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At least 451 records · Page 25Linked to original sources
PubMed2002

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

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

Binding, Competitive↗
PubMed2004

Mass spectrometric analysis of the HIV-1 integrase-pyridoxal 5'-phosphate complex reveals a new binding site for a nucleotide inhibitor.

HIV-1 integrase (IN) is an important target for designing new antiviral therapies. Screening of potential inhibitors using recombinant IN-based assays has revealed a number of promising leads including nucleotide analogs such as pyridoxal 5'-phosphate (PLP). Certain PLP derivatives were shown to also exhibit antiviral activities in cell-based assays. To identify an inhibitory binding site of PLP to IN, we used the intrinsic chemical property of this compound to form a Schiff base with a primary amine in the protein at the nucleotide binding site. The amino acid affected was then revealed by mass spectrometric analysis of the proteolytic peptide fragments of IN. We found that an IC(50) concentration (15 mum) of PLP modified a single IN residue, Lys(244), located in the C-terminal domain. In fact, we observed a correlation between interaction of PLP with Lys(244) and the compound's ability to impair formation of the IN.DNA complex. Site-directed mutagenesis studies confirmed an essential role of Lys(244) for catalytic activities of recombinant IN and viral replication. Molecular modeling revealed that Lys(244) together with several other DNA binding residues provides a plausible pocket for a nucleotide inhibitor-binding site. To our knowledge, this is the first report indicating that a small molecule inhibitor can impair IN activity through its binding to the protein C terminus. At the same time, our findings highlight the importance of structural analysis of the full-length protein.

Amino Acid Sequence↗
PubMed2005

Peptides derived from the reverse transcriptase of human immunodeficiency virus type 1 as novel inhibitors of the viral integrase.

Recent studies have shown that the integrase (IN) of HIV-1 is inhibited in vitro by HIV-1 reverse transcriptase (RT). We further investigated the specific protein sequences of RT that were involved in this inhibition by screening a complete library of RT-derived peptides for their inhibition of IN activities. Two 20-residue peptides, peptide 4286, derived from the RT DNA polymerase domain, and the one designated 4321, from the RT ribonuclease H domain, inhibit the enzymatic activities of IN in vitro. The former peptide inhibits all three IN-associated activities (3'-end processing, strand transfer, and disintegration), whereas the latter one inhibits primarily the first two functions. We showed the importance of the sequences and peptide length for the effective inhibition of IN activities. Binding assays of the peptides to IN (with no DNA substrate present) indicated that the two inhibitory peptides (as well as several non-inhibitory peptides) interact directly with IN. Moreover, the isolated catalytic core domain of IN also interacted directly with the two inhibitory peptides. Nevertheless, only peptide 4286 can inhibit the disintegration activity associated with the IN core domain, because this activity is the only one exhibited by this domain. This result was expected from the lack of inhibition of disintegration of full-length IN by peptide 4321. The data and the three-dimensional models presented suggested that the inhibition resulted from steric hindrance of the catalytic domain of IN. This information can substantially facilitate the development of novel drugs against HIV INs and thus contribute to the fight against AIDS.

Catalytic Domain↗
PubMed1996

Conserved sequences in the carboxyl terminus of integrase that are essential for human immunodeficiency virus type 1 replication.

We have previously identified a residue in the carboxyl terminus of human immunodeficiency virus type 1 integrase (HIV-1 IN), W-235, the requirement for which is only revealed in viral assays for integrase function (P. M. Cannon, W. Wilson, E. Byles, S. M. Kingsman, and A. J. Kingsman, J. Virol. 68:4768-4775, 1994). Our further analysis of this region of retroviral IN has now identified several sequence motifs which are conserved in all the retroviruses we examined, apart from human spumaretrovirus. We have made mutations within these motifs in HIV-1 IN and examined their phenotypes when reintroduced into an infectious proviral clone. The deleterious effects of several of these mutations demonstrate the importance of these regions for IN function in vivo. We observed a further discrepancy, at a motif that is only conserved in the lentiviruses, in the ability of mutants to function in in vitro and in vivo assays. Substitutions both in this region and at W-235 abolish HIV-1 infectivity but do not affect particle production, morphology, reverse transcription, or nuclear import in T-cell lines. Taken together with the in vitro data suggesting that neither of these residues is directly involved in the catalytic reactions of IN, it seems likely that we have identified regions of IN that are essential for interactions with other components of the integration machinery.

Amino Acid Sequence↗
PubMed1993

Expression of a processed and a non-processed form of the integrase protein of HIV-1 in the baculovirus system.

The integrase (IN) protein of HIV-1 was expressed as a processed and a non-processed protein in the eucaryotic baculovirus expression system. In immunoblots we could demonstrate that recombinant baculoviruses containing the complete gag and pol reading frames of HIV-1 expressed a gag/pol precursor polyprotein. The specific proteolytic activity of the recombinant protease on the gag and pol precursor proteins was used for the generation of processed gag (p 17, p 24, p 16) and pol (RT/RNaseH, IN) proteins. The non-processed IN protein, expressed as a polyhedrin fusion protein, was produced at much higher level than the processed protein.

Animals↗
PubMed1995

Cosalane analogues with enhanced potencies as inhibitors of HIV-1 protease and integrase.

Several new analogues of the novel anti-HIV agent cosalane have been synthesized and evaluated as inhibitors of HIV-1 integrase and protease, HIV-1 replication, HIV-1 and HIV-2 cytopathicity, HIV-1- and HIV-2-mediated syncytium formation, and cytopathicity of a variety of human pathogenic viruses. The congeners displayed enhanced potencies relative to cosalane itself as inhibitors of HIV-1 integrase and protease. The two most potent analogues against HIV-1 integrase displayed IC50 values of 2.2 microM, while the three most potent compounds against HIV-1 protease had IC50 values in the 0.35-0.39 microM range. In addition to its activity against HIV-1 and HIV-2 cytopathicity, cosalane inhibited the cytopathic effects of herpes simplex virus-1, herpes simplex virus-2, and human cytomegalovirus at concentrations that were well below the cytotoxic concentrations. Potentially useful antiviral activities were also revealed for some of the new cosalane congeners against influenza virus, Junin virus, and Tacaribe virus.

Amino Acid Sequence↗
PubMed2005

Active site binding modes of curcumin in HIV-1 protease and integrase.

Structure models for the interaction of curcumin with HIV-1 integrase (IN) and protease (PR) were investigated using computational docking. Curcumin was found to bind preferentially in similar ways to the active sites of both IN and PR. For IN, the binding site is formed by residues Asp64, His67, Thr66, Glu92, Thr93, Asp116, Ser119, Asn120, and Lys159. Docked curcumin contacts the catalytic residues adjacent to Asp116 and Asp64, and near the divalent metal (Mg2+). In the PR docking, the curcumin structure fitted well to the active site, interacting with residues Asp25, Asp29, Asp30, Gly27', Asp29', and Asp30'. The results suggest that o-hydroxyl and/or keto-enol structures are important for both IN and PR inhibitory actions. The symmetrical structure of curcumin seems to play an important role for binding to the PR protein, whereas the keto-enol and only one side of the terminal o-hydroxyl showed tight binding to the IN active site.

Binding Sites↗
PubMed2006

Current developments in HIV chemotherapy.

HIV infection is the leading cause of death worldwide and despite major advances in treatment, more new cases were diagnosed in 2004 than any previous year. Current treatment regimens are based on the use of two or more drugs from two or more classes of inhibitors termed highly active antiretroviral therapy (HAART). Although HAART is capable of suppressing viral loads to undetectable levels, problems of toxicity, patient adherence, and particularly the emergence of drug-resistant viruses continues to spur the development of new chemotherapeutics to combat HIV. Clinical candidates from the four existing classes of inhibitors are presented in this review along with lead compounds against new viral targets, with special emphasis on HIV integrase.

Anti-HIV Agents↗
PubMed1997

Complementation of integrase function in HIV-1 virions.

Proviral integration is essential for HIV-1 replication and represents an important potential target for antiviral drug design. Although much is known about the integration process from studies of purified integrase (IN) protein and synthetic target DNA, provirus formation in virally infected cells remains incompletely understood since reconstituted in vitro assays do not fully reproduce in vivo integration events. We have developed a novel experimental system in which IN-mutant HIV-1 molecular clones are complemented in trans by Vpr-IN fusion proteins, thereby enabling the study of IN function in replicating viruses. Using this approach we found that (i) Vpr-linked IN is efficiently packaged into virions independent of the Gag-Pol polyprotein, (ii) fusion proteins containing a natural RT/IN processing site are cleaved by the viral protease and (iii) only the cleaved IN protein complements IN-defective HIV-1 efficiently. Vpr-mediated packaging restored IN function to a wide variety of IN-deficient HIV-1 strains including zinc finger, catalytic core and C-terminal domain mutants as well as viruses from which IN was completely deleted. Furthermore, trans complemented IN protein mediated a bona fide integration reaction, as demonstrated by the precise processing of proviral ends (5'-TG...CA-3') and the generation of an HIV-1-specific (5 bp) duplication of adjoining host sequences. Intragenic complementation between IN mutants defective in different protein domains was also observed, thereby providing the first evidence for IN multimerization in vivo.

Amino Acid Sequence↗
PubMed2003

Anticancer and antiviral sulfonamides.

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.

Animals↗
PubMed2004

Antiviral sulfonamide derivatives.

The sulfonamides constitute an important class of drugs, with several types of pharmacological agents possessing antibacterial, anti-carbonic anhydrase, diuretic, hypoglycemic, antithyroid and anticancer activity among others. A large number of structurally novel sulfonamide derivatives have ultimately been reported to show substantial antiviral activity in vitro and in vivo. The review summarizes recent classes of sulfonamides and related sulfonyl derivatives disclosed as effective such agents. Thus, at least some HIV protease inhibitors used clinically (amprenavir) or compounds in advanced clinical trials (tipranavir, TMC-126, TMC-114, etc.) 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 sulfonamide 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. Finally, some small molecule chemokine antagonists acting as HIV entry inhibitors also possess sulfonamide functionalities in their scaffold.

Animals↗
PubMed1994

Monoclonal antibodies against HIV type 1 integrase: clues to molecular structure.

Eleven murine hybridoma clones were selected for their ability to produce anti-HIV-1 integrase (IN) antibodies. Competition and epitope mapping studies allowed segregation of the monoclonal antibodies (MAbs) into four distinct classes. The five MAbs that comprise the first class showed high affinity for epitopes within an N-terminal domain of 58 amino acids that includes a conserved zinc finger motif. The second class, with two MAbs, showed high affinity for epitopes within 29 amino acids at the C terminus. Another two MAbs, which constitute the third class, displayed moderate affinities for epitopes that mapped to regions within the highly conserved catalytic core referred to as the D,D(35)E domain. One of these MAbs showed significant cross-reactivity with HIV-2 IN and weak, but detectable, cross-reactivity with RSV IN. The remaining two MAbs, which comprise the fourth class, exhibited fairly low binding affinities and appeared to recognize epitopes in the zinc finger motif domain as well as the C-terminal half of the IN protein. The MAbs can be used for immunoprecipitation and immunoblotting procedures as well as for purification of HIV-1 IN protein by affinity chromatography. We show that several can also be used to immunostain viral IN sequences in HIV-1-infected T cells, presumably as a component of Gag-Pol precursors. Finally, analysis of our mapping and competition data suggests a structure for mature IN in which the C terminus approaches the central core domain, and the N and C termini touch or are proximal to each other. These MAbs should prove useful for further analyses of the structure and function of IN both in vitro and in vivo.

Amino Acid Sequence↗
PubMed2000

Similarities in the HIV-1 and ASV integrase active sites upon metal cofactor binding.

The HIV-1 integrase, which is essential for viral replication, catalyzes the insertion of viral DNA into the host chromosome thereby recruiting host cell machinery into making viral proteins. It represents the third main HIV enzyme target for inhibitor design, the first two being the reverse transcriptase and the protease. We report here a fully hydrated 2 ns molecular dynamics simulation performed using parallel NWChem3.2.1 with the AMBER95 force field. The HIV-1 integrase catalytic domain previously determined by crystallography (1B9D) and modeling including two Mg(2+) ions placed into the active site based on an alignment against an ASV integrase structure containing two divalent metals (1VSH), was used as the starting structure. The simulation reveals a high degree of flexibility in the region of residues 140-149 even in the presence of a second divalent metal ion and a dramatic conformational change of the side chain of E152 when the second metal ion is present. This study shows similarities in the behavior of the catalytic residues in the HIV-1 and ASV integrases upon metal binding. The present simulation also provides support to the hypothesis that the second metal ion is likely to be carried into the HIV-1 integrase active site by the substrate, a strand of DNA.

Avian Sarcoma Viruses↗
PubMed2005

Nef expressed from human immunodeficiency virus type 1 extrachromosomal DNA downregulates CD4 on primary CD4+ T lymphocytes: implications for integrase inhibitors.

Recently developed integrase inhibitors targeting the HIV-1 integrase (IN) protein block integration of HIV DNA in the target cell, preventing subsequent virus replication. In the absence of integration, viral DNA is shunted towards the formation of extrachromosomal DNA (E-DNA). Although HIV-1 E-DNA does not support productive replication, it is transcriptionally active and produces viral proteins. However, the significance of E-DNA in virus replication and pathogenesis is poorly understood. In this study, the functional activity of the HIV-1 Nef protein expressed in the absence of viral integration was analysed. Using both a recombinant HIV-1 IN defective virus and a diketo acid IN inhibitor, evidence was provided showing that Nef expressed from E-DNA downregulates CD4 surface expression on primary CD4(+) T lymphocytes. These results suggest that proteins expressed in the absence of integration may have potential clinical consequences, an issue that should be further explored with the introduction of IN inhibitors.

CD4 Antigens↗
PubMed1996

HIV as the cause of AIDS.

The two known types of HIV are members of a family of primate lentiviruses. HIV, like other retroviruses, contains a virus capsid, which consists of the major capsid protein, the nucleocapsid protein, the diploid single-stranded RNA genome, and the viral enzymes protease, reverse transcriptase, and integrase. HIV isolates show extensive genetic variability, resulting from the relatively low fidelity of reverse transcriptase in conjunction with the extremely high turnover of virions in vivo. These features of HIVs may have strong implications for vaccine development. Simian immunodeficiency viruses from naturally infected animals differ from HIV in one fundamental respect: they do not cause disease in their natural hosts. Study of these viruses may therefore lead to information about the interaction between lentiviruses and host immune response that could be exploited to combat AIDS.

Acquired Immunodeficiency Syndrome↗
PubMed2001

Antiviral Research. Retrovirus infections and antiviral research.

The retrovirus sections of the meeting focused on fundamental, preclinical aspects of the development of antiretroviral agents, although some clinical developments were also discussed. Novel nucleoside analogs, non-nucleoside reverse transcriptase inhibitors, protease inhibitors, as well as preclinical data on new molecules that target early stages of HIV-1 infection (HIV integrase), were discussed. The meeting comprised plenary presentations, short oral communications and over 180 oral or poster presentations, of which more than 70 dealt with inhibition of retrovirus replication with an emphasis on the human immunodeficiency virus (HIV).

Journal Article↗
PubMed2002

Slipping through the door: HIV entry into the nucleus.

HIV infection of non-dividing cellular targets like macrophages requires successful passage of the viral preintegration complex (PIC) across an intact nuclear envelope. Unique but redundant nuclear import signals reside within the HIV integrase, matrix, and Vpr proteins as well as the 'DNA flap'; these signals appear to facilitate PIC transport through the limiting nuclear pores. We discuss recent studies that have advanced our understanding of this key step in the HIV life cycle.

HIV Infections↗