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PubMed1997

Curcumin analogs with altered potencies against HIV-1 integrase as probes for biochemical mechanisms of drug action.

We have previously reported the inhibitory activity of curcumin against human immunodeficiency virus type one (HIV-1) integrase. In the present study, we have synthesized and tested analogs of curcumin to explore the structure-activity relationships and mechanism of action of this family of compounds in more detail. We found that two curcumin analogs, dicaffeoylmethane (6) and rosmarinic acid (9), inhibited both activities of integrase with IC50 values below 10 microM. We have previously demonstrated that lysine 136 may play a role in viral DNA binding. We demonstrated equivalent potencies of two curcumin analogs against both this integrase mutant and wild-type integrase, suggesting that the curcumin-binding site and the substrate-binding site may not overlap. Combining one curcumin analog with the recently described integrase inhibitor NSC 158393 resulted in integrase inhibition which was synergistic, reflective of drug-binding sites which may not overlap. We have also determined that these analogs can inhibit binding of the enzyme to the viral DNA but that this inhibition is independent of divalent metal ion. Furthermore, kinetic studies of these analogs suggest that they bind to the enzyme at a slow rate. These studies can provide mechanistic and structural information which may guide the future design of integrase inhibitors.

4-Hydroxycoumarins↗
PubMed1999

Chicoric acid analogues as HIV-1 integrase inhibitors.

The present study was undertaken to examine structural features of L-chicoric acid (3) which are important for potency against purified HIV-1 integrase and for reported cytoprotective effects in cell-based systems. Through a progressive series of analogues, it was shown that enantiomeric D-chicoric acid (4) retains inhibitory potency against purified integrase equal to its L-counterpart and further that removal of either one or both carboxylic functionalities results in essentially no loss of inhibitory potency. Additionally, while two caffeoyl moieties are required, attachment of caffeoyl groups to the central linking structure can be achieved via amide or mixed amide/ester linkages. More remarkable is the finding that blockage of the catechol functionality through conversion to tetraacetate esters results in almost no loss of potency, contingent on the presence of at least one carboxyl group on the central linker. Taken as a whole, the work has resulted in the identification of new integrase inhibitors which may be regarded as bis-caffeoyl derivatives of glycidic acid and amino acids such as serine and beta-aminoalanine. The present study also examined the reported ability of chicoric acid to exert cytoprotective effects in HIV-infected cells. It was demonstrated in target and cell-based assays that the chicoric acids do not significantly inhibit other targets associated with HIV-1 replication, including reverse transcription, protease function, NCp7 zinc finger function, or replication of virus from latently infected cells. In CEM cells, for both the parent chicoric acid and selected analogues, antiviral activity was observable under specific assay conditions and with high dependence on the multiplicity of viral infection. However, against HIV-1- and HIV-2-infected MT-4 cells, the chicoric acids and their tetraacetylated esters exhibited antiviral activity (50% effective concentration (EC50) ranging from 1.7 to 20 microM and 50% inhibitory concentration (IC50) ranging from 40 to 60 microM).

Animals↗
PubMed2006

Effect of DNA modifications on DNA processing by HIV-1 integrase and inhibitor binding: role of DNA backbone flexibility and an open catalytic site.

Integration of the viral cDNA into host chromosomes is required for viral replication. Human immunodeficiency virus integrase catalyzes two sequential reactions, 3'-processing (3'-P) and strand transfer (ST). The first integrase inhibitors are undergoing clinical trial, but interactions of inhibitors with integrase and DNA are not well understood in the absence of a co-crystal structure. To increase our understanding of integrase interactions with DNA, we examined integrase catalysis with oligonucleotides containing DNA backbone, base, and groove modifications placed at unique positions surrounding the 3'-processing site. 3'-Processing was blocked with substrates containing constrained sugars and alpha-anomeric residues, suggesting that integrase requires flexibility of the phosphodiester backbone at the 3'-P site. Of several benzo[a]pyrene 7,8-diol 9,10-epoxide (BaP DE) adducts tested, only the adduct in the minor groove at the 3'-P site inhibited 3'-P, suggesting the importance of the minor groove contacts for 3'-P. ST occurred in the presence of bulky BaP DE DNA adducts attached to the end of the viral DNA suggesting opening of the active site for ST. Position-specific effects of these BaP DE DNA adducts were found for inhibition of integrase by diketo acids. Together, these results demonstrate the importance of DNA structure and specific contacts with the viral DNA processing site for inhibition by integrase inhibitors.

Base Sequence↗
PubMed2005

Cluster analysis and three-dimensional QSAR studies of HIV-1 integrase inhibitors.

Three-dimensional quantitative structure-activity relationship (3D QSAR) and cluster analysis were applied to a variety of HIV-1 integrase inhibitors. One structure was chosen from each of 11 classes of inhibitors to represent the whole class in descriptor-based cluster analysis. The 11 classes of inhibitors were classified into two groups. The molecular field analysis (MFA) models for these two clusters had r2 values of 0.90 and 0.95 and q2 values of 0.85 and 0.91 that were noticeably enhanced from those of conventional QSAR models. The five test compounds, which were proposed to have a common binding site near the metal in HIV-1 integrase based on docking studies by Sotriffer et al., were utilized to compare the predictive capability of MFA and conventional QSAR models. Among these five compounds, only L-chicoric acid belongs to cluster 1 and the other four belong to cluster 2. MFA models give better overall predictions and more importantly the activity of these test compounds is better predicted by the MFA model derived from the cluster each test compound belongs to. The necessity of dividing the inhibitors into two groups to obtain predictive QSAR models supports the likelihood of two separate binding sites.

Cluster Analysis↗
PubMed2002

HIV-1 integrase interacts with yeast microtubule-associated proteins.

The human immunodeficiency virus type 1 (HIV-1) integrase (IN) mediates the insertion of viral DNA into the human genome. In addition to IN, cellular and viral proteins are associated to proviral DNA in the so-called preintegration complex (PIC). We previously reported that the expression of HIV-1 IN in yeast leads to the emergence of a lethal phenotype. This effect may be linked to the IN activity on infected human cells where integration requires the cleavage of genomic DNA. To isolate and characterize potential cellular partners of HIV-1 IN, we used it as a bait in a two-hybrid system with a yeast genomic library. IN interacted with proteins belonging to the microtubule network, or involved in the protein synthesis apparatus. We focused our interest on one of the selected inserts, L2, which corresponds to the C-end half of the yeast STU2p, a microtubule-associated protein (MAP). STU2p is an essential component of the yeast spindle pole body (SPB), which is able to bind microtubules in vitro. After expressing and purifying L2 as a recombinant protein, we showed its binding to IN by ELISA immunodetection. L2 was also able to inhibit IN activity in vitro. In addition, the effect of L2 was tested using the "lethal yeast phenotype". The coexpression of IN and the L2 peptide abolished the lethal phenotype, thus showing important in vivo interactions between IN and L2. The identification of components of the microtubule network associated with IN suggest a role of this complex in the transport of HIV-1 IN present in the PIC to the nucleus, as already described for other human viruses.

Binding Sites↗
PubMed2000

Oligomeric states of the HIV-1 integrase as measured by time-resolved fluorescence anisotropy.

Self-assembly properties of HIV-1 integrase were investigated by time-resolved fluorescence anisotropy using tryptophanyl residues as a probe. From simulation analyses, we show that suitable photon counting leads to an accurate determination of long rotational correlation times in the range of 20-80 ns, permitting the distinction of the monomer, dimer, and tetramer from higher oligomeric forms of integrase. The accuracy of correlation times higher than 100 ns is too low to distinguish the octamer from other larger species. The oligomeric states of the widely used detergent-solubilized integrase were then studied in solution under varying parameters known to influence the activity. In the micromolar range, integrase exists as high-order multimers such as an octamer and/or aggregates and a well-defined tetramer, at 25 and 35 degrees C, respectively. However, integrase is monomeric at catalytically active concentrations (in the sub-micromolar range). Detergents (NP-40 and CHAPS) and divalent cation cofactors (Mg(2+) and Mn(2+)) have a clear dissociative effect on the high multimeric forms of integrase. In addition, we observed that Mg(2+) and Mn(2+) have different effects on both the oligomeric state and the conformation of the monomer. This could explain in part why these two metal cations are not equivalent in terms of catalytic activity in vitro. In contrast, addition of Zn(2+) stimulates dimerization. Interestingly, this role of Zn(2+) in the multimerization process was evident only in the presence of Mg(2+) which by itself does not induce oligomerization. Finally, it is highly suggested that the presence of detergent during the purification procedure plays a negative role in the proper self-assembly of integrase. Accordingly, the accompanying paper [Leh, H., et al. (2000) Biochemistry 39, 9285-9294] shows that a detergent-free integrase preparation has self-assembly and catalytic properties different from those of the detergent-solubilized enzyme.

Cations, Divalent↗
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↗
PubMed2001

Specific inhibition of human immunodeficiency virus type 1 (HIV-1) integration in cell culture: putative inhibitors of HIV-1 integrase.

To study the effect of potential human immunodeficiency virus type 1 (HIV-1) integrase inhibitors during virus replication in cell culture, we used a modified nested Alu-PCR assay to quantify integrated HIV DNA in combination with the quantitative analysis of extrachromosomal HIV DNA. The two diketo acid integrase inhibitors (L-708,906 and L-731,988) blocked the accumulation of integrated HIV-1 DNA in T cells following infection but did not alter levels of newly synthesized extrachromosomal HIV DNA. In contrast, we demonstrated that L17 (a member of the bisaroyl hydrazine family of integrase inhibitors) and AR177 (an oligonucleotide inhibitor) blocked the HIV replication cycle at, or prior to, reverse transcription, although both drugs inhibited integrase activity in cell-free assays. Quercetin dihydrate (a flavone) was shown to not have any antiviral activity in our system despite reported anti-integration properties in cell-free assays. This refined Alu-PCR assay for HIV provirus is a useful tool for screening anti-integration compounds identified in biochemical assays for their ability to inhibit the accumulation of integrated HIV DNA in cell culture, and it may be useful for studying the effects of these inhibitors in clinical trials.

Acetoacetates↗
PubMedDate not supplied

Eigen value analysis of HIV-1 integrase inhibitors.

A three-dimensional quantitative structure activity relationship using the eigen value analysis (EVA) paradigm applied to 41 HIV-1 integrase inhibitors that inhibit integrase mediated cleavage (3'-processing step) and integration (3'-strand transfer step) in vitro was performed. The training set consisted of 35 molecules from five structurally diverse classes: salicylhydrazines, lichen acids, coumarins, quinones, and thiazolothiazepines. Models derived using semiempirical (MOPAC AM1 and PM3) calculated normal-mode frequencies were compared. The predictive ability of each resultant model was evaluated using a test set comprised of six molecules belonging to a different structural class: hydrazides. Models derived using AM1 method showed considerable internal as well as external predictivity (r(2)(cv) = 0.806, r(2)(pred) = 0.761 for 3'-processing and r(2)(cv) = 0.677, r(2)(pred) = 0.591 for 3'-strand transfer).

HIV Integrase↗
PubMed2005

Comparative molecular dynamics simulations of HIV-1 integrase and the T66I/M154I mutant: binding modes and drug resistance to a diketo acid inhibitor.

HIV-1 IN is an essential enzyme for viral replication and an interesting target for the design of new pharmaceuticals for use in multidrug therapy of AIDS. L-731,988 is one of the most active molecules of the class of beta-diketo acids. Individual and combined mutations of HIV-1 IN at residues T66, S153, and M154 confer important degrees of resistance to one or more inhibitors belonging to this class. In an effort to understand the molecular mechanism of the resistance of T66I/M154I IN to the inhibitor L-731,988 and its specific binding modes, we have carried out docking studies, explicit solvent MD simulations, and binding free energy calculations. The inhibitor was docked against different protein conformations chosen from prior MD trajectories, resulting in 2 major orientations within the active site. MD simulations have been carried out for the T66I/M154I DM IN, DM IN in complex with L-731,988 in 2 different orientations, and 1QS4 IN in complex with L-731,988. The results of these simulations show a similar dynamical behavior between T66I/M154I IN alone and in complex with L-731,988, while significant differences are observed in the mobility of the IN catalytic loop (residues 138-149). Water molecules bridging the inhibitor to residues from the active site have been identified, and residue Gln62 has been found to play an important role in the interactions between the inhibitor and the protein. This work provides information about the binding modes of L-731,988, as well as insight into the mechanism of inhibitor-resistance in HIV-1 integrase.

Amino Acid Substitution↗
PubMed1990

Expression of HIV-1 integrase in E. coli: immunological analysis of the recombinant protein.

Sequences encoding the human immunodeficiency virus type 1 (HIV-1) integrase gene have been cloned and expressed in Escherichia coli. The expressed protein is a lambda cII fusion protein of 37 kD containing the carboxyl-terminal 23 [corrected] amino acids of reverse transcriptase fused to the entire integrase sequence and is insoluble, a feature which allows partial purification away from soluble bacterial proteins. As judged by its reactivity with HIV positive sera in Western blot and in enzyme-linked immunosorbent assay (ELISA), the recombinant integrase retains antigenicity similar to native protein. Additionally, ELISA data obtained with the cloned protein indicate that patients infected with HIV-1 who are at different stages of progression to AIDS have antibodies reactive with the cloned integrase. HIV-2 positive human sera are also reactive with the cloned integrase. Rabbit antibodies produced against the recombinant protein react both by ELISA and Western blot with the homologous bacterially expressed protein, recognize both virion HIV-1 integrase and reverse transcriptase in Western blots, and immunoprecipitate an HIV-1 virion protein of 34 kD. Unlike human antisera from patients infected with HIV-1 or HIV-2 which are frequently reactive with both HIV-1 and HIV-2 integrase, the rabbit antibodies are type specific, reacting with HIV-1, but not with HIV-2 integrase by Western blot.

Acquired Immunodeficiency Syndrome↗
PubMed1998

Displacement of viral DNA termini from stable HIV-1 integrase nucleoprotein complexes induced by secondary DNA-binding interactions.

The human immunodeficiency virus type-1 (HIV-1) integrase is known to form a highly stable interaction with the termini of the linear, pre-integrated retroviral genome, where it catalyzes the 3'-OH processing and strand transfer processes required for their coordinated integration into host DNA. Here, we determine that the association of HIV-1 integrase with the viral DNA termini leads to the formation of two classes of nucleoprotein complexes with distinct properties in vitro. Both bound states are intrinsically stable and highly resistant to exonuclease digestion, but nonetheless they exhibit different stabilities in the presence of single-stranded polynucleotides. While a population of preassembled complexes tolerates elevated polynucleotide concentrations, the remainder forms an unstable ternary (integrase-substrate-polynucleotide) intermediate, leading to the rapid expulsion of the otherwise tightly bound substrate. The distribution of complexes between the two states is influenced by the preincubation time and temperature, increases in either of which favor the formation of the challenge-resistant species. Challenge-resistant complexes are formed more efficiently with Mn2+ than with Mg2+ and are sensitive to the length rather than the sequence of the DNA substrate. Due to the delayed appearance of the challenge-resistant form after the initial stable binding of the DNA substrate, our results may be indicative of a structural change in the preassembled complex which thereby modulates its response to exogenous DNA targets.

Base Sequence↗
PubMed2001

Activity of HIV-1 integrases recovered from subjects with varied rates of disease progression.

We recently described 102 HIV-1 integrase sequences that were amplified from blood cells or plasma obtained up to 18 years ago from 5 hemophiliacs who later died of AIDS and 5 hemophiliacs subsequently classified as slow or nonprogressors ( J Acquir Immune Defic Syndr Hum Retrovirol 1998;19:99-110). Although the region of the HIV-1 genome that encodes integrase was highly conserved, none of the deduced protein sequences of the patient-derived enzymes matched that of the clade B consensus or standard laboratory integrases. To test the hypothesis that the activity of HIV-1 integrases prevalent within an infected person contributes to the rate of disease progression, we have now expressed and purified these proteins and compared them in various assays. Most of the 75 unique full-length integrase proteins from the 102 clones were enzymatically active. Comparison of proteins derived from samples obtained soon after infection showed that the specificity and extent of viral DNA processing and the amount of DNA joining (the two biologically relevant activities of integrase) did not differ between the two groups of patients. In addition, the relative usage of alternative nucleophiles for processing and the amount of nonspecific nicking catalyzed by the proteins were indistinguishable between the patient groups. Although the patient-derived enzymes often exhibited different patterns of target site preferences compared with the laboratory integrase, there was no correlation with clinical course. Thus, the activities of HIV-1 integrases prevalent within these infected individuals, at least as reflected by standard assays, did not influence or predict the rate of disease progression.

Acquired Immunodeficiency Syndrome↗
PubMed1997

Depsides and depsidones as inhibitors of HIV-1 integrase: discovery of novel inhibitors through 3D database searching.

Seventeen lichen acids comprising despides, depsidones, and their synthetic derivatives have been examined for their inhibitory activity against HIV-1 integrase, and two pharmacophores associated with inhibition of this enzyme have been identified. A search of the NCI 3D database of approximately 200,000 structures yielded some 800 compounds which contain one or the other pharmacophore. Forty-two of these compounds were assayed for HIV-1 integrase inhibition, and of these, 27 had inhibitory IC50 values of less than 100 microM; 15 were below 50 microM. Several of these compounds were also examined for their activity against HIV-2 integrase and mammalian topoisomerase I.

Anti-HIV Agents↗
PubMed1999

Branched oligonucleotide-intercalator conjugate forming a parallel stranded structure inhibits HIV-1 integrase.

Integration of a DNA copy of the HIV-1 genome into chromosomal DNA of infected cells is a key step of viral replication. Integration is carried out by integrase, a viral protein which binds to both ends of viral DNA and catalyses reactions of the 3'-end processing and strand transfer. A 3'-3' branched oligonucleotide functionalised by the intercalator oxazolopyridocarbazole at each 5'-end was found to inhibit integration in vitro. We show that both a specific (G,A) sequence and the OPC intercalating agent contribute to the capability of the branched oligonucleotide to form a parallel stranded structure responsible for the inhibition.

Base Sequence↗
PubMed2003

Synthesis of styrylbenzofuran derivatives as styrylquinoline analogues for HIV-1 integrase inhibitor.

A series of styrylbenzofuran derivatives (8a-i) as styrylquinoline isosters were efficiently prepared by Wittig reaction and evaluated for inhibitory activity against HIV-1 integrase. In this series, compounds 8g, 8h and 8i containing a free catechol ring showed moderate inhibitory activities (IC50= approximately 40 microM) against HIV-1 integrase, while less than the corresponding styrylquinoline compound (I).

Benzofurans↗
PubMed2000

Selection of amino acid substitutions restoring activity of HIV-1 integrase mutated in its catalytic site using the yeast Saccharomyces cerevisiae.

The integration of proviral DNA into the genome of the host cell is an essential step in the replication of retroviruses. This reaction is catalyzed by a viral-encoded enzyme, the integrase (IN). We have previously shown that human immunodeficiency virus type 1 (HIV-1) IN causes a lethal effect when expressed in yeast cells. This system, called yeast lethal assay, was used as a tool to study IN activity in a cellular context. The yeast lethal assay allowed the selection and characterization of mutations affecting both the lethal phenotype and the in vitro IN activities. IN mutants were produced by random PCR mutagenesis in an IN gene bearing the inactivating D116A mutation in the catalytic site. The corresponding D116A substituted IN does not lead to lethality in yeast. Subsequent selection of mutants able to restore the lethal effect of IN was carried out using the yeast lethal assay. We isolated three mutants presenting a restored phenotype. The mutated IN genes were sequenced and the corresponding proteins were purified to characterize their in vitro activities. The three mutants presented restoration of the in vitro strand transfer activity, while 3' processing was only partially restored.The three mutants differ from D116A IN by at least one amino acid substitution located in the N-terminal domain of the protein, outside of the active site. These new mutated HIV-1 INs may therefore allow a better understanding of the N-terminal domain function in the integration reaction. In addition, these results support our hypothesis that explains the lethal effect as a consequence of the nuclear damage caused by wild-type IN in yeast cells. These data also indicate that the yeast lethal assay can be used as a tool to study the retroviral integration mechanism in a cellular context and to select specific inhibitors.

Amino Acid Substitution↗