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PubMed1997

Potent inhibitors of human immunodeficiency virus type 1 integrase: identification of a novel four-point pharmacophore and tetracyclines as novel inhibitors.

A four-point pharmacophore was constructed from energy-minimized structures of chicoric acid and dicaffeoylquinic acid. The search of 206,876 structures in the National Cancer Institute 3D database yielded 179 compounds that contain this pharmacophore. Thirty-nine of these compounds were tested in an in vitro assay specific for human immunodeficiency virus type 1 integrase (IN). Each retrieved structure was fit to the pharmacophore, and the conformation that afforded the best fit was identified. Twenty of the 39 compounds tested exhibited IC50 values of < 20 microM. Among the most potent inhibitors, tetracyclines emerged as a new class of inhibitors. Although the parent tetracycline exhibited marginal potency against purified IN, all substituted tetracyclines tested showed 5-100-fold increased potency. Disintegration assays with truncated IN mutants indicated that tetracyclines inhibit the IN catalytic core domain. To investigate whether chelation of divalent metals is implicated in differential potency of tetracyclines, enzyme assays were performed in the presence of both Mn2+ or Mg2+; no significance difference in potency was observed. Rolitetracycline inhibited IN/DNA complex formation in the presence of EDTA, which suggests that inhibition was metal independent. Rolitetracycline reversed DNA binding of IN after the complex was allowed to form before the addition of drug. Selectivity of tetracyclines was also examined in an assay specific for topoisomerase I, and none of the tetracyclines tested induced topoisomerase I-mediated cleavable complex or inhibited camptothecin-induced cleavable complex. Remarkable potency against the IN in the absence of divalent metals and the core enzyme coupled with water solubility makes tetracyclines potential candidates for X-ray crystal structure determination with IN.

Anti-HIV Agents↗
PubMed1998

Highly potent synthetic polyamides, bisdistamycins, and lexitropsins as inhibitors of human immunodeficiency virus type 1 integrase.

Alignment of the available human immunodeficiency virus type 1 (HIV-1) viral DNA termini [U5 and U3 long terminal repeats (LTRs)] shows a high degree of conservation and the presence of a stretch of five or six consecutive adenine and thymine (AT) sequences approximately 10 nucleotides away from each LTR end. A series of AT-selective minor-groove binders, including distamycin and bisdistamycins, bisnetropsins, novel lexitropsins, and the classic monomeric DNA binders Hoechst 33258, 4'-diamino-2-phenylindole, pentamidine, berenil, spermine, and spermidine, were tested for their inhibitory activities against HIV-1 integrase (IN). Although netropsin, distamycin, and all other monomeric DNA binders showed weak activities in the range of 50-200 microM, some of the polyamides, bisdistamycins, and lexitropsins were remarkably active at nanomolar concentrations. Bisdistamycins were 200 times less potent when the conserved AAAAT stretch present in the U5 LTR was replaced with GGGGG, consistent with the preferred binding of these drugs to AT sequences. DNase I footprinting of the U5 LTR further demonstrated the selectivity of these bisdistamycins for the conserved AT sequence. The tested compounds were more potent in Mg+2 than in Mn+2 and inhibited IN50-212 deletion mutant in disintegration assays and the formation of IN/DNA complexes. The lexitropsins also were active against HIV-2 IN. Some of the synthetic polyamides exhibited significant antiviral activity. Taken together, these data suggest that selective targeting of the U5 and U3 ends of the HIV-1 LTRs can inhibit IN function. Polyamides might represent new leads for the development of antiviral agents against acquired immune deficiency syndrome.

Anti-HIV Agents↗
PubMed2004

Mechanism of HIV-1 integrase inhibition by styrylquinoline derivatives in vitro.

Styrylquinoline derivatives (SQ) efficiently inhibit the 3'-processing activity of integrase (IN) with IC50 values of between 0.5 and 5 microM. We studied the mechanism of action of these compounds in vitro. First, we used steady-state fluorescence anisotropy to assay the effects of the SQ derivatives on the formation of IN-viral DNA complexes independently of the catalytic process. The IC50 values obtained in activity and DNA-binding tests were similar, suggesting that the inhibition of 3'-processing can be fully explained by the prevention of IN-DNA recognition. SQ compounds act in a competitive manner, with Ki values of between 400 and 900 nM. In contrast, SQs did not inhibit 3'-processing when IN-DNA complexes were preassembled. Computational docking followed or not by molecular dynamics using the catalytic core of HIV-1 IN suggested a competitive inhibition mechanism, which is consistent with our previous data obtained with the corresponding Rous sarcoma virus domain. Second, we used preassembled IN-preprocessed DNA complexes to assay the potency of SQs against the strand transfer reaction, independently of 3'-processing. Inhibition occurred even if the efficiency was decreased by about 5- to 10-fold. Our results suggest that two inhibitor-binding modes exist: the first one prevents the binding of the viral DNA and then the two subsequent reactions (i.e., 3'-processing and strand transfer), whereas the second one prevents the binding of target DNA, thus inhibiting strand transfer. SQ derivatives have a higher affinity for the first site, in contrast to that observed for the diketo acids, which preferentially bind to the second one.

Anti-HIV Agents↗
PubMed2006

Development of a human immunodeficiency virus vector-based, single-cycle assay for evaluation of anti-integrase compounds.

Therapeutic strategies aimed at inhibiting human immunodeficiency virus type 1 (HIV-1) replication employ a combination of drugs targeted to two viral enzymes (reverse transcriptase and protease) and to the viral entry/fusion step. However, the high propensity of HIV-1 to develop resistance makes the development of novel compounds targeting different steps of the HIV-1 life cycle essential. Among these, integrase (IN) inhibitors have successfully passed the early phases of clinical development. By preventing integration, IN inhibitors preclude viral replication while allowing production of extrachromosomal forms of viral DNA (E-DNA). Here, we describe an improved and standardized assay aimed at evaluating IN inhibitors by taking advantage of the transcriptional activity of E-DNA produced by HIV-derived vectors in the absence of replication-competent virus. In this context, the use of the firefly luciferase gene as a reporter gene provides a rapid and quantitative measure of viral-vector infectivity, thus making it a safe and cost-effective assay for evaluating novel IN inhibitors.

Cell Line↗
PubMed1997

Diarylsulfones, a novel class of human immunodeficiency virus type 1 integrase inhibitors.

A majority of reported human immunodeficiency virus type 1 integrase (HIV-1 IN) inhibitors are polyhydroxylated aromatic compounds containing two phenyl rings separated by aliphatic or aromatic linkers. Most inhibitors possessing a catechol moiety exhibit considerable toxicity in cellular assays. In an effort to identify nonhydroxylated analogs, a series of aromatic sulfones were tested for their ability to inhibit the 3' processing and strand transfer steps that are necessary for HIV replication. Several aromatic sulfones have previously been shown to have moderate activity against HIV-1 reverse transcriptase in cellular assays; however, their inhibitory potencies against IN have not been explored. In the present study, the inhibitory effect of a series of sulfones and sulfonamides against IN was determined. Among 52 diaryl sulfones tested, 4 were determined to be highly potent (50% inhibitory concentration [IC50], 0.8 to 10 micrograms/ml), 5 had good potencies (IC50, 11 to 50 micrograms/ml), 10 showed moderate potencies (IC50, 51 to 100 micrograms/ml), and 33 were inactive (IC50, > 100 micrograms/ml) against IN. All of the active compounds exhibited similar potencies against HIV-2 IN. Sulfa drugs, used extensively in treating Pneumocystis carinii pneumonia, a leading cause of morbidity and mortality in AIDs patients, were also examined. Among 19 sulfonamides tested, sulfasalazine (IC50, 50 micrograms/ml) was the most potent. We conclude that potent inhibitors of IN can be designed based on the results presented in this study.

Cell Line↗
PubMed2002

Inhibition of human immunodeficiency virus type 1 isolates by the integrase inhibitor L-731,988, a diketo Acid.

L-731,988 inhibits human immunodeficiency virus (HIV) replication through integrase. In this study, approximately 600 nM L-731,988 inhibited the replication of 12 HIV type 1 isolates from multiple clades, including primary isolates and cloned viruses. These data suggest that diketo acids or their derivatives may prove useful on a worldwide basis in treating HIV infection.

Acetoacetates↗
PubMed2005

Inhibition of human immunodeficiency virus type 1 reverse transcriptase, RNase H, and integrase activities by hydroxytropolones.

Human immunodeficiency virus type I reverse transcriptase (RT) possesses distinct DNA polymerase and RNase H sites, whereas integrase (IN) uses the same active site to perform 3'-end processing and strand transfer of the proviral DNA. These four enzymatic activities are essential for viral replication and require metal ions. Two Mg2+ ions are present in the RT polymerase site, and one or two Mg2+ ions are required for the catalytic activities of RNase H and IN. We tested the possibility of inhibition of the RT polymerase and RNase H as well as the IN 3'-end processing and transfer activities of purified enzymes by a series of 3,7-dihydroxytropolones designed to target two Mg2+ ions separated by approximately 3.7 angstroms. The RT polymerase and IN 3' processing and strand transfer activities were inhibited at submicromolar concentrations, while the RNase H activity was inhibited in the low micromolar range. In all cases, the lack of inhibition by tropolones and O-methylated 3,7-dihydroxytropolones was consistent with the active molecules binding the metal ions in the active site. In addition, inhibition of the DNA polymerase activity was shown to depend on the Mg2+ concentration. Furthermore, selective inhibitors were identified for several of the activities tested, leaving some potential for design of improved inhibitors. However, all tested compounds exhibited cellular toxicity that presently limits their applications.

Enzyme Inhibitors↗
PubMed2006

Preliminary mapping of a putative inhibitor-binding pocket for human immunodeficiency virus type 1 integrase inhibitors.

Molecular modeling studies have identified a putative human immunodeficiency virus (HIV) integrase (IN) inhibitor-binding pocket for l-chicoric acid (l-CA) and other inhibitors of IN (C. A. Sotriffer, H. Ni, and A. McCammon, J. Med. Chem. 43:4109-4117, 2000). By using site-directed mutagenesis of several amino acid residues identified by modeling studies, a common inhibitor-binding pocket on IN was confirmed for l-CA and the diketo acid L-731,988. Specifically, the single mutations E92K, Q148A, K156A, K156R, G140S, and G149S, as well as the double mutations C65S-K156N and H67D-G140A were evaluated for their effects on enzymatic activity and inhibitor susceptibility. Each recombinant IN was attenuated for 3'-end processing and strand transfer activities. Most proteins were also attenuated for disintegration; the IN that contained K156R and C65S-K156N, however, displayed disintegration activity similar to that of IN from HIV(NL4-3). All mutant IN proteins demonstrated decreased susceptibility to l-CA, while all mutant proteins except E92K and K156R demonstrated resistance to L-731,988. These data validate the computer modeling data and demonstrate that l-CA and L-731,988 share an overlapping inhibitor-binding pocket that involves amino acids Q148, C65, and H67. The resistance studies confirm that L-731,988 fills one-half of the inhibitor-binding pocket and binds to Q148 but excludes E92, while l-CA fills the entire binding groove and thus interacts with E92. These results provide "wet laboratory" evidence that molecular models of the HIV IN inhibitor-binding pocket can be used for drug discovery.

Binding Sites↗
PubMed1997

Subcellular localization of avian sarcoma virus and human immunodeficiency virus type 1 integrases.

The composition and subcellular trafficking of subviral preintegration complexes are reported to vary among the different retroviruses. The process by which the avian sarcoma virus (ASV) preintegration complex gains access to target chromatin remains unknown. Here we report that ASV integrase (IN) expressed as a fusion to beta-galactosidase accumulates in the nuclei of transfected COS-1 cells. In contrast, human immunodeficiency type 1 (HIV-1) IN-beta-galactosidase fusions expressed similarly are predominantly cytoplasmic. To identify the region of ASV IN that specifies nuclear localization, various subdomains of the protein were expressed as beta-galactosidase fusions and their subcellular locations were assessed cytochemically and by indirect immunofluorescence. These analyses showed that the ASV IN protein possesses a functional nuclear localization signal that spans amino acids 206 to 235 and displays limited homology with known nuclear transport signals.

Amino Acid Sequence↗
PubMed2004

Unintegrated lentivirus DNA persistence and accessibility to expression in nondividing cells: analysis with class I integrase mutants.

The circumstances under which unintegrated lentivirus DNA can persist and be a functional template for transcription and protein expression are not clear. We constructed and validated the first class I (nonpleiotropic) integrase (IN) mutants for a non-human lentivirus (feline immunodeficiency virus [FIV]) and analyzed both these and known class I human immunodeficiency virus type 1 IN mutants. The FIV IN mutants (D66V and D66V/D118A) had class I properties: Gag/Pol precursor expression, proteolytic processing, particle formation, and reverse transcriptase (RT) production were normal, while the transduction of dividing fibroblasts was prevented and integration was blocked. When injected into rat retinas, the wild-type (WT) vector produced extensive, persistent transgene expression, compared with only rare positive neuronal cells for the IN mutant vector. In contrast, both WT and mutant vectors produced entirely equivalent, effective transduction levels of primary rat neurons (retinal ganglion cells). By testing the hypothesis that the unexpected retinal neuron transduction was related to cell cycle status, we found that when fibroblasts were growth arrested, transduction and internally promoted transgene expression were not inhibited at all by the class I FIV or HIV-1 IN mutations. Cells were then transduced under aphidicolin arrest and were released from the block 48 h later. Vector expression was stable and durable during repeated passaging in WT vector-transduced cells, while the release of cells transduced with equivalent RT units of class I IN mutant FIV or HIV vector resulted in a steady decline of expression, from 97 to 0% of cells by day 10. Southern blot and PCR analyses showed a lack of integration, irrespective of cell cycle, for the class I mutants and an increase in one- and two-long terminal repeat circular and linear unintegrated DNAs in growth-arrested cells. We conclude that if cell division is prevented, unintegrated FIV and HIV-1 vector DNAs can produce high-level internally promoted transgene expression equivalent to WT vectors. The expression correlates with the unintegrated DNA levels. These observations may facilitate the study of the roles of IN and other preintegration complex components in preintegration phases of infection by (i) providing an alternative way to monitor unintegrated nuclear cDNA forms, (ii) restricting ascertainment to the transcriptionally functional subset of unintegrated DNA, (iii) enabling analysis in individual, nondividing cells, and (iv) uncoupling other potential functions of IN from integration.

Amino Acid Sequence↗
PubMed2004

Azido-containing diketo acid derivatives inhibit human immunodeficiency virus type 1 integrase in vivo and influence the frequency of deletions at two-long-terminal-repeat-circle junctions.

We previously found that azido-containing beta-diketo acid derivatives (DKAs) are potent inhibitors of human immunodeficiency virus type 1 (HIV-1) integrase (IN) (X. Zhang et al., Bioorg. Med. Chem. Lett., 13:1215-1219, 2003). To characterize the intracellular mechanisms of action of DKAs, we analyzed the antiviral activities of two potent azido-containing DKAs with either a monosubstitution or a disubstitution of azido groups, using single- and multiple-replication-cycle assays. Both azido-containing DKAs significantly inhibited HIV-1 infection in 293T, CEM-SS, and H9 cells (50% inhibitory concentration = 2 to 13 micro M) and exhibited low cytotoxicity (50% cytotoxic concentration = 60 to 600 micro M). Inhibition of HIV-1 IN in vivo was demonstrated by the observation that previously described L-708,906 resistance mutations in HIV-1 IN (T66I and T66I/S153Y) also conferred resistance to the azido-group-containing DKAs. In vitro assays and in vivo analysis indicated that the DKAs did not significantly inhibit the 3' processing and selectively inhibited the strand transfer reaction. In addition, quantitative PCR indicated that two-long-terminal-repeat (2-LTR) circles were elevated in the presence of the azido-containing DKAs, confirming that HIV-1 IN was the intracellular target of viral inhibition. To gain insight into the mechanism by which the DKAs increased 2-LTR-circle formation of 3'-processed viral DNAs, we performed extensive DNA sequencing analysis of 2-LTR-circle junctions. The results indicated that the frequency of deletions at the circle junctions was elevated from 19% for the untreated controls to 32 to 41% in the presence of monosubstituted (but not disubstituted) DKAs. These results indicate that the structure of the DKAs can influence the extent of degradation of viral DNA ends by host nucleases and the frequency of deletions at the 2-LTR-circle junctions. Thus, sequencing analysis of 2-LTR-circle junctions can elucidate the intracellular mechanisms of action of HIV-1 IN inhibitors.

Base Sequence↗
PubMed2004

Design and synthesis of novel dihydroxyindole-2-carboxylic acids as HIV-1 integrase inhibitors.

In a search for new HIV-1 integrase (IN) inhibitors, we synthesized and evaluated the biological activity of 5,6-dihydroxyindole-2-carboxylic acid (DHICA) and a series of its derivatives. These compounds were designed as conformationally constrained analogues of the acrylate moiety of caffeic acid phenethyl ester (CAPE). DHICA, an intermediate in the biosynthesis of melanins, was prepared as a monomeric unit by a novel synthetic route. In order to perform coherent SAR studies, two series of DHICA amides were synthesized. First, to validate the utility of a previously identified three-point pharmacophore based on CAPE in inhibitor design, we prepared a series of benzyl- or phenylethylamine substituted derivatives lacking and containing hydroxyl groups. Second, dimers of DHICA containing various aminoalkylamine linkers were also prepared with a goal to increase potency. All compounds were tested against purified IN and the C65S mutant in enzyme-based assays. They were also tested for cytotoxicity in an ovarian carcinoma cell line and antiviral activity in HIV-1-infected CEM cells. Seven compounds inhibited catalytic activities of purified IN with IC50 values below 10 microM. Further computational docking studies were performed to determine the title compounds' mode of interaction with the IN active site. The residues K156, K159 and D64 were the most important for potency against purified IN.

Catalysis↗
PubMed2005

Design of novel bioisosteres of beta-diketo acid inhibitors of HIV-1 integrase.

HIV-1 integrase (IN) is an attractive and validated target for the development of novel therapeutics against AIDS. Significant efforts have been devoted to the identification of IN inhibitors using various methods. In this context, through virtual screening of the NCI database and structure-based drug design strategies, we identified several pharmacophoric fragments and incorporated them on various aromatic or heteroaromatic rings. In addition, we designed and synthesized a series of 5-aryl(heteroaryl)-isoxazole-3-carboxylic acids as biological isosteric analogues of beta-diketo acid containing inhibitors of HIV-1 IN and their derivatives. Further computational docking studies were performed to investigate the mode of interactions of the most active ligands with the IN active site. Results suggested that some of the tested compounds could be considered as lead compounds and suitable for further optimization.

Acquired Immunodeficiency Syndrome↗
PubMed1998

Identification of HIV-1 integrase inhibitors based on a four-point pharmacophore.

The rapid emergence of human immunodeficiency virus (HIV) strains resistant to available drugs implies that effective treatment modalities will require the use of a combination of drugs targeting different sites of the HIV life cycle. Because the virus cannot replicate without integration into a host chromosome, HIV-1 integrase (IN) is an attractive therapeutic target. Thus, an effective IN inhibitor should provide additional benefit in combination chemotherapy. A four-point pharmacophore has been identified based on the structures of quinalizarin and purpurin, which were found to be potent IN inhibitors using both a preintegration complex assay and a purified enzyme assay in vitro. Searching with this four-point pharmacophore in the 'open' part of the National Cancer Institute three-dimensional structure database produced 234 compounds containing the pharmacophore. Sixty of these compounds were tested for their inhibitory activity against IN using the purified enzyme; 19 were found to be active against IN with IC50 values of less than 100 microM, among which 10 had IC50 values of less than 10 microM. These inhibitors can further serve as leads, and studies are in progress to design novel inhibitors based on the results presented in this study.

Anthraquinones↗
PubMed1998

Synthesis and anti-human immunodeficiency virus type 1 integrase activity of hydroxybenzoic and hydroxycinnamic acid flavon-3-yl esters.

A series of new hydroxybenzoic and hydroxycinnamic acid flavon-3-yl esters were synthesized in order to obtain compounds targeting the human immunodeficiency virus (HIV) type 1 integrase (IN). The esters were tested for anti-IN and anti-reverse transcriptase (RT) activity in enzyme assays and for anti-HIV-1, anti-proliferative and anti-topoisomerase activity in cell-based assays. In enzyme assays, the two gallic acid flavon-3-yl esters showed a notable IN inhibition (IC50 values were 8.3 and 9.1 microM, respectively), while the two caffeic acid flavon-3-yl esters exhibited a modest activity (IC50 75 and 60 microM, respectively). Replacement of hydroxyl groups resulted in loss of potency. Caffeic acid 3',4'-dichloroflavon-3-yl ester also inhibited the RT activity whereas it was not active on human topoisomerases. It therefore represents an interesting example of a compound specifically targeting more than one step of the virus replication cycle.

Anti-HIV Agents↗
PubMed2000

A new caffeoyl quinic acid from aster scaber and its inhibitory activity against human immunodeficiency virus-1 (HIV-1) integrase.

The phytochemical study of the aerial parts of Aster scaber Thunb. (Asteraceae) yielded a new caffeoyl quinic acid, (-) 3,5-dicaffeoyl-muco-quinic acid (2) and three known compounds, (-) 3,5-dicaffeoyl quinic acid (1), (-) 4,5-dicaffeoyl quinic acid (3), (-) 5-caffeoyl quinic acid (4). The structures were established by high resolution spectroscopic methods. The antiviral effects against HIV-1 integrase of the compounds was evaluated. (-) 3,5-Dicaffeoyl-muco-quinic acid (2) exhibited potent antiviral activity with an IC50 value of 7.0 +/- 1.3 microg/ml.

Asteraceae↗
PubMed2002

Sesquiterpenes and alkaloids from Lindera chunii and their inhibitory activities against HIV-1 integrase.

Three new eudesmane type sesquiterpenoid lindenanolides E (1), F (2) and G (3), and two new aporphine alkaloid lindechunines A (18) and B (20) were isolated from roots of Lindera chunii MERR., together with seven known sesquiterpenes including a new naturally-occurring lindenanolide H (4) and eight known aporphine alkaloids. The structures of these compounds were determined by spectroscopic means. Of the isolated compounds, hernandonine (14), laurolistine (16), 7-oxohernangerine (17) and lindechunine A (18) showed significant anti-human immunodeficiency virus type 1 (HIV-1) integrase activity with IC(50) values of 16.3, 7.7, 18.2 and 21.1 microM, respectively. The major alkaloids presented in the roots of L. chunii were quantitatively analyzed by an HPLC method.

Alkaloids↗