Showing papers on "Integrase published in 2003"

Anticancer and antiviral sulfonamides.

Abstract: The sulfonamides constitute an important class of drugs, with several types of pharmacological agents possessing antibacterial, anti- carbonic anhydrase, diuretic, hypoglycemic and antithyroid activity among others. A large number of structurally novel sulfonamide derivatives have ultimately been reported to show substantial antitumor activity in vitro and in vivo. Although they have a common chemical motif of aromatic/heterocyclic or amino acid sulfonamide, there are a variety of mechanisms of their antitumor action, such as carbonic anhydrase inhibition, cell cycle perturbation in the G1 phase, disruption of microtubule assembly, functional suppression of the transcriptional activator NF-Y, and angiogenesis (matrix metalloproteinase, MMP) inhibition among others. Some of these compounds selected via elaborate preclinical screenings or obtained through computer-based drug design, are currently being evaluated in clinical trials. The review summarizes recent classes of sulfonamides and related sulfonyl derivatives disclosed as effective tumor cell growth inhibitors, or for the treatment of different types of cancer. Another research line that progressed much in the last time regards different sulfonamides with remarkable antiviral activity. Thus, at least two clinically used HIV protease inhibitors possess sulfonamide moieties in their molecules, whereas a very large number of other derivatives are constantly being synthesized and evaluated in order to obtain compounds with less toxicity or activity against drug-resistant viruses. Several non nucleoside HIV reverse transcriptase or HIV integrase inhibitors containing sulfonamido groups were also reported. Another approach to inhibit the growth of retroviruses, including HIV, targets the ejection of zinc ions from critical zinc finger viral proteins, which has as a consequence the inhibition of viral replication in the absence of mutations leading to drug resistance phenotypes. Most compounds with antiviral activity possessing this mechanism of action incorporate in their molecules primary sulfonamide groups. Some small molecule chemokine antagonists acting as HIV entry inhibitors also possess sulfonamide functionalities in their scaffold.

Integration Site for Streptomyces Phage φBT1 and Development of Site-Specific Integrating Vectors

Abstract: Despite extensive similarities between the genomes of the Streptomyces temperate phages phiC31 and phiBT1, the attP-int loci are poorly conserved. Here we demonstrate that phiBT1 integrates into a different attachment site than phiC31. phiBT1 attB lies within SCO4848 encoding a 79-amino-acid putative integral membrane protein. Integration vectors based on phiBT1 integrase were shown to have a broad host range and are fully compatible with those based on the phiC31 attP-int locus.

Design and Synthesis of 8-Hydroxy-[1,6]Naphthyridines as Novel Inhibitors of HIV-1 Integrase in Vitro and in Infected Cells

Abstract: 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.

Site-specific cassette exchange and germline transmission with mouse ES cells expressing φC31 integrase

Abstract: Currently two site-specific recombinases are available for engineering the mouse genome: Cre from P1 phage and Flp from yeast. Both enzymes catalyze recombination between two 34-base pair recognition sites, lox and FRT, respectively, resulting in excision, inversion, or translocation of DNA sequences depending upon the location and the orientation of the recognition sites. Furthermore, strategies have been designed to achieve site-specific insertion or cassette exchange. The problem with both recombinase systems is that when they insert a circular DNA into the genome (trans event), two cis-positioned recognition sites are created, which are immediate substrates for excision. To stabilize the trans event, functional mutant recognition sites had to be identified. None of the systems, however, allowed efficient selection-free identification of insertion or cassette exchange. Recently, an integrase from Streptomyces phage phiC31 has been shown to function in Schizosaccharomyces pombe and mammalian cells. This enzyme recombines between two heterotypic sites: attB and attP. The product sites of the recombination event (attL and attR) are not substrates for the integrase. Therefore, the phiC31 integrase is ideal to facilitate site-specific insertions into the mammalian genome.

The Barrier-to-Autointegration Factor Is a Component of Functional Human Immunodeficiency Virus Type 1 Preintegration Complexes

Abstract: Retroviral integration in vivo is mediated by preintegration complexes (PICs) derived from infectious virions. In addition to the integrase enzyme and cDNA substrate, PICs contain a variety of viral and host cell proteins. Whereas two different cell proteins, high-mobility group protein A1 (HMGA1) and the barrier-to-autointegration factor (BAF), were identified as integration cofactors based on activities in in vitro PIC assays, only HMGA1 was previously identified as a PIC component. By using antibodies against known viral and cellular PIC components, we demonstrate here functional coimmunoprecipitation of endogenous BAF protein with human immunodeficiency virus type 1 (HIV-1) PICs. Since integrase protein and integration activity were also coimmunoprecipitated by anti-BAF antibodies, we conclude that BAF is a component of HIV-1 PICs. These data are consistent with the model that BAF functions as an integration cofactor in vivo.

Human Immunodeficiency Virus Type 1 Nucleocapsid Zn2+ Fingers Are Required for Efficient Reverse Transcription, Initial Integration Processes, and Protection of Newly Synthesized Viral DNA

Abstract: Human immunodeficiency virus type 1 (HIV-1) containing mutations in the nucleocapsid (NC) Zn2+ finger domains have greatly reduced infectivity, even though genome packaging is largely unaffected in certain cases. To examine replication defects, viral DNA (vDNA) was isolated from cells infected with viruses containing His-to-Cys changes in their Zn2+ fingers (NCH23C and NCH44C), an integrase mutant (IND116N), a double mutant (NCH23C/IND116N), or wild-type HIV-1. In vitro assays have established potential roles for NC in reverse transcription and integration. In vivo results for these processes were obtained by quantitative PCR, cloning of PCR products, and comparison of the quantity and composition of vDNA generated at discrete points during reverse transcription. Quantitative analysis of the reverse transcription intermediates for these species strongly suggests decreased stability of the DNA produced. Both Zn2+ finger mutants appear to be defective in DNA synthesis, with the minus- and plus-strand transfer processes being affected while interior portions of the vDNA remain more intact. Sequences obtained from PCR amplification and cloning of 2-LTR circle junction fragments revealed that the NC mutants had a phenotype similar to the IN mutant; removal of the terminal CA dinucleotides necessary for integration of the vDNA is disabled by the NC mutations. Thus, the loss of infectivity in these NC mutants in vivo appears to result from defective reverse transcription and integration processes stemming from decreased protection of the full-length vDNA. Finally, these results indicate that the chaperone activity of NC extends from the management of viral RNA through to the full-length vDNA.

Metal-Dependent Inhibition of HIV-1 Integrase by β-Diketo Acids and Resistance of the Soluble Double-Mutant (F185K/C280S)

Abstract: The beta-diketo acids (DKAs) represent a major advance for anti-HIV-1 integrase drug development. We compared the inhibition of HIV-1 integrase by six DKA derivatives using the wild-type enzyme or the double-mutant F185K/C280S, which has been previously used for crystal structure determinations. With the wild-type enzyme, we found that DKAs could be classified into two groups: those similarly potent in the presence of magnesium and manganese and those potent in manganese and relatively ineffective in the presence of magnesium. Both the aromatic and the carboxylic or tetrazole functions of DKAs determined their metal selectivity. The F185K/C280S enzyme was markedly more active in the presence of manganese than magnesium. The F185K/C280S integrase was also relatively resistant to the same group of DKAs that were potent in the presence of magnesium with the wild-type enzyme. Resistance was caused by a synergistic effect from both the F185K and C280S mutations. Molecular modeling and docking suggested metal-dependent differences for binding of DKAs. Molecular modeling also indicated that the tetrazole or the azido groups of some derivatives could directly chelate magnesium or manganese in the integrase catalytic site. Together, these experiments suggest that DKAs recognize conformational differences between wild-type and the double-mutant HIV-1 integrase, because they chelate the magnesium or manganese in the enzyme active site and compete for DNA binding.

Controlling integration specificity of a yeast retrotransposon

Abstract: Retrotransposons and retroviruses integrate nonrandomly into eukaryotic genomes. For the yeast retrotransposon Ty5, integration preferentially occurs within domains of heterochromatin. Targeting to these locations is determined by interactions between an amino acid sequence motif at the C terminus of Ty5 integrase (IN) called the targeting domain, and the heterochromatin protein Sir4p. Here we show that new Ty5 integration hot spots are created when Sir4p is tethered to ectopic DNA sites. Targeting to sites of tethered Sir4p is abrogated by single amino acid substitutions in either IN or Sir4p that prevent their interaction. Ty5 target specificity can be altered by replacing the IN-targeting domain with other peptide motifs that interact with known protein partners. Integration occurs at high efficiency and in close proximity to DNA sites where the protein partners are tethered. These findings define a mechanism by which retrotransposons shape their host genomes and suggest ways in which retroviral integration can be controlled.

Mycobacteriophage Bxb1 integrates into the Mycobacterium smegmatis groEL1 gene

Abstract: Mycobacteriophage Bxb1 is a temperate phage of Mycobacterium smegmatis and forms stable lysogens in which the Bxb1 genome is integrated into the host chromosome. Bxb1 encodes an integrase of the large serine recombinase family that catalyses integration and excision of the Bxb1 genome. We show here that Bxb1 integrates into a chromosomal attB site located within the 3' end of the groEL1 gene such that integration results in alteration of the C-terminal 21 amino acid residues. An integration-proficient plasmid vector containing the Bxb1 integrase gene and flanking DNA sequences efficiently transforms M. smegmatis via integration at attB. Bxb1-integrated recombinants are stable and fully compatible with L5 integration vectors. Strand exchange occurs within an 8 bp common core sequence present in attB and within an attP site situated immediately upstream of the phage integrase gene. Establishment of a defined in vitro system for Bxb1 integration shows that recombination occurs efficiently without requirement for high-energy cofactors, divalent metals, DNA supercoiling or additional proteins.

Sip, an integrase protein with excision, circularization and integration activities, defines a new family of mobile Staphylococcus aureus pathogenicity islands.

Abstract: We report the complete sequence of Staphylococcal pathogenicity island bovine 2 (SaPIbov2), encoding the biofilm-associated protein Bap. SaPIbov2 contains 24 open reading frames, including sip, which encodes a functional staphylococcal integrase protein. SaPIbov2 is bordered by 18 bp direct repeats. The integration site into the chromosome lies at the 3' end of a gene encoding GMP synthase. SaPIbov2 has extensive similarity to previously described pathogenicity islands of Staphylococcus aureus. The principal difference is that toxin genes present in the other pathogenicity islands are exchanged for a transposon-like element that carries the bap gene and genes encoding an ABC transporter and a transposase. Also, SaPIbov2 can be excised to form a circular element and can integrate site-specifically and RecA-independently at a chromosomal att site in a Sip-dependent manner. This was demonstrated both in S. aureus and with plasmid substrates ectopically in Escherichia coli. Thus, SaPIbov2 encodes a functional recombinase of the integrase family that promotes element excision and insertion/integration. In addition, we demonstrated that the presence of SaPIbov2 facilitated the persistence of S. aureus in an intramammary gland infection model. Finally, different bovine isolates of S. aureus were found to carry islands related to SaPIbov2, suggesting the existence of a family of related pathogenicity islands.

Evidence that the retroviral DNA integration process triggers an ATR-dependent DNA damage response

Abstract: Caffeine is an efficient inhibitor of cellular DNA repair, likely through its effects on ATM (ataxia telangiectasia mutated) and ATR (ATM and Rad3-related) kinases. Here, we show that caffeine treatment causes a dose-dependent reduction in the total amount of HIV-1 and avian sarcoma virus retroviral vector DNA that is joined to host DNA in the population of infected cells and also in the number of transduced cells. These changes were observed at caffeine concentrations that had little or no effect on overall cell growth, synthesis, and nuclear import of the viral DNA, or the activities of the viral integrase in vitro. Substantial reductions in the amount of host-viral-joined DNA in the infected population, and in the number of transductants, were also observed in the presence of a dominant-negative form of the ATR protein, ATRkd. After infection, a significant fraction of these cells undergoes cell death. In contrast, retroviral transduction is not impeded in ATM-deficient cells, and addition of caffeine leads to the same reduction that was observed in ATM-proficient cells. These results suggest that activity of the ATR kinase, but not the ATM kinase, is required for successful completion of the viral DNA integration process and/or survival of transduced cells. Components of the cellular DNA damage repair response may represent potential targets for antiretroviral drug development.

Human Immunodeficiency Virus Type 1 (HIV-1) Vpr Enhances Expression from Unintegrated HIV-1 DNA

Abstract: Retroviral DNA synthesized prior to integration, termed unintegrated viral DNA, is classically believed to be transcriptionally inert and to serve only as a precursor to the transcriptionally active integrated proviral DNA form. However, it has recently been found to be expressed under some circumstances during human immunodeficiency virus type 1 (HIV-1) replication and may play a significant role in HIV-1 pathogenesis. HIV-1 Vpr is a virion-associated accessory protein that is critical for HIV-1 replication in nondividing cells and induces cell cycle arrest and apoptosis. We find that Vpr, either expressed de novo or released from virions following viral entry, is essential for unintegrated viral DNA expression. HIV-1 mutants defective for integration in either the integrase catalytic domain or the cis-acting att sites can express unintegrated viral DNA at levels similar to that of wild-type HIV-1, but only in the presence of Vpr. In the absence of Vpr, the expression of unintegrated viral DNA decreases 10- to 20-fold. Vpr does not affect the efficiency of integration from integrase-defective HIV-1. Vpr-mediated enhancement of expression from integrase-defective HIV-1 requires that the viral DNA be generated in cells through infection and is mediated via a template that declines over time. Vpr activation of expression does not require exclusive nuclear localization of Vpr nor does it correlate with Vpr-mediated cell cycle arrest. These results attribute a new function to HIV-1 Vpr and implicate Vpr as a critical component in expression from unintegrated HIV-1 DNA.

Development of Resistance against Diketo Derivatives of Human Immunodeficiency Virus Type 1 by Progressive Accumulation of Integrase Mutations

Abstract: 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.

Isolation, structure, and HIV-1-integrase inhibitory activity of structurally diverse fungal metabolites

Abstract: HIV-1 integrase is a critical enzyme for replication of HIV, and its inhibition is one of the most promising new drug strategies for anti-retroviral therapy, with potentially significant advantages over existing therapies. In this report, a series of HIV-1 inhibitors isolated from the organic extract of fermentations from terrestrial fungi is described. These fungal species, belonging to a variety of genera, were collected from throughout the world following the strict guidelines of Rio Convention on Biodiversity. The polyketide- and terpenoid-derived inhibitors are represented by two naphthoquinones, a biphenyl and two triphenyls, a benzophenone, four aromatics with or without catechol units, a linear aliphatic terpenoid, a diterpenoid, and a sesterterpenoid. These compounds inhibited the coupled and strand-transfer reaction of HIV-1 integrase with an IC(50) value of 0.5-120 micro M. The bioassay-directed isolation, structure elucidation, and HIV-1 inhibitory activity of these compounds are described.

The Human Polycomb Group EED Protein Interacts with the Integrase of Human Immunodeficiency Virus Type 1

Abstract: Human EED, a member of the superfamily of WD-40 repeat proteins and of the Polycomb group proteins, has been identified as a cellular partner of the human immunodeficiency virus type 1 (HIV-1) matrix (MA) protein (R. Peytavi et al., J. Biol. Chem. 274:1635-1645, 1999). In the present study, EED was found to interact with HIV-1 integrase (IN) both in vitro and in vivo in yeast. In vitro, data from mutagenesis studies, pull-down assays, and phage biopanning suggested that EED-binding site(s) are located in the C-terminal domain of IN, between residues 212 and 264. In EED, two putative discrete IN-binding sites were mapped to its N-terminal moiety, at a distance from the MA-binding site, but EED-IN interaction also required the integrity of the EED last two WD repeats. EED showed an apparent positive effect on IN-mediated DNA integration reaction in vitro, in a dose-dependent manner. In situ analysis by immunoelectron microscopy (IEM) of cellular distribution of IN and EED in HIV-1-infected cells (HeLa CD4+ cells or MT4 lymphoid cells) showed that IN and EED colocalized in the nucleus and near nuclear pores, with maximum colocalization events occurring at 6 h postinfection (p.i.). Triple colocalizations of IN, EED, and MA were also observed in the nucleoplasm of infected cells at 6 h p.i., suggesting the ocurrence of multiprotein complexes involving these three proteins at early steps of the HIV-1 virus life cycle. Such IEM patterns were not observed with a noninfectious, envelope deletion mutant of HIV-1.

φC31 Integrase-Mediated Nonviral Genetic Correction of Junctional Epidermolysis Bullosa

Abstract: Patients afflicted with severe laminin 5-deficient junctional epidermolysis bullosa (JEB) often die in infancy with massive cutaneous blistering. Prior approaches to genetically correct this disorder have relied on stable integration of wild-type LAMB3 sequences, using retroviral vectors. To develop a nonviral approach to JEB gene therapy, we used the phiC31 integrase, which mediates unidirectional genomic integration of plasmids containing a specific attB site. An attB-containing laminin 5 beta3 expression plasmid was integrated into the genomes of primary keratinocytes from four unrelated, genetically characterized JEB patients. phiC31 integrase supported genomic integration into epidermal progenitor cells. Regeneration of human skin on immunedeficient mice, using these cells, produced human skin tissue with restored laminin 5 expression. Furthermore, corrected JEB tissue restored hemidesmosome formation and abolished histologic evidence of subepidermal blistering. These findings provide an approach to durable nonviral correction of JEB.

S-1360 Shionogi-GlaxoSmithKline.

Abstract: Shionogi and GlaxoSmithKline, as the joint venture company Shionogi-GlaxoSmithKline Pharmaceuticals, is developing S-1360, an HIV integrase inhibitor as a potential treatment for HIV infection. As of October 2002, phase II trials were ongoing and launch is expected in 2004/2005.

Phage integrases for the construction and manipulation of transgenic mammals

Abstract: Phage integrases catalyze site-specific, unidirectional recombination between two short att recognition sites. Recombination results in integration when the att sites are present on two different DNA molecules and deletion or inversion when the att sites are on the same molecule. Here we demonstrate the ability of the φC31 integrase to integrate DNA into endogenous sequences in the mouse genome following microinjection of donor plasmid and integrase mRNA into mouse single-cell embryos. Transgenic early embryos and a mid-gestation mouse are reported. We also demonstrate the ability of the φC31, R4, and TP901-1 phage integrases to recombine two introduced att sites on the same chromosome in human cells, resulting in deletion of the intervening material. We compare the frequencies of mammalian chromosomal deletion catalyzed by these three integrases in different chromosomal locations. The results reviewed here introduce these bacteriophage integrases as tools for site-specific modification of the genome for the creation and manipulation of transgenic mammals.

Unusual Integrase Gene Expression on the clc Genomic Island in Pseudomonas sp. Strain B13

Abstract: An unusual type of gene expression from an integrase promoter was found in cultures of the bacterium Pseudomonas sp. strain B13. The promoter controls expression of the intB13 integrase gene, which is present near the right end of a 105-kb conjugative genomic island (the clc element) encoding catabolism of aromatic compounds. The enzymatic activity of integrase IntB13 is essential for site-specific integration of the clc element into the bacterial host’s chromosome. By creating transcription fusions between the intB13 promoter and the gfp gene, we showed that integrase expression in strain B13 was inducible under stationary-phase conditions but, strangely, occurred in only a small proportion of individual bacterial cells rather than equally in the whole population. Integrase expression was significantly stimulated by growing cultures on 3-chlorobenzoate. High cell density, heat shock, osmotic shock, UV irradiation, and treatment with alcohol did not result in measurable integrase expression. The occurrence of the excised form of the clc element and an increase in the rates of clc element transfer in conjugation experiments correlated with the observed induction of the intB13-gfp fusion in stationary phase and in the presence of 3-chlorobenzoate. This suggested that activation of the intB13 promoter is the first step in stimulation of clc transfer. To our knowledge, this is the first report of a chlorinated compound’s stimulating horizontal transfer of the genes encoding its very metabolism.

Intracellular transport of human immunodeficiency virus type 1 integrase.

Abstract: The human immunodeficiency virus type 1 integrase protein has karyophilic properties; that is, it localizes to the cell nucleus according to a range of assays. As an essential component of the preintegration complex, it has been suggested that the karyophilic properties of integrase might facilitate transport of the preintegration complex through the nuclear pore complexes of nondividing cells. However, no experiments have satisfactorily identified a nuclear localization signal within integrase. In this work, we investigated the karyophilic properties of integrase in intact cells with hopes of identifying a genuine transferable nuclear localization signal. Our results confirm that integrase tightly binds chromosomal DNA in vivo. However, our analysis determined that large integrase fusion proteins are unable to access the nucleus, indicating that integrase might lack a transferable nuclear localization signal. In addition, we present several lines of evidence to indicate that DNA binding might facilitate integrase nuclear accumulation. Furthermore, our data indicate integrase is degraded in the cytoplasm by a proteasome-dependent process, an event that probably contributes to the apparent nuclear accumulation of integrase. These results provide new insight into human immunodeficiency virus type 1 integrase intracellular dynamics.

Small-molecule HIV-1 integrase inhibitors: the 2001-2002 update.

Abstract: Integration of viral DNA into host cell chromosomal DNA to form a provirus is an essential step in the viral life cycle. This process is mediated by integrase (IN), a 32 KDa viral enzyme. The unique properties of IN makes it an ideal target for drug design. First, there arc no cellular homologues to IN and the reactions catalyzed by IN are unique. Second, IN is absolutely required for viral replication and mutations in a number of key residues dramatically block viral replication. Third, IN has been validated as a legitimate target and the results from S-1360 (1) the only available IN inhibitor under clinical trials suggest synergistic effect with reverse transcriptase (RT) and protease (PR) inhibitors. During the past 10 years a plethora of inhibitors have been identified and some were shown to be selective against IN and block viral replication. The two most predominant classes of inhibitors have been the catechol containing hydroxylated aromatics and more recently the diketoacid containing aromatics. Herein, we review all small molecule compounds reported to inhibit recombinant HIV-1 IN with IC 50 values < 20 μM during the past two years. It is important to bear in mind that the true mechanism of action and antiviral activities of many of the compounds are currently not established. However, based on the growing body of literature certain classes of compounds can be easily excluded as bona fide IN inhibitors.

Modeling HIV-1 integrase complexes based on their hydrodynamic properties.

Abstract: We present a model structure of a candidate tetramer for HIV-1 integrase. The model was built in three steps using data from fluorescence anisotropy, structures of the individual integrase domains, cross-linking data, and other biochemical data. First, the structure of the full-length integrase monomer was modeled using the individual domain structures and the hydrodynamic properties of the full-length protein that were recently measured by fluorescence depolarization. We calculated the rotational correlation times for different arrangements of three integrase domains, revealing that only structures with close proximity among the domains satisfied the experimental data. The orientations of the domains were constrained by iterative tests against the data on cross-linking and footprinting in integrase–DNA complexes. Second, the structure of an integrase dimer was obtained by joining the model monomers in accordance with the available dimeric crystal structures of the catalytic core. The hydrodynamic properties of the dimer were in agreement with the experimental values. Third, the active sites of the two model dimers were placed in agreement with the spacing between the sites of integration on target DNA as well as the integrase–DNA cross-linking data, resulting in twofold symmetry of a tetrameric complex. The model is consistent with the experimental data indicating that the F185K substitution, which is found in the model at a tetramerization interface, selectively disrupts correct complex formation in vitro and HIV replication in vivo. Our model of the integrase tetramer bound to DNA may help to design anti-integrase inhibitors. © 2002 Wiley Periodicals, Inc. Biopolymers 68: 110–120, 2003

Dihydroxypyridopyrazine-1,6-dione compounds useful as hiv integrase inhibitors

Abstract: 8,9-Dihydroxydihydropyridopyrazine-1,6-diones and 8,9-dihydroxypyridopyrazine-1,6-diones are inhibitors of HIV integrase and inhibitors of HIV replication. In one embodiment, the pyridopyrazinediones are of Formula (I): (I),wherein R1, R2, R3, R4 and R5 are defined herein. The compounds are useful in the prevention and treatment of infection by HIV and in the prevention, delay in the onset, and treatment of AIDS. The compounds are employed against HIV infection and AIDS as compounds per se or in the form of pharmaceutically acceptable salts. The compounds and their salts can be employed as ingredients in pharmaceutical compositions, optionally in combination with other antivirals, immunomodulators, antibiotics or vaccines. Methods of preventing, treating or delaying the onset of AIDS and methods of preventing or treating infection by HIV are described.