing reaction. The change in fluorescence intensity is linear over time and proportional to the rate of the reaction. This assay demonstrates the potential utility of this new class of fluorophore for continuous monitoring of protein/DNA interactions.

Base Sequence↗
PubMed1997

A metal-induced conformational change and activation of HIV-1 integrase.

Retroviral integrases are composed of three independently folding domains whose organization relevant to one another is largely unknown. As an approach to understanding its structure, we have investigated the effect of the required metal cofactor(s), Mn2+ or Mg2+, on the conformation of human immunodeficiency virus type 1 (HIV-1) integrase (IN) using monoclonal antibodies (mAbs) that are specific for each of these three domains. Upon the addition of increasing concentrations of the divalent cations to immobilized HIV-1 IN in ELISA assays, binding of mAbs specific for either the C-terminal domain or for an epitope in the catalytic core domain was lost, whereas binding of an N terminus-specific mAb was unaffected. Size exclusion chromatography of a nonaggregating derivative of HIV-1 IN showed that the oligomeric state of the protein did not change under conditions in which recognition of the core and C terminus-specific mAbs was lost. Preincubation with Mn2+ increased the resistance of HIV-1 IN to proteolytic digestion and produced a digestion pattern that was significantly different from that observed with the apoprotein. A derivative that lacked the N-terminal domain, IN(50-288), exhibited the same metal-dependent changes observed with the full-length protein, whereas the isolated catalytic core domain IN(50-212) did not. From this we conclude that the metal-induced conformational change comprises a reorganization of the core and C-terminal domains. Preincubation with Mn2+ increased the specific activity of HIV-1 IN 5-fold. Enzymatic activity was inhibited by the conformation-sensitive C terminus-specific mAb, but this inhibition was reduced greatly if the enzyme was first preincubated with metal ions. Thus, it appears that apo-HIV-1 IN exists predominantly in an inactive conformation that is converted into a catalytically competent form upon the addition of metal ions.

Antibodies, Monoclonal↗
PubMed2001

Molecular electrostatic potentials as input for the alignment of HIV-1 integrase inhibitors in 3D QSAR.

Comparative molecular similarity indices analysis (CoMSIA), a three-dimensional quantitative structure activity relationship (3D QSAR) paradigm, was used to examine the correlations between the calculated physicochemical properties and the in vitro activities (3'-processing and 3'-strand transfer inhibition) of a series of human immunodeficiency virus type 1 (HIV-1) integrase inhibitors. The training set consisted of 34 molecules from five structurally diverse classes: salicylpyrazolinones, dioxepinones, coumarins, quinones, and benzoic hydrazides. The data set was aligned using extrema of molecular electrostatic potentials (MEPs). The predictive ability of the resultant model was evaluated using a test set comprised of 7 molecules belonging to a different structural class of thiazepinediones. A CoMSIA model using an MEP-based alignment showed considerable internal as well external predictive ability (r2(cv) = 0.821, r2(pred) = 0.608 for 3'-processing; and r2(cv) = 0.759, r2(pred.) = 0.660 for 3'-strand transfer).

Computer Simulation↗
PubMed1996

Assaying the activity of HIV-1 integrase with DNA-coated plates.

Integration of reverse transcribed viral DNA of HIV into host chromosomes is mediated by the viral enzyme, integrase. This enzymatic activity can be monitored in vitro by integration of a small labeled DNA (donor) into a second unlabeled DNA (target). The methodology usually involves isotope labeling and gel electrophoresis. To simplify the measurement, a method mimicking enzyme-linked immunosorbent assay (ELISA) procedures was developed. Fragments of DNA were adsorbed directly on 96-well plates and used as the target DNA. The donor was a synthetic 21-bp DNA duplex of HIV-1 U5 LTR; biotin was incorporated into the 5' end of one strand whose two nucleotides at the 3' end were specifically removed during the integration. As a result of integration, the biotin-labeled donor DNA was joined with the target DNA and became immobilized on plates. These integration products were then measured by binding of avidin-alkaline phosphatase on plates. The method is simple and straightforward and can easily be adapted for high throughput screening of integrase inhibitors.

DNA↗