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PubMed2006

Designing HIV integrase inhibitors--shooting the last arrow.

The arsenal of drugs in the fight against AIDS is rapidly diminishing as the HIV becomes resistant to the available reverse transcriptase and protease inhibitors. After killing millions all over the world, the virus is still on the rampage and hence the pharmaceutical industry is resorting to the development of inhibitors of integrase. This seems to be the last arrow in the quiver of potential drug leads to combat the deadly infection. Several classes of HIV integrase inhibitors have been reported to date; however, none is clinically useful. This review details the existing knowledge of the biological functions of the HIV-1 integrase with the focus on its available inhibitors, their disadvantages, and the current trends in designing novel compounds as anti-integrase.

Combinatorial Chemistry Techniques↗
PubMed2004

HIV-1 integrase: a target for new AIDS chemotherapeutics.

Since the beginning of the HIV epidemic almost 70 million people have been infected with HIV. It is estimated that 42 million people are currently living with HIV/AIDS. The spread of HIV continues throughout the world and current estimates indicate that in 2002, 5 million people were newly infected with HIV and 3 million people died. Current treatments employ a combination of therapeutic agents that target the viral reverse transcriptase and protease enzymes and viral entry. However the clinical benefit of these agents is often limited due to issues of regimen compliance, significant side effects, and the emergence of viral strains that are drug resistant. The introduction of novel agents that interfere with alternate stages in the viral life cycle represent potential solutions to these problems. The integration of the HIV genome into the cellular chromosome, a process catalyzed by the viral enzyme integrase, has been shown to be essential for viral replication. Since HIV integrase has no direct cellular counterpart it presents itself as an attractive target for therapeutic intervention. This review summarizes recent and promising developments both in the HIV integrase field and the global quest for therapeutically useful inhibitors of HIV integrase.

Acquired Immunodeficiency Syndrome↗
PubMed2006

Phage phiC31 integrase-mediated genomic integration of the common cytokine receptor gamma chain in human T-cell lines.

BACKGROUND: X-linked severe combined immunodeficiency (SCID-X1, X-SCID) is a life-threatening disease caused by a mutated common cytokine receptor gamma chain (gammac) gene. Although ex vivo gene therapy, i.e., transduction of the gammac gene into autologous CD34(+) cells, has been successful for treating SCID-X1, the retrovirus vector-mediated transfer allowed dysregulated integration, causing leukemias. Here, to explore an alternative gene transfer methodology that may offer less risk of insertional mutagenesis, we employed the phiC31 integrase-based integration system using human T-cell lines, including the gammac-deficient ED40515(-). METHODS: A phiC31 integrase and a neo(r) gene expression plasmid containing the phiC31 attB sequence were co-delivered by electroporation into Jurkat cells. After G418 selection, integration site analyses were performed using linear amplification mediated-polymerase chain reaction (LAM-PCR). ED40515(-) cells were also transfected with a gammac expression plasmid containing attB, and the integration sites were determined. IL-2 stimulation was used to assess the functionality of the transduced gammac in an ED40515(-)-derived clone. RESULTS: Following co-introduction of the phiC31 integrase expression plasmid and the plasmid carrying attB, the efficiency of integration into the unmodified human genome was assessed. Several integration sites were characterized, including new integration sites in intergenic regions on chromosomes 13 and 18 that may be preferred in hematopoietic cells. An ED40515(-) line bearing the integrated gammac gene exhibited stable expression of the gammac protein, with normal IL-2 signaling, as assessed by STAT5 activation. CONCLUSIONS: This study supports the possible future use of this phiC31 integrase-mediated genomic integration strategy as an alternative gene therapy approach for treating SCID-X1.

Bacteriophages↗
PubMed1994

Gene transfer using purified retroviral integrase.

We present a new method of gene transfer into cultured cells using a purified retroviral integrase protein with liposomes. The acceleration rate of transfection by the integrase was increased by a few to ten times. The integrase target sequence containing the 3' end of LTR on the introduced plasmid was necessary for the acceleration, and the orientation of this sequence determined the level of acceleration activity. The analyses of the chromosomal DNAs of each transfectant demonstrated the integration of the introduced plasmid DNA within the integrase-target sequence.

Animals↗
PubMed1998

Cobalamin inhibition of HIV-1 integrase and integration of HIV-1 DNA into cellular DNA.

Our prior studies showed that certain cobalamins inhibit productive HIV-1 infection of primary cultures of blood lymphocytes and monocytes. We demonstrate here that this antiviral activity may be mediated by an inhibition of HIV-1 integrase, an enzyme required for productive infection. Purified recombinant HIV-1 integrase activity was inhibited in vitro by hydroxocobalamin (OH-Cbl), methylcobalamin (Me-Cbl), adenosylcobalamin (Ado-Cbl), and dicyanocobinamide (CN2-Cbi) with IC50 values of approximately 17, 17, 17, and 4 microM, respectively. The agents inhibited HIV-1 infection of cultured monocytes (IC50 values for OH-Cbl, Me-Cbl, Ado-Cbl, and CN2-Cbi of 6, 7, 4, and 1 microM, respectively) and of cultured lymphocytes (IC50 values of 60, 50, 60, and 11 microM, respectively). Experiments using cultured monocytes or lymphocytes demonstrated that OH-Cbl inhibited integration of HIV-1 DNA into cellular DNA. Thus, cobalamins and cobinamides represent novel inhibitors of HIV-1 integrase. These or related agents may be useful as anti-viral treatments that target HIV-1 integrase.

Base Sequence↗
PubMed1995

Identifying determinants of recombination specificity: construction and characterization of chimeric bacteriophage integrases.

Bacteriophage integrases are members of a family of structurally related enzymes that promote recombination between DNA molecules that carry specific sites. Phages lambda and HK022 encode closely related integrases that recognize different sets of sequences within the core regions of their respective attachment sites. To locate the amino acid residues that determine this difference in specificity, we isolated recombinant phages that produce chimeric integrases and measured the ability of these chimeras to promote recombination of lambda and HK022 sites in vivo. A chimera that is of lambda origin except for one HK022 residue at position 99 and 12 HK022 residues located between positions 279 and 329 had wild-type HK022 specificity and activity for both integrative and excisive recombination. Chimeras containing certain subsets of these 13 residues had incomplete specificity. The region around position 99 is not well-conserved in other members of the integrase family, but the 279-329 segment includes residues that are highly conserved and believed to be directly involved in catalysis. Many chimeras were inactive in recombining either HK022 or lambda sites. Selection for mutants that restored activity to these chimeras revealed sets of residues that are likely to interact with each other.

Amino Acid Sequence↗
PubMed1999

A newly identified, essential catalytic residue in a critical secondary structure element in the integrase family of site-specific recombinases is conserved in a similar element in eucaryotic type IB topoisomerases.

The integrase family of site-specific recombinases catalyzes conservative rearrangements between defined segments of DNA. A highly conserved tetrad (RHRY) of catalytic residues is essential for this process. This tetrad is dispersed in two motifs in the linear sequence, but is configured appropriately in the catalytic pocket to execute the strand cleavage and rejoining reactions. A third conserved motif has been identified in the Xer subgroup of the integrase family. Mutational analysis of 12 conserved residues in this motif in the XerD protein from Salmonella typhimurium led to the identification of an essential fifth catalytic residue (lysine 172) which is implicated in strand cleavage or exchange. This lysine residue occupies part of the turn of an antiparallel beta-hairpin which forms one side of the catalytic cleft in XerD, and is found at similar positions among evolutionarily diverse integrase family members. Related antiparallel beta-hairpins are present in eucaryotic type IB topoisomerase enzymes which also contain a critical lysine residue in the turn of the hairpin. In both the integrase family and eucaryotic type IB topoisomerases, the catalytic lysine residues are in close contact with the substrates and may play similar roles in influencing the reactivity of the phosphotyrosine intermediates formed during reactions catalyzed by both enzymes.

Amino Acid Sequence↗
PubMed2000

Evolution of the family of pRN plasmids and their integrase-mediated insertion into the chromosome of the crenarchaeon Sulfolobus solfataricus.

Plasmid pHEN7 from Sulfolobus islandicus was sequenced (7.83 kb) and shown to belong to the archaeal pRN family, which includes plasmids pRN1, pRN2, pSSVx and pDL10 that share a large conserved sequence region. pHEN7 is most closely related to pRN1 in this conserved region. It also shares a large variant region containing several homologous genes with pDL10, which is absent from the other plasmids. The variant region is flanked by the sequence motif TTAGAATGGGGATTC and similar duplicated motifs occur in plasmids pRN1 and pRN2, separated by a few bases. It is inferred that recombination at these sites produces the main genetic variability in the plasmid family. The conserved region of the plasmid, and duplicated copies of the motif, are also present in the genome of Sulfolobus solfataricus P2. Moreover, they are bordered by a partitioned integrase gene (int) and by a 45 bp perfect direct repeat corresponding to the downstream half of a tRNA(Val) gene. The integrase and the direct repeat are highly similar in sequence to the integrase and the chromosomal integration site (att), respectively, of the SSV1 virus, which integrates into the chromosome of Sulfolobus shibatae. Recombination at the att repeats in S. solfataricus would produce a novel plasmid, pXQ1, which carries both an intact integrase gene and a single integration site (att). This strongly suggests that the same mechanism of site-specific integration at a tRNA gene is used for both viruses and plasmids in Sulfolobus.

Attachment Sites, Microbiological↗
PubMed2005

Site-specific gene integration in cultured silkworm cells mediated by phiC31 integrase.

The integrase from the Streptomyces bacteriophage phiC31 carries out efficient recombination between an attP site in the phage genome and an attB site in the host chromosome. In the present study, we have used the phiC31 integrase system to mediate site-specific recombination in the cultured silkworm cell line BmN4. A plasmid containing a cDNA encoding DsRed flanked by two phiC31 attP sites was co-transfected together with a helper plasmid encoding the phiC31 integrase into a cell line in which phiC31 attB sites inserted between a baculovirus IE2 promoter, and a polyadenylation signal are present in one chromosome. Seven days after transfection, expression of DsRed was observed in transformed cells. Nucleotide sequence analysis demonstrated that the expected recombination between the attB and attP sites had been precisely carried out by the phiC31 integrase. These results indicate that the phiC31 site-specific recombination system should be widely applicable for efficient site-specific gene integration into silkworm chromosomes.

Animals↗
PubMed1992

A frog virus 3 gene codes for a protein containing the motif characteristic of the INT family of integrases.

The integrase (INT) family of bacteriophage coded integrase-recombinase proteins are responsible for catalyzing strand exchange between DNA molecules and play an important role in the DNA replication of many bacteriophages. Within the frog virus 3 (FV3) genome we have identified an open reading frame (ORF) of which the deduced amino acid sequence contains a motif characteristic of the INT family of integrases-recombinases. The ORF consists of 825 bp which codes for a protein of 275 amino acids with a predicted Mr of 29,945. RNA transcribed from this ORF during virus infection was detected by Northern blot analysis and it is a delayed early message of approximately 1100 bases. The 5' and 3' ends of the putative FV3 integrase-recombinase transcript were mapped. The transcriptional start site is preceded by a presumptive TATA box, and a region of hyphenated dyad symmetry is present at the 3' end of the message. A protein with an Mr of approximately 30,500 was synthesized by a rabbit reticulocyte lysate programmed with capped runoff transcripts from the cloned gene, indicating that the ORF can be transcribed into a message coding for a viral protein. In the FV3 life cycle, DNA replication occurs in a large complex formed through the recombination of small viral DNA molecules. Thus, at this stage, DNA replication and recombination are interlinked. Resolution of concatameric DNA is required for the packaging of genomes into virus particles. The putative FV3 INT gene may be involved in one or more of these functions.

Amino Acid Sequence↗
PubMed2005

Integration specificity of phage phiC31 integrase in the human genome.

The site-specific integrase from bacteriophage phiC31 functions in mammalian cells and is being applied for genetic engineering, including gene therapy. The phiC31 integrase catalyzes precise, unidirectional recombination between its 30-40-bp attP and attB recognition sites. In mammalian cells, the enzyme also mediates integration of plasmids bearing attB into native sequences that have partial sequence identity with attP, termed pseudo attP sites. Here, we analyzed the features of phiC31-mediated integration into pseudo attP sites in the human genome. Sequence analysis of 196 independent integration events derived from three cell lines revealed approximately 101 integration sites: 56% of the events were recurrent integrations distributed among 19 pseudo attP sequences. Bioinformatics analysis revealed a approximately 30-bp palindromic consensus sequence motif shared by all of the repeat occurrences and most of the single occurrence sites, verifying that phiC31-mediated integration into pseudo attP sites is significantly guided by DNA sequence recognition. The most favored unique sequence in these cell lines occurred at chromosome 19q13.31 and accounted for 7.5% of integration events. Other frequent integration sites were in three specific sequences in subfamilies of ERVL and L1 repetitive sequences, accounting for an additional 17.9% of integration events. Integrations could occur in either orientation at a pseudo attP site, were often accompanied by small deletions, and typically occurred in a single copy per cell. A number of aberrant events were also described, including large deletions and chromosome rearrangements. phiC31 integrase-mediated integration only slightly favored genes and did not favor promoter regions. Gene density and expression studies suggested chromatin context effects. An analysis of the safety of integration sites in terms of proximity to cancer genes suggested minimal cancer risk. We conclude that integration systems derived from phiC31 integrase have great potential utility.

Animals↗
PubMed2006

Characterization of HIV-1 integrase N-terminal mutant viruses.

During infection, human immunodeficiency virus type 1 integrase engages a number of molecules and mechanisms, both of viral and cellular origin. In one of such instances, integrase is thought to be degraded by the N-end rule proteasome pathway a process that targets the N-terminal residue of its substrates. Here we describe the properties of HIV-1 viruses in which the first amino acid residue of integrase has been substituted to render it resistant to the N-end rule pathway. As result of this exchange, we observe a set of class I and class II defects that result in a large decrease of viral replication efficiency. Specifically, reverse transcription and integration are the steps that appear to be affected. We propose that the severe deficiency of these mutants exert a strong selective pressure that leads to the near total conservation of the N-terminal residue of integrase in HIV-1, HIV-2 and SIV.

Amino Acid Substitution↗
PubMed2004

Nucleofection of muscle-derived stem cells and myoblasts with phiC31 integrase: stable expression of a full-length-dystrophin fusion gene by human myoblasts.

Ex vivo gene therapy offers a potential treatment for Duchenne muscular dystrophy by transfection of the dystrophin gene into the patient's own myogenic precursor cells, followed by transplantation. We used nucleofection to introduce DNA plasmids coding for enhanced green fluorescent protein (eGFP) or eGFP-dystrophin fusion protein and the phage phiC31 integrase into myogenic cells and to integrate these genes into a limited number of sites in the genome. Using a plasmid expressing eGFP, we transfected 50% of a mouse muscle-derived stem cell line and 60% of normal human myoblasts. Co-nucleofection of a plasmid expressing the phiC31 integrase and an eGFP expression plasmid containing an attB sequence produced 15 times more frequent stable expression, because of site-specific integration of the transgene. Co-nucleofection of the phiC31 integrase plasmid and a large plasmid containing the attB sequence and the gene for an eGFP-full-length dystrophin fusion protein produced fluorescent human myoblasts that were able to form more intensely fluorescent myotubes after 1 month of culture. A nonviral approach combining nucleofection and the phiC31 integrase may eventually permit safe autotransplantation of genetically modified cells to patients.

Animals↗
PubMed2005

Site-specific integration with phiC31 integrase for prolonged expression of therapeutic genes.

Need of a site-specific integrating vector in gene therapy has become pressing, as recent work has shown that many of the current integrating vectors used preferentially integrate in the vicinity of genes. A site-specific integrating vector would reduce the risk of insertional mutagenesis posed by randomly integrating vectors, and a non-viral vector would reduce the safety and immunogenicity problems associated with viral vectors. The phiC31 integrase is a protein from Streptomyces phage phiC31 that has been developed as a non-viral site-specific gene therapy vector. The phiC31 integrase catalyzes the integration of a plasmid containing attB into pseudo attP sites in mammalian genomes. It has been shown to function in tissue culture cells as well as in mice. Vectors based on the phiC31 integrase were able to treat tyrosinemia type I in a mouse model and two forms of epidermolysis bullosa in keratinocytes from patients, demonstrating its effectiveness as a gene therapy vector. Development of phiC31 integrase-based vectors is still underway, but it has already been shown to provide long-term expression through site-specific integration.

Animals↗
PubMed2000

Combinations of reverse transcriptase, protease, and integrase inhibitors can be synergistic in vitro against drug-sensitive and RT inhibitor-resistant molecular clones of HIV-1.

Combinations of anti-HIV agents including one or two reverse transcriptase inhibitors with a protease inhibitor are potent and effective. However, toxicities, costs and the emergence of drug-resistant organisms have compromised their long-term efficacy in people. A next, likely, target for anti-HIV therapy is HIV-1 integrase. Viral integration, catalyzed by integrase, is absolutely required for HIV replication. L-chicoric acid is a potent and selective inhibitor of HIV-1 integrase that also inhibits HIV-1 replication in cell culture. As a first step in understanding the potential role for integrase inhibitors in clinical medicine, the activities of L-chicoric acid alone and in combination with 2', 3'-dideoxycytidine, zidovudine, and a protease inhibitor, nelfinavir, were tested in vitro against molecular clones of HIV-1 resistant to reverse transcriptase inhibitors. L-chicoric acid was equally effective against a wild-type clone of HIV-1, HIV(NL4-3), or against HIV-1 resistant to either zidovudine or dideoxycytidine. L-chicoric acid was largely synergistic with zidovudine and synergistic with both dideoxycytidine and nelfinavir.

Anti-HIV Agents↗
PubMed1998

L-chicoric acid, an inhibitor of human immunodeficiency virus type 1 (HIV-1) integrase, improves on the in vitro anti-HIV-1 effect of Zidovudine plus a protease inhibitor (AG1350).

Combinations of anti-human immunodeficiency virus (HIV) drugs, including reverse transcriptase inhibitors and protease inhibitors, have proven immensely potent in the therapy of acquired immune deficiency syndrome (AIDS). To determine whether HIV integrase is a suitable target for combination therapy, the ability of an HIV integrase inhibitor, L-chicoric acid, to work in combination with a protease inhibitor and Zidovudine was tested in vitro. The addition of L-chicoric acid to either Zidovudine or protease inhibitor improved upon the observed anti-HIV activity of either compound alone. When all three drugs were combined, the anti-HIV activity was substantially better than either of the three compounds alone or any combination of two inhibitors. Doses of both Zidovudine and protease inhibitor could be reduced by more than 33% for an equivalent anti-HIV effect if L-chicoric acid was added. The improved anti-HIV activity was observed with a tissue culture adapted strain of HIV (HIV(LAI)) and with limited passage clinical isolates of HIV (HIV(R19) and HIV(R45)). These data demonstrate that a first generation HIV integrase inhibitor, L-chicoric acid, is at least additive in combination with existing multi-drug regimens and suggest that HIV integrase will be an excellent target for combination therapy of HIV infection.

Anti-HIV Agents↗
PubMed1998

A highly efficient method for the site-specific integration of transfected plasmids into the genome of mammalian cells using purified retroviral integrase.

Using purified bovine leukemia virus (BLV) integrase with liposome, we developed a highly efficient method for the site-specific integration of plasmid vectors into the genome of cultured mammalian cells. The presence of the BLV integrase recognition sequence (IRS) in both the host genome and the plasmid vector to be transfected was required for this integration. The integration occurred within the IRS pre-introduced into the host genome and resulted in a complete or partial deletion of the sequence and an adjacent drug-resistant gene. This site-specific integration was not observed upon transfection without the integrase or with vectors harboring no IRS. This novel method may be useful for manipulating a mammalian genome or for targeting a retroviral genome integrated into a virus-infected cell by using the virus-specific integrase and LTR sequence.

Binding Sites↗
PubMed2003

Four novel bis-(naphtho-gamma-pyrones) isolated from Fusarium species as inhibitors of HIV-1 integrase.

Integration of viral DNA into host cell DNA is an essential step in retroviral (HIV-1) replication and is catalyzed by HIV-1 integrase. HIV-1 integrase is a novel therapeutic target and is the focus of efforts to identify effective inhibitors that will prevent/or cure HIV infections. Four novel naphtho-gamma-pyrones, belonging to the chaetochromin and ustilaginoidin family, were discovered as inhibitors of HIV-1 integrase from the screening of fungal extracts using a recombinant in vitro assay. These compounds inhibit both the coupled and strand transfer activity of HIV-1 integrase with IC(50) values of 1-3 and 4-12 microM, respectively. The discovery, structure elucidation, chemical modification and the structure-activity relationship of these compounds are described.

Anti-HIV Agents↗