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PubMed2002

ILG1 : a new integrase-like gene that is a marker of bacterial contamination by the laboratory Escherichia coli strain TOP10F'.

BACKGROUND: Identification of differentially expressed genes between normal and diseased states is an area of intense current medical research that can lead to the discovery of new therapeutic targets. However, isolation of differentially expressed genes by subtraction often suffers from unreported contamination of the resulting subtraction library with clones containing DNA sequences not from the original RNA samples. MATERIALS AND METHODS: Subtraction using cDNA representational difference analysis (RDA) was performed on human B cells from normal or common variable immunodeficiency patients. The material remaining after the subtraction was cloned and individual clones were sequenced. The sequence of one clone with similarity to integrases (ILG1, integrase-like gene-1) was used to obtain the full length cDNA sequence and as a probe for the presence of this sequence in RNA or genomic DNA samples. RESULTS: After five rounds of cDNA RDA, 23.3% of the clones from the resulting subtraction library contained Escherichia coli DNA. In addition, three clones contained the sequence of a new integrase, ILG1. The full length cDNA sequence of ILG1 exhibits prokaryotic, but not eukaryotic, features. At the DNA level, ILG1 is not similar to any known gene. At the protein level, ILG1 has 58% similarity to integrases from the cryptic P4 bacteriophage family (S clade). The catalytic domain of ILG1 contains the conserved features found in site-specific recombinases. The critical residues that form the catalytic active site pocket are conserved, including the highly conserved R-H-R-Y hallmark of these recombinases. Interestingly, ILG1 was not present in the original B cell populations. By probing genomic DNA, ILG1 could only be detected in the E. coli TOP10F' strain used in our laboratory for molecular cloning, but not in any of its precursor strains, including TOP10. Furthermore, bacteria cultured from the mouth of the laboratory worker who performed cDNA RDA were also positive for ILG1. CONCLUSIONS: In the course of our studies using cDNA RDA, we have isolated and identified ILG1, a likely active site-specific recombinase and new member of the bacteriophage P4 family of integrases. This family of integrases is implicated in the horizontal DNA transfer of pathogenic genes between bacterial species, such as those found in pathogenic strains of E. coli, Shigella, Yersinia, and Vibrio cholera. Using ILG1 as a marker of our laboratory E. coli strain TOP10F', our evidence suggests that contaminating bacterial DNA in our subtraction experiment is due to this laboratory bacterial strain, which colonized exposed surfaces of the laboratory worker. Thus, identification of differentially expressed genes between normal and diseased states could be dramatically improved by using extra precaution to prevent bacterial contamination of samples.

Amino Acid Sequence↗

PubMed1994

Viral long terminal repeat substrate binding characteristics of the human immunodeficiency virus type 1 integrase.

A DNA binding assay was developed for the human immunodeficiency virus type 1 (HIV-1) integrase. The assay was capable of defining discrete complexes between the enzyme and the viral long terminal repeat (LTR) substrate. DNA binding reflected the sequence requirements previously demonstrated for the enzyme's 3'-end processing activity. Binding exhibited a nonlinear dependence on integrase concentration, suggesting that the enzyme functions as a multimer. The oligomeric state was investigated by UV-photo-cross-linking of integrase-LTR oligonucleotide complexes using DNA substrates substituted with 5-bromo-2'-deoxycytidine within the integrase recognition sequence. In the absence of divalent cation, integrase cross-linked to the LTR oligonucleotide as a single species whose mobility by SDS-polyacrylamide gel electrophoresis was consistent with the formation of tetramers. Using these techniques, analysis of the binding properties of integrase mutants demonstrated that the catalytic and sequence-specific DNA binding activities of the enzyme are distinct, involving residues within the conserved "DD(35)E" and zinc finger motifs, respectively.

Base Sequence↗

PubMed2000

Crystal structure of an active two-domain derivative of Rous sarcoma virus integrase.

Integration of retroviral cDNA is a necessary step in viral replication. The virally encoded integrase protein and DNA sequences at the ends of the linear viral cDNA are required for this reaction. Previous studies revealed that truncated forms of Rous sarcoma virus integrase containing two of the three protein domains can carry out integration reactions in vitro. Here, we describe the crystal structure at 2.5 A resolution of a fragment of the integrase of Rous sarcoma virus (residues 49-286) containing both the conserved catalytic domain and a modulatory DNA-binding domain (C domain). The catalytic domains form a symmetric dimer, but the C domains associate asymmetrically with each other and together adopt a canted conformation relative to the catalytic domains. A binding path for the viral cDNA is evident spanning both domain surfaces, allowing modeling of the larger integration complexes that are known to be active in vivo. The modeling suggests that formation of an integrase tetramer (a dimer of dimers) is necessary and sufficient for joining both viral cDNA ends at neighboring sites in the target DNA. The observed asymmetric arrangement of C domains suggests that they could form a rotationally symmetric tetramer that may be important for bridging integrase complexes at each cDNA end.

Amino Acid Sequence↗

PubMed2000

Carbonyl J Derivatives: A New Class of HIV-1 Integrase Inhibitors.

Integration of a DNA copy of the HIV-1 genome is required for viral replication and pathogenicity, and this highly specific molecular process is mediated by the virus-encoded integrase protein. The requirement for integration, combined with the lack of a known analogous process in mammalian cells, makes integrase an attractive target for therapeutic inhibitors of HIV-1 replication. While many reports of HIV-1 IN inhibitors exist, no such compounds have yet emerged to treat HIV-1 infection. As such, new classes of integrase inhibitors are needed. We have combined molecular modeling and combinatorial chemistry to identify and develop a new class of HIV-1 integrase inhibitors, the Carbonyl J [N,N'-bis(2-(5-hydroxy-7-naphthalenesulfonic acid)urea] derivatives. This new class includes a number of compounds with sub-micromolar IC(50) values for inhibiting purified HIV-1 integrase in vitro. Herein we describe the chemical characteristics that are important for integrase inhibition and cell toxicity within the Carbonyl J derivatives. Copyright 2000 Academic Press.

Journal Article↗

PubMed2001

Targeting HIV-1 integrase.

Human immunodeficiency virus Type 1 (HIV-1) integrase is an essential enzyme for the obligatory integration of the viral DNA into the infected cell chromosome. As no cellular homologue of HIV integrase has been identified, this unique HIV-1 enzyme is an attractive target for the development of new therapeutics. Treatment of HIV-1 infection and AIDS currently consists of the use of combinations of HIV-1 inhibitors directed against reverse transcriptase (RT) and protease. However, their numerous side effects and the rapid emergence of drug-resistant variants limit greatly their use in many AIDS patients. In principle, inhibitors of the HIV-1 integrase should be relatively non-toxic and provide additional benefits for AIDS chemotherapy. There have been many major advances in our understanding of the molecular mechanism of the integration reaction, although some critical aspects remain obscure. Several classes of compounds have been screened and further scrutinised for their inhibitory properties against the HIV integrase; however, there are currently no useful inhibitors available clinically for the treatment of AIDS patients. This review describes the current knowledge of the biological functions of the HIV-1 integrase and reports the major classes of integrase inhibitors identified to date.

Journal Article↗

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↗

PubMedDate not supplied

HIV integrase as a target for antiviral chemotherapy.

One of the three key enzymes encoded by the pol gene of HIV is a M(r) 32 000 protein called HIV integrase. This viral enzyme is involved in the integration of HIV DNA into host chromosomal DNA. There appears to be no functional equivalent of the enzyme in human cells. The biochemical mechanism of integration of HIV DNA into the host cell genome involves a carefully defined sequence of DNA tailoring (3'-processing) and coupling (joining or integration) reactions. In spite of some effort in this area targeted at the discovery of therapeutically useful inhibitors of this viral enzyme, there are no drugs for HIV/AIDS in clinical use where the mechanism of action is inhibition of HIV integrase. Thus, new knowledge on inhibitors of this enzyme is of critical importance in the anti-HIV drug discovery area. The focus of this review will be on several classes of compounds, including nucleotides, dinucleotides, oligonucleotides and miscellaneous small molecules such as heterocyclic systems, natural products, diketo acids and sulfones, that have been discovered as inhibitors of HIV integrase. Special emphasis in the review will be placed on discoveries from my laboratory on HIV integrase inhibitors that are non-natural, nuclease-resistant dinucleotides. Comments on future directions and the prospects for developing integrase inhibitors as therapeutic antiviral agents are discussed.

Anti-HIV Agents↗

PubMed2000

X-ray structure of simian immunodeficiency virus integrase containing the core and C-terminal domain (residues 50-293)--an initial glance of the viral DNA binding platform.

The crystal structure of simian immunodeficiency virus (SIV) integrase that contains in a single polypeptide the core and the C-terminal deoxyoligonucleotide binding domain has been determined at 3 A resolution with an R-value of 0.203 in the space group P2(1)2(1)2(1). Four integrase core domains and one C-terminal domain are found to be well defined in the asymmetric unit. The segment extending from residues 114 to 121 assumes the same position as seen in the integrase core domain of avian sarcoma virus as well as human immunodeficiency virus type-1 (HIV-1) crystallized in the absence of sodium cacodylate. The flexible loop in the active site, composed of residues 141-151, remains incompletely defined, but the location of the essential Glu152 residue is unambiguous. The residues from 210-218 that link the core and C-terminal domains can be traced as an extension from the core with a short gap at residues 214-215. The C(alpha) folding of the C-terminal domain is similar to the solution structure of this domain from HIV-1 integrase. However, the dimeric form seen in the NMR structure cannot exist as related by the non-crystallographic symmetry in the SIV integrase crystal. The two flexible loops of the C-terminal domain, residues 228-236 and residues 244-249, are much better fixed in the crystal structure than in the NMR structure with the former in the immediate vicinity of the flexible loop of the core domain. The interface between the two domains encompasses a solvent-exclusion area of 1500 A(2). Residues from both domains purportedly involved in DNA binding are narrowly distributed on the same face of the molecule. They include Asp64, Asp116, Glu152 and Lys159 from the core and Arg231, Leu234, Arg262, Arg263 and Lys264 from the C-terminal domain. A model for DNA binding is proposed to bridge the two domains by tethering the 228-236 loop of the C-terminal domain and the flexible loop of the core.

Amino Acid Sequence↗

PubMed2004

Inhibition of human immunodeficiency virus type I integrase by naphthamidines and 2-aminobenzimidazoles.

Retroviral integrases catalyze two of the steps of insertion of proviral DNA into the host genomic DNA. Inhibitors that target the second step, strand transfer into the host DNA, have been demonstrated to have antiviral activity in cell culture. We describe two classes of HIV-1 integrase inhibitors that block strand transfer, one based on a naphthamidine core and one on a benzimidazole core. While the naphthamidine compounds showed some propensity to interact with the DNA substrate, both classes were shown to bind directly to integrase. The naphthamidine compounds showed activity in cell culture, and a direct effect on integrase was indicated by an increase in 2-LTR products in the presence of a naphthamidine compound. These two classes of compounds represent potential starting points for the development of new classes of integrase inhibitors.

Base Sequence↗

PubMed2006

Conformationally restrained carbazolone-containing alpha,gamma-diketo acids as inhibitors of HIV integrase.

Since alpha,gamma-diketo acid (DKA) compounds were identified as potent and selective inhibitors for HIV integrase, numerous structural modification studies have been carried out to search for a clinical candidate as a supplement for the highly active antiretroviral therapy regimen. Due to the lack of structural information on inhibitor-integrase interactions, a comprehensive structure-activity relationship study is necessary. Most of the reported modification studies on the key alpha,gamma-diketo acid pharmacophore focused on substituting the carboxylate moiety with its bioisosteres or other electron-pair bearing heterocycles. We were interested in studying the conformation and geometry of the central diketo moiety. A series of carbazolone-containing alpha,gamma-diketo acids were designed and synthesized by applying conformational restraint onto the open-chain form of the diketo acid. These compounds showed anti-integrase activity in the low micromolar range, and integrase assay results indicated that the geometry of the diketo acid moiety is crucial to potency. Carbazol-1-one containing DKA analogs (7-8) showed a 2- to 3-fold increase in activity compared with those of carbazol-4-one containing DKA analogs (5 and 6). Alkylation of carbazol-4-one DKA nitrogen (6a-c) led to a loss of activity, suggesting this nitrogen atom may directly interact with the active site of integrase. The halogens (7b-d) and para-fluorobenzyl substituents (8a-d) on carbazol-1-one ring had little effect on potency.

Acylation↗

PubMed2006

Synthesis and biological evaluation of purine derivatives incorporating metal chelating ligands as HIV integrase inhibitors.

Because of its essential role in HIV replication and lack of human counterpart, HIV integrase is an attractive target for the development of novel anti-AIDS agents. Among the recently developed integrase inhibitors, only the alpha,gamma-diketo acid (DKA) compounds were biologically validated as potent and selective integrase inhibitors. The general structure of DKAs contains a diketo acid moiety as the Mg(2+) chelating pharmacophore, and an adjacent aryl group to provide selectivity. Numerous structure-activity relationship (SAR) studies on DKAs have been conducted, which generally involved substituting the carboxylate group or the aryl group. Our objective was to investigate the SARs of the DKA molecule by incorporating a purine ring in the aryl moiety and replacing the labile diketo acid moiety with other divalent metal (Me(2+)) chelating ligands. A series of amide substituted purine derivatives were synthesized via palladium-catalyzed amidation reactions, and their biological activities against HIV integrase were evaluated. These purine derivatives showed anti-integrase activity at low micromolar range. The biological results indicated that the type of Me(2+) ligands, two-point ligand picolinamide or three-point ligand 8-hydroxy-quinoline-7-carboxamide, affected inhibitory potency depending on the substitution position of the para-fluorobenzyl group. The C(6)-,C(8)-dipicolinamide substituted purine (32) exhibited the best potency among this series.

Amides↗

PubMed2002

DNA aptamers derived from HIV-1 RNase H inhibitors are strong anti-integrase agents.

HIV-1 integrase, the retroviral-encoded enzyme involved in the integration of the retrotranscribed viral genome into the host nuclear DNA, is an attractive and still unexploited target. To date, very few inhibitors of this enzyme with a potential therapeutic value have been described. During the search for new HIV-1 targets, we recently described DNA oligodeoxynucleotide aptamers (ODN 93 and ODN 112) that are strong inhibitors of the RNase H activity associated with HIV-1 reverse transcriptase. The striking structural homology between RNase H and integrase led us to study the effect of the RNase H inhibitors on the integrase. Shorter DNA aptamers derived from ODNs 93 and 112 (ODNs 93del and 112del) were able to inhibit HIV-1 integrase in the nanomolar range. They had G-rich sequences able to form G-quartets stabilized by the presence of K(+). The presence of these ions increased the inhibitory efficiency of these agents dramatically. Inhibition of enzymatic activities by ODN 93del and ODN 112del was observed in a cell-free assay system using a recombinant integrase and HIV-1 replication was abolished in infected human cells. Moreover, cell fusion assays showed that these agents do not block viral cell entry at concentrations where viral replication is stopped.

Anti-HIV Agents↗

PubMed2001

A comparison of HIV-1 integrase inhibition by aqueous and methanol extracts of Chinese medicinal herbs.

The aqueous and methanol extracts of twenty herbs traditionally used in Chinese medicine were screened for anti-HIV-1 integrase activity in a non-radioactive ELISA-based HIV-1 integrase assay. The screening was performed at an herb extract concentration of 50 microg/ml. It was found that most of the aqueous and methanol herb extracts could elicit strong inhibition of HIV-I integrase activity. The inhibition was most likely due to tannins or polyphenolics in the herb extracts. In most of the herb extracts, 40-80% of the anti-HIV-1 integrase activity could be removed after passing through a minicolumn of polyamide resin. After removal of polyphenolic compounds, the methanol extract of Paeonia suffruticosa still exerted potent inhibition of HIV-1 integrase (EC50 = 15 microg/ml) and the aqueous extract of Prunella vulgaris caused moderate inhibition (EC50 = 45 microg/ml). The results support the view that herbs represent a rich source of anti-HIV compounds.

Drugs, Chinese Herbal↗

PubMed2002

Thalassiolins A-C: new marine-derived inhibitors of HIV cDNA integrase.

Human immunodeficiency virus (HIV) replication requires integration of viral cDNA into the host genome, a process mediated by the viral enzyme integrase. We describe a new series of HIV integrase inhibitors, thalassiolins A-C (1-3), isolated from the Caribbean sea grass Thalassia testudinum. The thalassiolins are distinguished from other flavones previously studied by the substitution of a sulfated beta-D-glucose at the 7-position, a substituent that imparts increased potency against integrase in biochemical assays. The most active of these molecules, thalassiolin A (1), displays in vitro inhibition of the integrase catalyzed strand transfer reaction (IC50=0.4 microM) and an antiviral IC50 of 30 microM. Molecular modeling studies indicate a favorable binding mode is probable at the catalytic core domain of HIV-1 integrase.

Binding Sites↗

PubMed2002

QSAR studies of HIV-1 integrase inhibition.

Compounds from a wide variety of structural classes inhibit HIV-1 integrase. However, a single unified understanding of the relationship between the structures and activities of these compounds still eludes researchers. We report herein the development of QSAR models for integrase inhibition. The genetic function approximation (GFA) was utilized to select descriptors for the development of the QSAR models. The best QSAR model derived for the complete set of 11 structural classes had a correlation coefficient (r(2)) of only 0.54 and a cross-validated correlation coefficient (q(2)) of only 0.42. This indicated that the compounds studied may differ in the exact relationship between structure and inhibition, perhaps through interactions with different subsets of amino acids in the binding pocket, or through the presence of non-overlapping binding pockets. Descriptor-based cluster analysis indicated that the 11 structural classes of integrase inhibitors studied belonged to two clusters, one consisting of five structural classes, and the other six. QSAR models for these two clusters had r(2) values of 0.79 and 0.82 and q(2) values of 0.71 and 0.74, a significant improvement over models obtained for the complete set of compounds. The two models were applied to predict the activities of compounds from the same structural classes as those used to build the models, giving r(2) values of 0.65 and 0.78. The models were also used to predict the activities of compounds shown in crystallographic or docking studies to interact near the active site metal ion. The model describing the larger cluster of structural classes was better able to reproduce the biological activities of these five structures with an average percent residual error of 7.9 compared with the 19.3% residual error for predictions from the other model. This indicated that the six structural classes comprising the larger cluster may bind near the metal ion in a fashion similar to that observed in one publicly available co-crystal structure of an inhibitor bound to HIV-1 integrase. Flexible alignment of inhibitors in the two clusters found different pharmacophores that are consistent with previously published pharmacophores developed on the basis of individual structural classes that have produced novel inhibitory compounds. Thus we expect that these two QSAR models can be used in the search for novel HIV-1 integrase inhibitors as well as to provide insight into the binding modes of such diverse chemical compounds.

Anti-HIV Agents↗

PubMed1997

Discovery of HIV-1 integrase inhibitors by pharmacophore searching.

Based upon a class of known HIV-1 integrase inhibitors, several pharmacophore models were proposed from molecular modeling studies and validated using a 3D database of 152, compounds for which integrase assay data are known. Using the most probable pharmacophore model as the query, the NCI 3D database of 206,876 compounds was searched, and 340 compounds that contain the pharmacophore query were identified. Twenty-nine of these compounds were selected and tested in the HIV-1 integrase assay. This led to the discovery of 10 novel, structurally diverse HIV-1 integrase inhibitors, four of which have an IC50 value less than 30 microM and are promising lead compounds for further HIV-1 integrase inhibitor development.

4-Hydroxycoumarins↗

PubMed1997

Hydrazide-containing inhibitors of HIV-1 integrase.

Inhibitors of HIV integrase are currently being sought as potential new therapeutics for the treatment of AIDS. A large number of inhibitors discovered to date contain the o-bis-hydroxy catechol structure. In an effort to discover structural leads for the development of new HIV integrase inhibitors which do not rely on this potentially cytotoxic catechol substructure, NSC 310217 was identified using a three-point pharmacophore search based on its assigned structure N-(2-hydroxybenzoyl)-N-(2-hydroxy-3-phenoxypropyl)hydrazine (1). When a sample of NSC 310217 was obtained from the NCI repository, it was shown to exhibit potent inhibition of HIV-1 integrase (3'-processing IC50 = 0.6 microgram/mL). In work reported herein, we demonstrate that NSC 310217, rather than containing 1, which has no inhibitory potency against HIV-1 integrase, is comprised of roughly a 1:1 mixture of N-(2-hydroxybenzoyl)-N'-(2-hydroxy-3-phenoxypropyl)hydrazine (6) and N,N'-bis-salicylhydrazine 7, with all inhibitory potency residing with compound 7(IC50 = 0.7 microM for strand transfer). In subsequent structure-activity studies on 7, it is shown that removing a single amide carbonyl (compound 14, IC50 = 5.2 microM) or replacing one aromatic ring system with a naphthyl ring (compound 19, IC50 = 1.1 microM) can be accomplished with little loss of inhibitory potency. Additionally, replacing a single hydroxyl with a sulfhydryl (compound 23, IC50 = 5.8 microM) results in only moderate loss of potency. All other modifications examined, including the replacement of a single hydroxyl with an amino group (compound 22), resulted in complete loss of potency. Being potent, structurally simple, and non-catechol-containing, compounds such as 7 and 14 may provide useful leads for the development of a new class of HIV integrase inhibitor.

Databases, Factual↗

PubMed2002

Functional oligomeric state of avian sarcoma virus integrase.

Retroviral integrase, one of only three enzymes encoded by the virus, catalyzes the essential step of inserting a DNA copy of the viral genome into the host during infection. Using the avian sarcoma virus integrase, we demonstrate that the enzyme functions as a tetramer. In presteady-state active site titrations, four integrase protomers were required for a single catalytic turnover. Volumetric determination of integrase-DNA complexes imaged by atomic force microscopy during the initial turnover additionally revealed substrate-induced assembly of a tetramer. These results suggest that tetramer formation may be a requisite step during catalysis with ramifications for antiviral design strategies targeting the structurally homologous human immunodeficiency virus, type 1 (HIV-1) integrase.

Antiviral Agents↗