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PubMed2005

Discovery of HIV-1 integrase inhibitors through a novel combination of ligand and structure-based drug design.

Over the past 10 years, classical computer-aided molecular design methods have not been frequently applied for the discovery of novel HIV-1 integrase (IN) inhibitors, due to the intrinsic challenges that this enzyme presents. Therefore, a novel approach that combines the chemical information of known integrase inhibitors with the enzyme's detailed 3D structure in a stepwise fashion is proposed: (I) use of a pharmacophore model (PM), which takes into account in a weighted fashion the chemical features of known ligands, in analogous manner to the to search the Maybridge and the NCI 3D databases; (II) drug-likeness optimization; (III) virtual high-throughput screening of the hits matching the PM query against 1QS4 wild-type IN structure using different Docking/Scoring combinations; (IV) visual inspection and selection of the hits in function of: binding free energies; binding mode type within the active site; retrieval among the best 20% hits in more than 6 Docking/Scoring protocols at the same time. This approach aims at a rational selection of new potential HIV-1 integrase inhibitors.

Computer Simulation↗
PubMed2006

Mode of inhibition of HIV-1 Integrase by a C-terminal domain-specific monoclonal antibody.

BACKGROUND: To further our understanding of the structure and function of HIV-1 integrase (IN) we developed and characterized a library of monoclonal antibodies (mAbs) directed against this protein. One of these antibodies, mAb33, which is specific for the C-terminal domain, was found to inhibit HIV-1 IN processing activity in vitro; a corresponding Fv fragment was able to inhibit HIV-1 integration in vivo. Our subsequent studies, using heteronuclear nuclear magnetic resonance spectroscopy, identified six solvent accessible residues on the surface of the C-terminal domain that were immobilized upon binding of the antibody, which were proposed to comprise the epitope. Here we test this hypothesis by measuring the affinity of mAb33 to HIV-1 proteins that contain Ala substitutions in each of these positions. To gain additional insight into the mode of inhibition we also measured the DNA binding capacity and enzymatic activities of the Ala substituted proteins. RESULTS: We found that Ala substitution of any one of five of the putative epitope residues, F223, R224, Y226, I267, and I268, caused a decrease in the affinity of the mAb33 for HIV-1 IN, confirming the prediction from NMR data. Although IN derivatives with Ala substitutions in or near the mAb33 epitope exhibited decreased enzymatic activity, none of the epitope substitutions compromised DNA binding to full length HIV-1 IN, as measured by surface plasmon resonance spectroscopy. Two of these derivatives, IN (I276A) and IN (I267A/I268A), exhibited both increased DNA binding affinity and uncharacteristic dissociation kinetics; these proteins also exhibited non-specific nuclease activity. Results from these investigations are discussed in the context of current models for how the C-terminal domain interacts with substrate DNA. CONCLUSION: It is unlikely that inhibition of HIV-1 IN activity by mAb33 is caused by direct interaction with residues that are essential for substrate binding. Rather our findings are most consistent with a model whereby mAb33 binding distorts or constrains the structure of the C-terminal domain and/or blocks substrate binding indirectly. The DNA binding properties and non-specific nuclease activity of the I267A derivatives suggest that the C-terminal domain of IN normally plays an important role in aligning the viral DNA end for proper processing.

Alanine↗
PubMed2000

An inhibitory monoclonal antibody binds at the turn of the helix-turn-helix motif in the N-terminal domain of HIV-1 integrase.

With the increase in our understanding of its structure and enzymatic mechanism, HIV-1 integrase (IN) has become a promising target for designing drugs to treat patients with AIDS. To investigate the structure and function of IN, a panel of monoclonal antibodies (mAbs) directed against HIV-1 IN was raised and characterized previously in this laboratory. Among them, mAbs17, -4, and -33 were found to inhibit IN activity in vitro. In this study, we investigated the interaction of N-terminal-specific mAb17 and its isolated Fab fragment with full-length HIV-1 IN(1-288) and its isolated N-terminal, Zn(2+)-binding domain IN(1-49). Our results show that binding of Zn(2+) to IN(1-49) stabilizes the mAb17-IN complex and that dimer dissociation is not required for binding of the Fab. To identify the epitope recognized by mAb17, we developed a protein footprinting technique based on controlled proteolysis and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Binding was mapped to a region within amino acids Asp(25)-Glu(35). This peptide corresponds to the end of a helix-turn-helix motif in the IN(1-55) NMR structure and contributes to the dimerization of the N-terminal domain. Antibody binding also appears to destabilize the N-terminal helix in this domain. A molecular model of the [IN(1-49)](2).(Fab)(1) complex shows Fab binding across the dimer protein and suggests a potential target for drug design. These data also suggest that mAb17 inhibits integrase activity by blocking critical protein-protein interactions and/or by distorting the orientation of the N-terminal alpha-helix. The relevance of our results to an understanding of IN function is discussed.

Amino Acid Sequence↗
PubMed2004

HIV-1 integrase can process a 3'-end crosslinked substrate.

Integrase of the human immunodeficiency virus type-1 (HIV-1) recognizes specific sequences located in the U3 and U5 regions at the ends of viral DNA. We synthesized DNA duplexes mimicking the U5 region and containing either 2'-aminonucleosides or non-nucleoside 1,3-propanediol insertions at the third and terminal positions and studied their interactions with HIV-1 integrase. Both modifications introduced a local structural distortion in the DNA double helix. Replacement of the terminal nucleosides by corresponding 2'-aminonucleosides had no significant effect on integrase activity. We used an integrase substrate bearing terminal 2'-aminonucleosides in both strands to synthesize a duplex with cross-linked strands. This duplex was then used to determine whether terminal base pair disruption is an obligatory step of retroviral DNA 3'-processing. Processing of the cross-linked analog of the integrase substrate yielded a product of the same length as 3'-processing of the wild-type substrate but the reaction efficiency was lower. Replacement of the third adenosine in the processed strand by a corresponding 2'-aminonucleoside did not affect integrase activity, whereas, its replacement by 1,3-propanediol completely inhibited 3'-processing. Both modifications of the complementary thymidine in the nonprocessed strand increased the initial rate of 3'-processing. The same effect was observed when both nucleosides, at the third position, were replaced by corresponding 2'-aminonucleosides. This indicates that the local duplex distortion facilitated the cleavage of the phosphodiester bond. Thus, a localized destabilization of the third A-T base pair is necessary for efficient 3'-processing, whereas 3'-end-fraying is important but not absolutely required.

Base Pairing↗
PubMed2002

Styrylquinazoline derivatives as HIV-1 integrase inhibitors.

Styrylquinazoline derivatives were prepared by Perkin condensation and evaluated for inhibitory activity against HIV-1 integrase. Among them, compound 5c containing a free catechol ring was the most potent (IC(50)=0.8 +/- 1.9 microM)and showed 6-fold more potency than the corresponding styrylquinoline compound (IC(50) = 130.7 +/- 8.6 microM).

HIV Integrase Inhibitors↗
PubMed2002

Integramides A and B, two novel non-ribosomal linear peptides containing nine C(alpha)-methyl amino acids produced by fungal fermentations that are inhibitors of HIV-1 integrase.

[structure: see text]. Integramides A and B are two novel 16-mer linear peptides rich in C(alpha)-methyl amino acids that were isolated from fungal extracts of Dendrodochium sp. by employing a bioassay-guided isolation procedure using recombinant HIV-1 integrase. The structure and stereochemistry were elucidated by a combination of 2D NMR and ESI- and FAB-MS including MS/MS studies and by Marfey's method. Integramides A and B inhibited the coupled reaction of HIV-1 integrase with IC50 values of 17 and 10 microM, respectively.

Chromatography, High Pressure Liquid↗
PubMed2001

Self-association of an amphipathic helix peptide inhibitor of HIV-1 integrase assessed by electro spray ionization mass spectrometry in trifluoroethanol/water mixtures.

Establishing the auto-associative properties of a molecule in solution can be important for determination of its structure and function. EAA26 (VESMNEELKKIIAQVRAQAEHLKTAY) has been designed to inhibit HIV-1 integrase via formation of a stable coiled-coil structure with a nearly homologous segment in the enzyme. The latter catalyzes the permanent incorporation of a DNA copy of the retrovirus genome into host cell DNA, and is thus essential to the life of the retrovirus. This makes integrase an obvious drug target in the therapy of AIDS. The present work has demonstrated, using electrospray ionization mass spectrometry (ESI-MS), that EAA26 is monomeric in pure water, and tetrameric and dimeric at respectively low and medium concentrations of 2,2,2-trifluoroethanol (TFE), and again monomeric at higher TFE concentrations. Thus, the apolar solvent TFE may contribute to either stabilization or disruption of the intermolecular hydrophobic contacts depending on its concentration in aqueous solution. Previous NMR and ultracentifugation results are thus confirmed, indicating the reliability of ESI-MS for defining the self-association state of biologically relevant peptides in both water and organic-water solutions.

Amino Acid Sequence↗
PubMed2005

Design and optimization of tricyclic phtalimide analogues as novel inhibitors of HIV-1 integrase.

Human immunodeficiency virus type-1 integrase is an essential enzyme for effective viral replication and hence a valid target for the design of inhibitors. We report here on the design and synthesis of a novel series of phthalimide analogues as integrase inhibitors. The short synthetic pathway enabled us to synthesize a series of analogues with a defined structure diversity. The presence of a single carbonyl-hydroxy-aromatic nitrogen motif was shown to be essential for the enzymatic activity and this was confirmed by molecular docking studies. The enzymatically most active compound from this series is 7-(3,4-dichlorobenzyl)-5,9-dihydroxypyrrolo[3,4-g]quinoxaline-6,8-dione (15l) with an IC(50) value of 112 nM on the HIV-1 integrase enzyme, while ((7-(4-chlorobenzyl)-5,9-dihydroxy-pyrrolo[3,4-g]quinoxaline-6,8-dione (15k)) showed an EC(50) of 270 nM against HIV-1 in a cell-based assay.

Anti-HIV Agents↗
PubMed1998

Analysis of a large collection of natural HIV-1 integrase sequences, including those from long-term nonprogressors.

A large collection of natural HIV-1 integrase (IN) sequences has not previously been described. We reasoned that analysis of such sequences would address whether natural variation of HIV-1 IN contributes to the pathogenesis of AIDS and might also identify amino acid residues important for IN function. Sequences encoding HIV-1 IN were amplified from cryopreserved lymphocytes or plasma obtained at different times from 10 hemophilia patients who had been observed for up to 17 years. The region of the HIV-1 genome that encodes the 288-amino acid IN protein was sequenced from a total of 102 clones; information was obtained for 99.97% of 29,478 amino acid positions. Phylogenetic analysis indicated that patient samples were unique. Interpatient nucleic acid distances ranged from 0.8% to 4.9%, highlighting the tight conservation of this genomic region. No major differences were found between DNA and RNA or between early and late time points from the same patient. Significantly, no amino acid changes that might account for the variable rate of disease progression between patients were evident. Only one amino acid substitution involved a highly conserved residue known to be important for enzymatic activity. However, several interesting amino acid substitutions were noted, including residues within the C-terminal region of the protein for which sequence comparisons between animal retroviruses have not been very informative. These results should encourage the pursuit of anti-integrase therapies, especially inasmuch as the apparent biologic constraints on the IN sequence may deter the development of drug resistance.

Acquired Immunodeficiency Syndrome↗
PubMed1996

A soluble active mutant of HIV-1 integrase: involvement of both the core and carboxyl-terminal domains in multimerization.

Structural studies of human immunodeficiency virus type 1 (HIV-1) integrase have been impeded by the low solubility of the protein. By systematic replacement of hydrophobic residues, we previously identified a single amino acid change (F185K) that dramatically improved the solubility of the catalytic domain of HIV-1 integrase and enabled the structure to be determined by x-ray crystallography. We have introduced the same mutation into full-length HIV-1 integrase. The resulting recombinant protein is soluble and fully active in vitro, whereas, HIV-1 carrying the mutation is replication-defective due to improper virus assembly. Analysis of the recombinant protein by gel filtration and sedimentation equilibrium demonstrate a dimer-tetramer self-association. We find that the regions involved in multimerization map to both the catalytic core and carboxyl-terminal domains. The dramatically improved solubility of this protein make it a good candidate for structural studies.

Amino Acid Sequence↗
PubMed2005

Evaluation of the activity of HIV-1 integrase over-expressed in eukaryotic cells.

Since the integration of viral DNA in the host genome is an essential step in the replication cycle of HIV-1, an active search for inhibitors of the integration step is ongoing. Our laboratory has been working on the development of a cellular integration system. Such a system would be helpful in the study of the HIV-1 integration process and, eventually, could be used in the search for new inhibitors that selectively interfere with HIV integration. We have previously selected stable cell lines (293T-INS) that constitutively express high levels of HIV-1 integrase (IN) from a synthetic gene [FASEB J. 14 (2000) 1389]. We have now constructed linear DNA substrates containing the terminal HIV LTR sequences (so called 'mini-HIV') and EGFP as reporter gene to evaluate whether IN can improve the integration of transfected linear DNA. After electroporation of this mini-HIV we observed a 2- to 3-fold increase in EGFP expression in IN expressing cell lines relative to control cells. The increase in EGFP expression was still evident after passaging of the cells. The effect was observed with linear DNA but not with circular DNA, thus excluding an effect on DNA uptake. The increase was the highest in the 293T-INS(D64V) cell line due to an increase in the amount of total mini-HIV DNA and 2-LTR circles as quantified by Q-PCR. Our data suggest that IN over-expressed in our cell lines interacts with the incoming DNA, protects it from nuclease degradation but does not catalyze the integration as such.

Aspartic Acid↗
PubMed2001

Structure of a two-domain fragment of HIV-1 integrase: implications for domain organization in the intact protein.

Retroviral integrase, an essential enzyme for replication of human immunodeficiency virus type-1 (HIV-1) and other retroviruses, contains three structurally distinct domains, an N-terminal domain, the catalytic core and a C-terminal domain. To elucidate their spatial arrangement, we have solved the structure of a fragment of HIV-1 integrase comprising the N-terminal and catalytic core domains. This structure reveals a dimer interface between the N-terminal domains different from that observed for the isolated domain. It also complements the previously determined structure of the C-terminal two domains of HIV-1 integrase; superposition of the conserved catalytic core of the two structures results in a plausible full-length integrase dimer. Furthermore, an integrase tetramer formed by crystal lattice contacts bears structural resemblance to a related bacterial transposase, Tn5, and exhibits positively charged channels suitable for DNA binding.

Catalytic Domain↗
PubMed2004

CD8+ T lymphocyte responses target functionally important regions of Protease and Integrase in HIV-1 infected subjects.

BACKGROUND: CD8+ T cell responses are known to be important to the control of HIV-1 infection. While responses to reverse transcriptase and most structural and accessory proteins have been extensively studied, CD8 T cell responses specifically directed to the HIV-1 enzymes Protease and Integrase have not been well characterized, and few epitopes have been described in detail. METHODS: We assessed comprehensively the CD8 T cell responses to synthetic peptides spanning Protease and Integrase in 56 HIV-1 infected subjects with acute, chronic, or controlled infection using IFN-gamma-Elispot assays and intracellular cytokine staining. Fine-characterization of novel CTL epitopes was performed on peptide-specific CTL lines in Elispot and 51Chromium-release assays. RESULTS: Thirteen (23%) and 38 (68%) of the 56 subjects had detectable responses to Protease and Integrase, respectively, and together these targeted most regions within both proteins. Sequence variability analysis confirmed that responses cluster largely around conserved regions of Integrase, but responses against a large, highly conserved region of the N-terminal DNA-binding domain of Integrase were not readily detected. CD8 T cell responses targeted regions of Protease that contain known Protease inhibitor mutation residues, but strong Protease-specific CD8 T cell responses were rare. Fine-mapping of targeted epitopes allowed the identification of three novel, HLA class I-restricted, frequently-targeted optimal epitopes. There were no significant correlations between CD8 T cell responses to Protease and Integrase and clinical disease category in the study subjects, nor was there a correlation with viral load. CONCLUSIONS: These findings confirm that CD8 T cell responses directed against HIV-1 include potentially important functional regions of Protease and Integrase, and that pharmacologic targeting of these enzymes will place them under both drug and immune selection pressure.

Journal Article↗
PubMed2006

Relationship between the oligomeric status of HIV-1 integrase on DNA and enzymatic activity.

The 3'-processing of the extremities of viral DNA is the first of two reactions catalyzed by HIV-1 integrase (IN). High order IN multimers (tetramers) are required for complete integration, but it remains unclear which oligomer is responsible for the 3'-processing reaction. Moreover, IN tends to aggregate, and it is unknown whether the polymerization or aggregation of this enzyme on DNA is detrimental or beneficial for activity. We have developed a fluorescence assay based on anisotropy for monitoring release of the terminal dinucleotide product in real-time. Because the initial anisotropy value obtained after DNA binding and before catalysis depends on the fractional saturation of DNA sites and the size of IN.DNA complexes, this approach can be used to study the relationship between activity and binding/multimerization parameters in the same assay. By increasing the IN:DNA ratio, we found that the anisotropy increased but the 3'-processing activity displayed a characteristic bell-shaped behavior. The anisotropy values obtained in the first phase were predictive of subsequent activity and accounted for the number of complexes. Interestingly, activity peaked and then decreased in the second phase, whereas anisotropy continued to increase. Time-resolved fluorescence anisotropy studies showed that the most competent form for catalysis corresponds to a dimer bound to one viral DNA end, whereas higher order complexes such as aggregates predominate during the second phase when activity drops off. We conclude that a single IN dimer at each extremity of viral DNA molecules is required for 3'-processing, with a dimer of dimers responsible for the subsequent full integration.

Anisotropy↗
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↗
PubMed1997

The core domain of HIV-1 integrase recognizes key features of its DNA substrates.

We investigated which features of the substrate specificity of human immunodeficiency virus type 1 (HIV-1) integrase could be assigned to the central domain of the 288-residue HIV-1 integrase protein, composed of amino acids 50-212. This domain contains the active site and shares structural homology with a large family of polynucleotidyl transferases. Using model substrates with defined alterations in critical features we found that this domain alone is sufficient for recognition of: 1) the phylogenetically conserved CA/TG base pairs near the viral DNA end; 2) the 5'-terminal dinucleotide that is left unpaired after end processing; and 3) target DNA flanking the site of joining. Future efforts aimed at identifying specific amino acids involved in recognition of these key substrate features can now be targeted at this domain.

Catalysis↗
PubMed1995

Solution structure of the DNA binding domain of HIV-1 integrase.

The solution structure of the DNA binding domain of HIV-1 integrase (residues 220-270) has been determined by multidimensional NMR spectroscopy. The protein is a dimer in solution, and each subunit is composed of a five-stranded beta-barrel with a topology very similar to that of the SH3 domain. The dimer is formed by a stacked beta-interface comprising strands 2, 3, and 4, with the two triple-stranded antiparallel beta-sheets, one from each subunit, oriented antiparallel to each other. One surface of the dimer, bounded by the loop between strands beta 1 and beta 2, forms a saddle-shaped groove with dimensions of approximately 24 x 23 x 12 A in cross section. Lys264, which has been shown from mutational data to be involved in DNA binding, protrudes from this surface, implicating the saddle-shaped groove as the potential DNA binding site.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Modeling HIV-1 integrase complexes based on their hydrodynamic properties.

We present a model structure of a candidate tetramer for HIV-1 integrase. The model was built in three steps using data from fluorescence anisotropy, structures of the individual integrase domains, cross-linking data, and other biochemical data. First, the structure of the full-length integrase monomer was modeled using the individual domain structures and the hydrodynamic properties of the full-length protein that were recently measured by fluorescence depolarization. We calculated the rotational correlation times for different arrangements of three integrase domains, revealing that only structures with close proximity among the domains satisfied the experimental data. The orientations of the domains were constrained by iterative tests against the data on cross-linking and footprinting in integrase-DNA complexes. Second, the structure of an integrase dimer was obtained by joining the model monomers in accordance with the available dimeric crystal structures of the catalytic core. The hydrodynamic properties of the dimer were in agreement with the experimental values. Third, the active sites of the two model dimers were placed in agreement with the spacing between the sites of integration on target DNA as well as the integrase-DNA cross-linking data, resulting in twofold symmetry of a tetrameric complex. The model is consistent with the experimental data indicating that the F185K substitution, which is found in the model at a tetramerization interface, selectively disrupts correct complex formation in vitro and HIV replication in vivo. Our model of the integrase tetramer bound to DNA may help to design anti-integrase inhibitors.

Base Sequence↗