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PubMed2002

HIV-1 integrase and RNase H activities as therapeutic targets.

The retroviruses are a large, diverse family of enveloped RNA viruses defined by their structure, composition and replicative properties. The hallmark of the family is its replicative strategy, essential steps of which include reverse transcription of the viral RNA and the subsequent integration of this DNA into the genome of the cell. These steps are performed by two viral-encoded enzymes, reverse transcriptase (RT), which possesses DNA polymerase and ribonuclease H (RNase H) activities, and integrase (IN). These enzymes are excellent targets for retroviral therapy since they are essential for viral replication. Numerous substances capable of inhibiting the DNA polymerase activity of HIV-1 RT are available, while few specific inhibitors of RNase H activity have been described. IN is absolutely necessary for stable and productive infection of cells. Some IN inhibitors have been recently reported and are available demonstrating the potential of IN as an antiviral target. This paper is an overview of the inhibitors of RNase H and IN and describes the most promising inhibitors.

Anti-HIV Agents↗
PubMed2002

Structure-based inhibitor design targeting HIV-1 integrase.

HIV integrase (IN) is a viral-encoded protein that catalyzes the breaking and joining reactions that mediate integration of viral DNA into the host genome. Therefore, IN offers a unique target for the development of novel anti-HIV and anti-AIDS therapeutics. To take advantage of this potential, drug discovery efforts via structure-based design approaches have been undertaken. Presented is a review of computer-aided drug design efforts targeting HIV IN. Included is an overview of the life-cycle of HIV, with emphasis on the mechanism of action of IN, biological assays for measuring IN activity and identifying IN inhibitors, and the appropriate cell-based assays required for determining the antiviral activity of IN inhibitors. This is followed by a review of the available three-dimensional structures of HIV IN. Structure-based drug design efforts are then critiqued, including both ligand-based (e.g. pharmacophore) and target-based (e.g. docking) methods. Results from recent computational chemistry studies of IN are also discussed.

Catalysis↗
PubMed2003

The integrase of the human immunodeficiency virus as a novel target for the antiviral therapy of AIDS.

Integration of the proviral DNA is a crucial step in the life cycle of the human immunodeficiency virus (HIV). Basic understanding of the cell biology of HIV integration will help the development of new antiviral drugs to complement the currently used cocktails of reverse transcriptase and protease inhibitors. Our research team has made major advancement in the field of antiviral research in recent years. The scientific-technological achievements were possible by the collaboration of a group of researchers with complementary research expertise. The integrase team is headed by Myriam Witvrouw and Zeger Debyser. After a general overview we summarized our most relevant publications. In the perspectives we referred to the ongoing work.

Acquired Immunodeficiency Syndrome↗
PubMed1999

Refined solution structure of the C-terminal DNA-binding domain of human immunovirus-1 integrase.

The structure of the C-terminal DNA-binding domain of human immunovirus-1 integrase has been refined using nuclear magnetic resonance spectroscopy. The protein is a dimer in solution and shows a well-defined dimer interface. The folding topology of the monomer consists of a five-stranded beta-barrel that resembles that of Src homology 3 domains. Compared with our previously reported structure, the structure is now defined far better. The final 42 structures display a back-bone root mean square deviation versus the average of 0.46 A. Correlation of the structure with recent mutagenesis studies suggests two possible models for DNA binding. Proteins 1999;36:556-564.

Binding Sites↗
PubMed2000

Mapping epitopes of monoclonal antibodies against HIV-1 integrase with limited proteolysis and matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

Monoclonal antibodies (mAbs) have been used extensively in the biochemical analysis of proteins. Molecular identification of a specific epitope can enhance our understanding of the relationship between the structure and function of a protein. We recently developed a protein footprint technique for mapping mAb epitopes that employs limited proteolysis followed by peptide analysis with matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Here we describe the rational for the technique and illustrate its use in mapping the epitopes of two mAbs that bind to the C-terminal domain of human immunodeficiency virus type-1 integrase. The results provide a plausible explanation for the fact that one mAb inhibits enzyme activity while the second does not.

Amino Acid Sequence↗
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↗
PubMed1994

Molecular dynamics simulation of a leucine zipper motif predicted for the integrase of human immunodeficiency virus type 1.

We have used the molecular dynamics (MD) simulation package AMBER4 to search the conformation of a peptide predicted as a leucine zipper motif for the human immunodeficiency virus type 1 integrase protein (HIV IN-LZM). The peptide is composed of 22 amino acid residues and its location is from Val 151 to Leu 172. The searching procedure also includes two known alpha-helices that served as positive controls--namely, a 22-residue GCN4-p1 (LZM) and a 20-residue poly (L-alanine) (PLA). A 21-residue peptide extracted from a cytochrome C crystal (CCC-t) with determined conformation as a beta-turn is also included as a negative control. At the beginning of the search, two starting conformations--namely, the standard right-handed alpha-helix and the fully stretched conformations--are generated for each peptide. Structures generated as standard alpha-helix are equilibrated at room temperature for 90 ps while structures generated as a fully stretched one are equilibrated at 600 K for 120 ps. The CCC-t and PLA helices are nearly destroyed from the beginning of equilibration. However, for both the HIV IN-LZM and the GCN4-p1 LZM structures, there is substantial helicity being retained throughout the entire course of equilibration. Although helix propagation profiles calculated indicate that both peptides possess about the same propensity to form an alpha-helix, the HIV IN-LZM helix appears to be more stable than the GCN4-p1 one as judged by a variety of analyses on both structures generated during the equilibration course. The fact that predicted HIV IN-LZM can exist as an alpha-helix is also supported by the results of high temperature equilibration run on the fully stretched structures generated. In this run, the RMS deviations between the backbone atoms of the structures with the lowest potential energy (PE) identified within every 2 ps and the structure with the lowest PE searched in the same course of simulation are calculated. For both the HIV IN-LZM and the GCN4-p1 LZM, these rms values decrease with the decrease of PE, which indicates that both structures are closer in conformations as their PEs are moved deeper into the PE well.

Amino Acid Sequence↗
PubMed2003

Site-specific recombination in human cells catalyzed by the wild-type integrase protein of coliphage HK022.

The activity of the Integrase (Int) protein encoded by coliphage HK022 was tested in a human cell culture. Plasmids were constructed as substrates that carry the sites of the integration reaction (attP and attB) or the sites of excision (attL and attR). The site-specific recombination reactions were monitored in cis and in trans configurations by the expression of the green fluorescent protein (GFP) as a reporter. Cells cotransfected with the substrate plasmid(s) and with a plasmid that expresses the wild-type Int show efficient integration as well as excision in both configurations. The wild-type Int was active in the human cells without the need to supply the accessory proteins integration host factor (IHF) and excisionase (Xis) that are indispensable for the reaction in the bacterial host.

Catalysis↗
PubMed1993

Complementation between HIV integrase proteins mutated in different domains.

HIV integrase (IN) cleaves two nucleotides off the 3' end of viral DNA and integrates viral DNA into target DNA. Previously, three functional domains in the HIV IN protein have been identified: (i) the central catalytic domain, (ii) the C-terminal DNA binding domain, and (iii) the N-terminal region, which is also necessary for activity. We have now investigated whether IN proteins mutated in different domains can complement each other. Mutant D116I does not contain an intact active site, but does bind DNA, whereas the C-terminal deletion mutant C delta 73 does not bind DNA, but does have an intact active site. Neither mutant protein mediates site-specific cleavage or integration. However, a mixture of both proteins is active, suggesting that IN functions as an oligomer, and that two subunits can have different functions; one subunit binds the (viral) DNA and another subunit provides the active site. We found three classes of mutants, corresponding to the three domains mentioned above. Mutants from different classes, but not from the same class, can complement each other. However, complementation is most efficient when the N- and C-termini are present on the same molecule.

Binding Sites↗
PubMed1993

Synaptic intermediates in bacteriophage lambda site-specific recombination: integrase can align pairs of attachment sites.

Bacteriophage lambda uses site-specific recombination to move its DNA into and out of the Escherichia coli genome. The recombination event is mediated by the recombinase integrase (Int) together with several accessory proteins through short specific DNA sequences known as attachment sites. A gel mobility shift assay has been used to show that, in the absence of accessory proteins, Int can align and hold together two DNA molecules, each with an attachment site, to form stable non-covalent 'bimolecular complexes'. Each attachment site must have both core and arm binding sites for Int to participate in a bimolecular complex. These stable structures can be formed between pairs of attL and attP attachment sites, but cannot include attB or attR sites; they are inhibited by integration host factor (IHF) protein. The bimolecular complexes are shown to represent a synaptic intermediate in the reaction in which Int protein promotes the IHF-independent recombination of two attL sites. These complexes should enable a detailed analysis of synapsis for this pathway.

Bacteriophage lambda↗
PubMed1994

Lambda integrase cleaves DNA in cis.

In the Int family of site-specific recombinases, DNA cleavage is accomplished by nucleophilic attack on the activated scissile phosphodiester bond by a specific tyrosine residue. It has been proposed that this tyrosine is contributed by a protomer bound to a site other than the one being cleaved ('trans' cleavage). To test this hypothesis, the difference in DNA binding specificity between closely related integrases (Ints) from phages lambda and HK022 was exploited to direct wild type Ints and cleavage- or activation-defective mutants to particular sites on bispecific substrates. Analysis of Int cleavage at individual sites strongly indicates that DNA cleavage is catalyzed by the Int bound to the cleaved site ('cis' cleavage). This conclusion contrasts with those from previous experiments with two members of the Int family, FLP and lambda Int, that supported the hypothesis of trans cleavage. We suggest explanations for this difference and discuss the implications of the surprising finding that Int-family recombinases appear capable of both cis and trans mechanisms of DNA cleavage.

Bacteriophage lambda↗
PubMed2004

Expression of HIV-1 integrase in CEM cells inhibits HIV-1 replication.

BACKGROUND: HIV-1 integrase (IN) is an interesting target for the gene therapy of AIDS. Although the in vivo functions are not well characterized, it is thought that IN has pleiotropic effects and plays a central role in the interplay between the virus and the host cell. Expression of IN in mammalian cells has proven difficult. We have previously established a 293T-derived cell line that stably expresses high levels of HIV-1 IN from a synthetic gene. We now have constructed CEM-derived cell lines stably expressing the enzyme or its different domains and studied the impact of IN expression on HIV-1 replication. METHODS: The CEM cell lines were selected following transduction with a retroviral vector encoding the full-length IN, the N-terminal domain, the catalytic core or the C-terminal domain. Stable IN expression in CEM cell lines was verified by Western blotting. The impact of IN expression on HIV-1 replication and HIV-1 vector transduction was studied. RESULTS: A marked inhibitory effect on HIV-1 replication was observed in CEM cells expressing IN. Expression of IN interfered with both particle production and integration. Expression of the N-terminal domain alone was sufficient for the inhibiting of HIV-1 replication. CONCLUSIONS: Expression of IN in CEM cells inhibits HIV-1 replication by a cumulative inhibitory effect on integration and particle production, in accord with the known pleiotropic interactions of IN. The inhibition of HIV-1 replication in CEM cells expressing the N-terminal domain of IN may lead to a novel approach for the gene therapy of AIDS.

Acquired Immunodeficiency Syndrome↗
PubMedDate not supplied

The role of flexibility and hydration on the sequence-specific DNA recognition by the Tn916 integrase protein: a molecular dynamics analysis.

The N-terminal domain of the Tn916 integrase protein (INT-DBD) is responsible for DNA binding in the process of strand cleavage and joining reactions required for transposition of the Tn916 conjugative transposon. Site-specific association is facilitated by numerous protein-DNA contacts from the face of a three-stranded beta-sheet inserted into the major groove. The protein undergoes a subtle conformational transition and is slightly unfolded in the protein-DNA complex. The conformation of many charged residues is poorly defined by NMR data but mutational studies have indicated that removal of polar side chains decreases binding affinity, while non-polar contacts are malleable. Based on analysis of the binding enthalpy and binding heat capacity, we have reasoned that dehydration of the protein-DNA interface is incomplete. This study presents results from a molecular dynamics investigation of the INT-DBD-DNA complex aimed at a more detailed understanding of the role of conformational dynamics and hydration in site-specific binding. Comparison of simulations (total of 13 ns) of the free protein and of the bound protein conformation (in isolation or DNA-bound) reveals intrinsic flexibility in certain parts of the molecule. Conformational adaptation linked to partial unfolding appears to be induced by protein-DNA contacts. The protein-DNA hydrogen-bonding network is highly dynamic. The simulation identifies protein-DNA interactions that are poorly resolved or only surmised from the NMR ensemble. Single water molecules and water clusters dynamically optimize the complementarity of polar interactions at the 'wet' protein-DNA interface. The simulation results are useful to establish a qualitative link between experimental data on individual residue's contribution to binding affinity and thermodynamic properties of INT-DBD alone and in complex with DNA.

DNA-Binding Proteins↗
PubMed2006

HIV-1 integrase inhibitors: 2003-2004 update.

The integration of viral cDNA into the host genome is an essential step in the HIV-1-life cycle and is mediated by the virally encoded enzyme, integrase (IN). Inhibition of this process provides an attractive strategy for antiviral drug design. The discovery of beta-diketo acid inhibitors played a major role in validating IN as a legitimate antiretroviral drug target. Over a decade of research, a plethora of IN inhibitors have been discovered and some showed antiviral activity consistent with their effect on IN. To date, at least two compounds have been tested in human but none are close to the FDA approval. In this review, we provide a comprehensive report of all small-molecule IN inhibitors discovered during the years 2003 and 2004. Compilation of such data will prove beneficial in developing QSAR, virtual screening, pharmacophore hypothesis generation, and validation.

Anti-Retroviral Agents↗
PubMed1994

Folding of the multidomain human immunodeficiency virus type-I integrase.

Protein folding conditions were established for human immunodeficiency virus integrase (IN) obtained from purified bacterial inclusion bodies. IN was denatured by 6 M guanidine.HCl-5 mM dithiothreitol, purified by gel filtration, and precipitated by ammonium sulfate. The reversible solvation of precipitated IN by 6 M guanidine.HCl allowed for wide variation of protein concentration in the folding reaction. A 6-fold dilution of denatured IN by 1 M NaCl buffer followed by dialysis produced enzymatically active IN capable of 3' OH end processing, strand transfer, and disintegration using various human immunodeficiency virus-1 (HIV-1) long terminal repeat DNA substrates. The specific activities of folded IN preparations for these enzymatic reactions were comparable to those of soluble IN purified directly from bacteria. The subunit composition and enzymatic activities of IN were affected by the folding conditions. Standard folding conditions were defined in which monomers and protein aggregates sedimenting as dimers and tetramers wree produced. These protein aggregates were enzymatically active, whereas monomers had reduced strand transfer activity. Temperature modifications of the folding conditions permitted formation of mainly monomers. Upon assaying, these monomers were efficient for strand transfer and disintegration, but the oligomeric state of IN under the conditions of the assay is determinate. Our results suggest that monomers of the multidomain HIV-1 IN are folded correctly for various catalytic activities, but the conditions for specific oligomerization in the absence of catalytic activity are undefined.

Ammonium Sulfate↗
PubMed1998

Solution structure of the His12 --> Cys mutant of the N-terminal zinc binding domain of HIV-1 integrase complexed to cadmium.

The solution structure of His12 --> Cys mutant of the N-terminal zinc binding domain (residues 1-55; IN(1-55)) of HIV-1 integrase complexed to cadmium has been solved by multidimensional heteronuclear NMR spectroscopy. The overall structure is very similar to that of the wild-type N-terminal domain complexed to zinc. In contrast to the wild-type domain, however, which exists in two interconverting conformational states arising from different modes of coordination of the two histidine side chains to the metal, the cadmium complex of the His12 --> Cys mutant exists in only a single form at low pH. The conformation of the polypeptide chain encompassing residues 10-18 is intermediate between the two forms of the wild-type complex.

Binding Sites↗
PubMed2002

Hamming distance geometry of a protein conformational space: application to the clustering of a 4-ns molecular dynamics trajectory of the HIV-1 integrase catalytic core.

Protein structures can be encoded into binary sequences (Gabarro-Arpa et al., Comput Chem 2000;24:693-698) these are used to define a Hamming distance in conformational space: the distance between two different molecular conformations is the number of different bits in their sequences. Each bit in the sequence arises from a partition of conformational space in two halves. Thus, the information encoded in the binary sequences is also used to characterize the regions of conformational space visited by the system. We apply this distance and their associated geometric structures to the clustering and analysis of conformations sampled during a 4-ns molecular dynamics simulation of the HIV-1 integrase catalytic core. The cluster analysis of the simulation shows a division of the trajectory into two segments of 2.6 and 1.4 ns length, which are qualitatively different: the data points to the fact that equilibration is only reached at the end of the first segment. The Hamming distance is compared also to the r.m.s. deviation measure. The analysis of the cases studied so far shows that under the same conditions the two measures behave quite differently, and that the Hamming distance appears to be more robust than the r.m.s. deviation.

Algorithms↗
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↗