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At least 253 records · Page 14Linked to original sources

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↗

PubMed2005

Pharmacophore-based design of HIV-1 integrase strand-transfer inhibitors.

Using a training set of diketo-like acid HIV-1 integrase (IN) strand-transfer inhibitors, a 3D pharmacophore model was derived having quantitative predictive ability in terms of activity. The best statistical hypothesis consisted of four features (one hydrophobic aromatic region, two hydrogen-bond acceptors, and one hydrogen-bond donor) with r of 0.96. The resulting pharmacophore model guided the rational design of benzylindoles as new potent IN inhibitors, whose microwave-assisted synthesis and biological evaluation are reported.

Anti-HIV Agents↗

PubMed2006

Triketoacid inhibitors of HIV-integrase: a new chemotype useful for probing the integrase pharmacophore.

Integrase is one of three enzymes expressed by HIV and represents a validated target for therapy. This study reports on the discovery of a new triketoacid-based chemotype that selectively inhibits the strand transfer reaction of HIV-integrase. SAR studies showed that the template binds to integrase in a manner similar to the diketoacid-based inhibitors. Moreover, comparison of the new chemotype to two different diketoacid templates led us to propose two aryl-binding domains in the inhibitor binding site. This information was used to design a new diketoacid template with improved activity against the enzyme.

HIV Integrase↗

PubMed2005

Rational design of novel diketoacid-containing ferrocene inhibitors of HIV-1 integrase.

Molecular interaction field, density functional, and docking studies of novel potential ferrocene inhibitors of HIV-1 integrase (IN) are reported. The high docking scores, analysis of the ligand-receptor interactions in the active site as well as the molecular interaction potential calculations at the binding site of the receptor indicate important features for novel HIV-1 IN inhibitors. We also confirm in this work a novel binding trench in HIV-1 integrase, recently reported in a theoretical work by other authors. This observation may be interesting since the lack of detailed structural information about IN-ligand interactions has hampered the design of IN inhibitors. Our proposed ligands are open to experimental synthesis and testing.

Acids↗

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↗

PubMed2004

Resistance towards exonucleases of dinucleotides with stereochemically altered internucleotide phosphate bonds.

Kinetic constants for the hydrolytic susceptibility of the internucleotide phosphate bond in normal dinucleotides [e.g., 2'-deoxycytidylyl-(3'>5')-2'-deoxyuridine (dCpdU) and 2'-deoxyadenylyl-(3'-->5')-2'-deoxycytidine (dApdC)] and isomeric dinucleotides [e.g., 2'-deoxycytidylyl-(3'-->5')-1'-deoxy-2'-isouridine (dCpisodU) and 1'-deoxy-2'-isoadenylyl-(3'-->5')-2'-deoxycytidine (isodApdC)], toward 5'- and 3'-exonucleases, phosphodiesterase I (PDE I) and phosphodiesterase II (PDE II) were experimentally determined and remarkable differences emerged. The study is of importance in the discovery of nuclease-stable inhibitors of HIV integrase, but may also have ramifications in the area of anti-sense oligonucleotides of therapeutic interest.

Dinucleoside Phosphates↗

PubMed2003

Reversible inhibitors of lambda integrase-mediated recombination efficiently trap Holliday junction intermediates and form the basis of a novel assay for junction resolution.

The bacteriophage lambda integrase catalyzes four site-specific recombination pathways with distinct protein and DNA requirements and nucleoprotein intermediates. Some of these intermediates are very transient and difficult to obtain in significant amounts, due to the high efficiency and processivity of integrase, the lack of requirements for external energy factors or metal ions, and the highly reversible nature of each of the intermediates. We have previously used mixture-based combinatorial libraries to identify hexapeptides that trap 40-60% of recombination substrates at the Holliday junction stage of the reaction. These inhibitors discriminate between the four pathways, blocking one of them (bent-L recombination) more severely than the others and blocking the excision pathway least. We presume that these differences reflect specific conformational differences of the nucleoprotein intermediates in each pathway. We have now identified new inhibitors of the excision pathway. One of these, WRWYCR, is over 50-fold more potent at inhibiting excision than the previously identified peptides. This peptide stably traps Holliday junction complexes in all recombination pathways mediated by integrase as well as Cre. This finding and other data presented indicate that the peptide's target is a common feature shared by the Holliday junction complexes assembled by tyrosine recombinases. We have taken advantage of reversible inhibition by the active peptides to develop a new assay for Holliday junction resolution. This assay is particularly useful for determining junction resolution rates in cases where complexes directly assembled on junction substrates undergo little or no catalysis.

Bacteriophage lambda↗

PubMed2002

[HIV-1 integrase enzyme linked immunosorbent assay and inhibitors].

BACKGROUND: To establish an ELISA method for detecting the activity of HIV-1 integrase and screening of inhibitors. METHODS: HIV recombinant plasmid F185K/C280S IN1-288 was transformed into the E.coli strain BL21(DE3) integrase with a histag was induced by adding isopropyl-?-D?thiogalactopyranoside (IPTG)and purified by nickel affinity chromatography. The synthesized donor substrate oligonucleotide representing the HIV-1 U5LTR was immobilized onto covalink polystyrene microtiter plates, and a synthesized biotinlated 20 bp oligonucleotide was used as the target substrate. The products were detected and quantified using a colorimetic avidin?linked alkaline phosphatase reporter system,and identified by 32? autoradiography. Some natural products and chemically synthesized compounds were screened for HIV-1 integrase inhibitors. RESULTS: The purified integrase was identified by SDS?PAGE and showed integration activity by ELISA and?32P radioisotopic assay.?Coefficients of variation (CV)of ELISA in a lot and among the lots were 4.63% and 5.89% respectively, the mean of P/N was 2.836 0.161 under the optimal experimental condition. Some plant extracts were found as potent integrase inhibitors. The IC50s for CEH and CEHL were (20.41 5.68)?g/ml and (7.56 1.86)?g/ml respectively. CONCLUSIONS: The authors have established a simple and rapid ELISA method with stable repeatability for detecting integrase activity, which can be used for screening and studying of specific inhibitors of HIV-1 integrase.

Antiviral Agents↗

PubMed1997

Discovery and analysis of inhibitors of the human immunodeficiency integrase.

An essential step in the replication of retroviruses is the integration of a DNA copy of the viral genome into the genome of the host cell. Integration encompasses a series of ordered endonucleolytic and DNA strand transfer reactions catalyzed by the viral enzyme, integrase. The requirement for integrase activity in the propagation of HIV-1 in cell culture defines the enzyme as a potential target for chemotherapeutic intervention. We have therefore developed a non-radioisotopic microtiter plate assay which can be used to identify novel inhibitors of integrase from random chemical screens and for the bioassay driven isolation of inhibitors from natural products. This assay uncouples various steps in the reaction pathway and therefore can be exploited to characterize inhibitors. In this monograph we describe a series of modifications to the method which facilitate such mechanistic studies using as an example a series of previously described integrase inhibitors.

Antiviral Agents↗

PubMed2005

Diketo acid pharmacophore. 2. Discovery of structurally diverse inhibitors of HIV-1 integrase.

Because of its unique role in the viral replication process, HIV-1 integrase (IN) is an important antiretroviral drug target. The beta-diketo acid class of IN inhibitors has played a major role in validating IN as a legitimate target for antiretroviral drug design. S-1360 (1) and L-870,810 (2) are examples of beta-diketo acid related compounds to enter clinical trials. With an aim to discover novel lead compounds with diverse structural scaffolds, we employed common feature pharmacophore models using four known beta-diketo acid analogues including S-1360 (J. Med. Chem. 2005, 1, 111-120). The best-ranked pharmacophore model (Hypo1) contained a hydrophobic (HYA), an H-bond acceptor (HBA), and two H-bond donor (HBD) features. A search of a 3D database containing approximately 150,000 small molecules using Hypo1 found 1700 compounds that satisfied all the features of the pharmacophore query. Of the 1700 compounds, 110 were selected for in vitro screening studies on the basis of their docking scores, predicted binding location inside the active site of IN, and their druglike properties. Forty-eight compounds inhibited IN catalytic activities with an IC50 value less than 100 microM. Twenty-seven structurally diverse inhibitors are reported here. Out of the 27 compounds, 13 compounds inhibited strand transfer activity of IN with an IC50 value less than 30 microM. These compounds are novel, druglike, and readily amenable for synthetic optimization.

Cell Line↗

PubMed1997

HIV-1 integrase pharmacophore: discovery of inhibitors through three-dimensional database searching.

Starting from a known inhibitor of human immunodeficiency virus type 1 (HIV-1) integrase (IN); caffeic acid phenethyl ester (CAPE), a putative three-point pharmacophore for binding of inhibitors to IN was derived. This pharmacophore was used to search the National Cancer Institute three-dimensional (3D) structural database. Out of the open, nonproprietary part of this database, comprising approximately 200000 compounds, 267 structures were found to match the pharmacophore in at least one conformation, and 60 of those were tested in an in vitro assay against HIV-1 IN. Out of these, 19 were found to inhibit both the 3'-processing and strand transfer of IN at micromolar concentrations. In order to test the validity of this pharmacophore, a small 3D database of 152 published IN inhibitors was built. A search in this database yielded a statistically significant correlation of the presence of this pharmacophore and the potency of the compounds. An automated pharmacophore identification procedure performed on this set of compounds provided additional support for the importance of this pharmacophore for binding of inhibitors to IN and hinted at a possible second pharmacophore. The role of aromatic moieties in the binding of ligands to HIV-1 IN through interactions with divalent metal cations, which are known to be necessary for activity of the enzyme, was explored in ab initio calculations.

Caffeic Acids↗

PubMed1998

Geometrically and conformationally restrained cinnamoyl compounds as inhibitors of HIV-1 integrase: synthesis, biological evaluation, and molecular modeling.

Various cinnammoyl-based structures were synthesized and tested in enzyme assays as inhibitors of the HIV-1 integrase (IN). The majority of compounds were designed as geometrically or conformationally constrained analogues of caffeic acid phenethyl ester (CAPE) and were characterized by a syn disposition of the carbonyl group with respect to the vinylic double bond. Since the cinnamoyl moiety present in flavones such as quercetin (inactive on HIV-1-infected cells) is frozen in an anti arrangement, it was hoped that fixing our compounds in a syn disposition could favor anti-HIV-1 activity in cell-based assays. Geometrical and conformational properties of the designed compounds were taken into account through analysis of X-ray structures available from the Cambridge Structural Database. The polyhydroxylated analogues were prepared by reacting 3,4-bis(tetrahydropyran-2-yloxy)benzaldehyde with various compounds having active methylene groups such as 2-propanone, cyclopentanone, cyclohexanone, 1,3-diacetylbenzene, 2, 4-dihydroxyacetophenone, 2,3-dihydro-1-indanone, 2,3-dihydro-1, 3-indandione, and others. While active against both 3'-processing and strand-transfer reactions, the new compounds, curcumin included, failed to inhibit the HIV-1 multiplication in acutely infected MT-4 cells. Nevertheless, they specifically inhibited the enzymatic reactions associated with IN, being totally inactive against other viral (HIV-1 reverse transcriptase) and cellular (RNA polymerase II) nucleic acid-processing enzymes. On the other hand, title compounds were endowed with remarkable antiproliferative activity, whose potency correlated neither with the presence of catechols (possible source of reactive quinones) nor with inhibition of topoisomerases. The SARs developed for our compounds led to novel findings concerning the molecular determinants of IN inhibitory activity within the class of cinnamoyl-based structures. We hypothesize that these compounds bind to IN featuring the cinnamoyl residue C=C-C=O in a syn disposition, differently from flavone derivatives characterized by an anti arrangement about the same fragment. Certain inhibitors, lacking one of the two pharmacophoric catechol hydroxyls, retain moderate potency thanks to nonpharmacophoric fragments (i.e., a m-methoxy group in curcumin) which favorably interact with an "accessory" region of IN. This region is supposed to be located adjacent to the binding site accommodating the pharmacophoric dihydroxycinnamoyl moiety. Disruption of coplanarity in the inhibitor structure abolishes activity owing to poor shape complementarity with the target or an exceedingly high strain energy of the coplanar conformation.

Anti-HIV Agents↗

PubMed2002

Structure-based design of a novel peptide inhibitor of HIV-1 integrase: a computer modeling approach.

The insertion of viral DNA into the host chromosome is an essential step in the replication of HIV-1, and is carried out by an enzyme, HIV-1 integrase (IN). Since the latter has no human cellular counterpart, it is an attractive target for antiviral drug design. Several IN inhibitors having activities in the micromolar range have been reported to date. However, no clinically useful inhibitors have yet been developed. Recently reported diketo acids represent a novel and selective class of IN inhibitors. These are the only class which appear to selectively target integrase and two of the inhibitors, L-708,906 and L-731,988, are the most potent inhibitors of preintegration complexes described to date. The X-ray crystal structure of the IN catalytic domain complexed with a diketo acid derivative inhibitor, 5CITEP, has recently been determined. Although the structure is of great value as a platform for drug design, experimental data suggest that crystal packing effects influence the observed inhibitor position. This has been confirmed by computational docking studies using the latest version (3.0) of the AutoDock program, which has been shown to give results largely consistent with available experimental data. Using AutoDock 3.0 and SYBYL6.6 we have modeled the complexes of IN with the diketo acid inhibitors so as to identify the enzyme binding site. In the quest for novel, potent and selective small molecule inhibitors, we present here a new approach to peptide inhibitor design using a, b- unsaturated (dehydro) residues, which confer a unique conformation on a peptide sequence. Based on the above models, we selected a tetrapeptide sequence containing a dehydro-Phe residue, which was found to have an open conformation as ascertained from its X-ray crystal structure. Docking results on this peptide led us to propose a modification at the C-terminal end. The modified peptide was found to dock in a similar position as the diketo acid inhibitors and was predicted to have a comparable potency.

Acetoacetates↗

PubMed2004

A naphthyridine carboxamide provides evidence for discordant resistance between mechanistically identical inhibitors of HIV-1 integrase.

The increasing incidence of resistance to current HIV-1 therapy underscores the need to develop antiretroviral agents with new mechanisms of action. Integrase, one of three viral enzymes essential for HIV-1 replication, presents an important yet unexploited opportunity for drug development. We describe here the identification and characterization of L-870,810, a small-molecule inhibitor of HIV-1 integrase with potent antiviral activity in cell culture and good pharmacokinetic properties. L-870,810 is an inhibitor with an 8-hydroxy-(1,6)-naphthyridine-7-carboxamide pharmacophore. The compound inhibits HIV-1 integrase-mediated strand transfer, and its antiviral activity in vitro is a direct consequence of this ascribed effect on integration. L-870,810 is mechanistically identical to previously described inhibitors from the diketo acid series; however, viruses selected for resistance to L-870,810 contain mutations (integrase residues 72, 121, and 125) that uniquely confer resistance to the naphthyridine. Conversely, mutations associated with resistance to the diketo acid do not engender naphthyridine resistance. Importantly, the mutations associated with resistance to each of these inhibitors map to distinct regions within the integrase active site. Therefore, we propose a model of the two inhibitors that is consistent with this observation and suggests specific interactions with discrete binding sites for each ligand. These studies provide a structural basis and rationale for developing integrase inhibitors with the potential for unique and nonoverlapping resistance profiles.

Animals↗

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↗

PubMed2004

L-chicoric acid inhibits human immunodeficiency virus type 1 integration in vivo and is a noncompetitive but reversible inhibitor of HIV-1 integrase in vitro.

The human immunodeficiency virus (HIV) integrase (IN) must covalently join the viral cDNA into a host chromosome for productive HIV infection. l-Chicoric acid (l-CA) enters cells poorly but is a potent inhibitor of IN in vitro. Using quantitative real-time polymerase chain reaction (PCR), l-CA inhibits integration at concentrations from 500 nM to 10 microM but also inhibits entry at concentrations above 1 microM. Using recombinant HIV IN, steady-state kinetic analyses with l-CA were consistent with a noncompetitive or irreversible mechanism of inhibition. IN, in the presence or absence of l-CA, was successively washed. Inhibition of IN diminished, demonstrating that l-CA was reversibly bound to the protein. These data demonstrate that l-CA is a noncompetitive but reversible inhibitor of IN in vitro and of HIV integration in vivo. Thus, l-CA likely interacts with amino acids other than those which bind substrate.

Acetoacetates↗

PubMed2004

Identification of an inhibitor-binding site to HIV-1 integrase with affinity acetylation and mass spectrometry.

We report a methodology that combines affinity acetylation with MS analysis for accurate mapping of an inhibitor-binding site to a target protein. For this purpose, we used a known HIV-1 integrase inhibitor containing aryl di-O-acetyl groups (Acetylated-Inhibitor). In addition, we designed a control compound (Acetylated-Control) that also contained an aryl di-O-acetyl group but did not inhibit HIV-1 integrase. Examination of the reactivity of these compounds with a model peptide library, which collectively contained all 20 natural amino acids, revealed that aryl di-O-acetyl compounds effectively acetylate Cys, Lys, and Tyr residues. Acetylated-Inhibitor and Acetylated-Control exhibited comparable chemical reactivity with respect to these small peptides. However, these two compounds differed markedly in their interactions with HIV-1 integrase. In particular, Acetylated-Inhibitor specifically acetylated K173 at its inhibitory concentration (3 microM) whereas this site remained unrecognized by Acetylated-Control. Our data enabled creation of a detailed model for the integrase:Acetylated-Inhibitor complex, which indicated that the inhibitor selectively binds at an architecturally critical region of the protein. The methodology reported herein has a generic application for systems involving a variety of ligand-protein interactions.

Acetylation↗

PubMed1999

Thiazolothiazepine inhibitors of HIV-1 integrase.

A series of thiazolothiazepines were prepared and tested against purified human immunodeficiency virus type-1 integrase (HIV-1 IN) and viral replication. Structure-activity studies reveal that the compounds possessing the pentatomic moiety SC(O)CNC(O) with two carbonyl groups are in general more potent against purified IN than those containing only one carbonyl group. Substitution with electron-donating or -withdrawing groups did not enhance nor abolish potency against purified IN. By contrast, compounds with a naphthalene ring system showed enhanced potency, suggesting that a hydrophobic pocket in the IN active site might accommodate an aromatic system rather than a halogen. The position of sulfur in the thiazole ring appears important for potency against IN, as its replacement with an oxygen or carbon abolished activity. Further extension of the thiazole ring diminished potency. Compounds 1, 19, and 20 showed antiviral activity and inhibited IN within similar concentrations. These compounds inhibited IN when Mn(2+) or Mg(2+) was used as cofactor. None of these compounds showed detectable activities against HIV-1 reverse transcriptase, protease, virus attachment, or nucleocapsid protein zinc fingers. Therefore, thiazolothiazepines are potentially important lead compounds for development as inhibitors of IN and HIV replication.

Cell Adhesion↗