Binding aspects of baicalein to HIV-1 integrase

Human immunodeficiency virus type 1 (HIV-1) integrase is an essential enzyme in the life cycle of the virus. It is responsible for catalyzing the insertion of the viral genome into the host cell chromosome. This integrase is an attractive target for the design of a HIV antiviral drug, because integrase has no human counterpart. In order to know the interaction mode of HIV-1 integrase with its inhibitor, we investigated the effect of the inhibitor, baicalein, on the conformation of the HIV-1 integrase catalytic domain [IN-(50-212/F185K)] using fluorescence and circular dichroism (CD) spectroscopy. We found that baicalein binds to the hydrophobic region of the HIV-1 integrase catalytic core domain. This binding of baicalein induces the conformational change of the enzyme. We also found that the binding ratio of baicalein to the HIV-1 integrase catalytic domain is 2:1.     

Molecular dynamics studies of the wild-type and double mutant HIV-1 integrase complexed with the 5CITEP inhibitor: mechanism for inhibition and drug resistance

The human immunodeficiency virus type 1 (HIV-1) integrase (IN) is an essential enzyme in the life cycle of the virus and is an attractive target for the development of new drugs useful in acquired immunodeficiency syndrome multidrug therapy. Starting from the crystal structure of the 5CITEP inhibitor bound to the active site in the catalytic domain of the HIV-1 IN, two different molecular dynamics simulations in water have been carried out. In the first simulation the wild-type IN was used, whereas in the second one the double mutation T66I/M154I, described to lead to drug resistance, was introduced in the protein. Compelling differences have been observed in these two structures during analyses of the molecular dynamics trajectories, particularly in the inhibitor binding modes and in the conformational flexibility of the loop (residues 138-149) located near the three catalytic residues in the active site (Asp(64), Asp(116), Glu(152)). Because the conformational flexibility of this region is important for efficient biological activity and its behavior is quite different in the two models, we suggest a hypothetical mechanism for the inhibition and drug resistance of HIV-1 IN. These results can be useful for the rational design of more potent and selective integrase inhibitors and may allow for the design of inhibitors that will be more robust against known resistance mutations.     

Functional oligomeric state of avian sarcoma virus integrase

Retroviral integrase, one of only three enzymes encoded by the virus, catalyzes the essential step of inserting a DNA copy of the viral genome into the host during infection. Using the avian sarcoma virus integrase, we demonstrate that the enzyme functions as a tetramer. In presteady-state active site titrations, four integrase protomers were required for a single catalytic turnover. Volumetric determination of integrase-DNA complexes imaged by atomic force microscopy during the initial turnover additionally revealed substrate-induced assembly of a tetramer. These results suggest that tetramer formation may be a requisite step during catalysis with ramifications for antiviral design strategies targeting the structurally homologous human immunodeficiency virus, type 1 (HIV-1) integrase.     

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.     

Docking dinucleotides to HIV-1 integrase carboxyl-terminal domain to find possible DNA binding sites

HIV-1 integrase mediates a processed viral DNA into the host genome DNA. The binding modes between HIV-1 integrase and viral/host DNA have not been clarified until now. The C-terminal domain of integrase has been found to be a DNA-binding domain. In this work, we have explored the possible DNA binding sites of dimeric C-terminal domain by docking dinucleotides to HIV-1 integrase. The docking results suggest that two symmetrical DNA-binding sites are likely located on the outside surface of the dimeric C-terminal domain and not located in the groove. Those sites are in agreement with the experimental data.     

Large-scale conformational dynamics of the HIV-1 integrase core domain and its catalytic loop mutants

HIV-1 integrase is one of the three essential enzymes required for viral replication and has great potential as a novel target for anti-HIV drugs. Although tremendous efforts have been devoted to understanding this protein, the conformation of the catalytic core domain around the active site, particularly the catalytic loop overhanging the active site, is still not well characterized by experimental methods due to its high degree of flexibility. Recent studies have suggested that this conformational dynamics is directly correlated with enzymatic activity, but the details of this dynamics is not known. In this study, we conducted a series of extended-time molecular dynamics simulations and locally enhanced sampling simulations of the wild-type and three loop hinge mutants to investigate the conformational dynamics of the core domain. A combined total of >480 ns of simulation data was collected which allowed us to study the conformational changes that were not possible to observe in the previously reported short-time molecular dynamics simulations. Among the main findings are a major conformational change (>20 A) in the catalytic loop, which revealed a gatinglike dynamics, and a transient intraloop structure, which provided a rationale for the mutational effects of several residues on the loop including Q(148), P(145), and Y(143). Further, clustering analyses have identified seven major conformational states of the wild-type catalytic loop. Their implications for catalytic function and ligand interaction are discussed. The findings reported here provide a detailed view of the active site conformational dynamics and should be useful for structure-based inhibitor design for integrase.     

HIV-1整合酶四聚体结构模拟及其活性位点分析

HIV-1复制需要HIV-1整合酶将其环状DNA整合进宿主DNA中,这其中包括2个重要反应,即“3′-加工”和“链转移”,两者均由HIV-1整合酶催化完成.阻断其中的任一反应,都能达到抑制HIV-1复制的目的.因此,了解HIV-1整合酶的完整结构和聚合状态,对深入探讨其作用机理及设计新型抑制剂具有重要的指导作用.然而,迄今为止仅有HIV-1整合酶单独结构域的晶体结构可供参考,而其全酶晶体结构尚未获得解析.本研究利用分子模拟技术,通过蛋白质 蛋白质/DNA分子对接、动力学模拟等方法,构建了全长整合酶四聚体的结构模型、HIV-1 DNA与整合酶复合物的结构模型,进一步从理论上证实HIV-1整合酶是以四聚体形态发挥催化作用,明确“3′-加工”和“链转移”在HIV-1整合酶上的催化位点.同时,通过与作用机理相似的细菌转座子Tn5转座酶等的结构比对,推测HIV-1整合酶的核心结构域中应有第2个Mg^２＋存在,其位置螯合于Asp64与Glu152之间.在HIV-1整合酶结构研究的基础上,有望进一步设计出新的抗艾滋病药物.     

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.     

Human immunodeficiency virus type 1 integrase: effects of mutations on viral ability to integrate, direct viral gene expression from unintegrated viral DNA templates, and sustain viral propagation in primary cells.

Integrase is the only viral protein necessary for integration of retroviral DNA into chromosomal DNA of the host cell. Biochemical analysis of human immunodeficiency virus type 1 (HIV-1) integrase with purified protein and synthetic DNA substrates has revealed extensive information regarding the mechanism of action of the enzyme, as well as identification of critical residues and functional domains. Since in vitro reactions are carried out in the absence of other viral proteins and they analyze strand transfer of only one end of the donor substrate, they do not define completely the process of integration as it occurs during the course of viral infection. In an effort to further understand the role of integrase during viral infection, we initially constructed a panel of 24 HIV-1 mutants with specific alanine substitutions throughout the integrase coding region and analyzed them in a human T-cell line infection. Of these mutant viruses, 12 were capable of sustained viral replication, 11 were replication defective, and 1 was temperature sensitive for viral growth. The replication defective viruses express and correctly process the integrase and Gag proteins. Using this panel of mutants and an additional set of 18 mutant viruses, we identified nine amino acids which, when replaced with alanine, destroy integrase activity. Although none of the replication-defective mutants are able to integrate into the host genome, a subset of them with alterations in the catalytic triad are capable of Tat-mediated transactivation of an indicator gene linked to the viral long terminal repeat promoter. We present evidence that integration of the HIV-1 provirus is essential not only for productive infection of T cells but also for virus passage in both cultured peripheral blood lymphocytes and macrophage cells.     

Molecular dynamics studies of the full-length integrase-DNA complex

We have carried out a molecular dynamics (MD) simulation of full-length HIV-1 integrase (IN) dimer complexed with viral DNA with the aim of gaining information about the enzyme motion and investigating the movement of the catalytic flexible loop (residues 140-149) thought to be essential in the catalytic mechanism of IN. During the simulation, we observed quite a different behavior of this region in the presence or absence of the viral DNA. In particular, the MD results underline the crucial role of the residue Tyr143 in the mechanism of integration of viral DNA into the host chromosome. The present findings confirm the experimental data (e.g., site-directed mutagenesis experiments) showing that the loop is involved in the integration reactions and its mobility is correlated with the catalytic activity of HIV-1 integrase.     

Recognition and inhibition of HIV integrase by a novel dinucleotide

The viral enzyme, HIV integrase, is involved in the integration of viral DNA into host cell DNA. In the quest for a small nucleotide system with nuclease stability of the internucleotide phosphate bond and critical structural features for recognition and inhibition of HIV-1 integrase, we have discovered a conceptually novel dinucleotide, pIsodApdC, which is a potent inhibitor of this key viral enzyme.     

Modulation of HIV-1 integrase activity by single-stranded oligonucleotides and their conjugates with eosin

Integration of the DNA copy of the genomic RNA into an infected cell genome is one of the key steps of the replication cycle of all retroviruses. It is catalyzed by the viral enzyme, integrase. We have shown that conjugates of short single-stranded oligonucleotides with eosin efficiently inhibit the catalytic activity of the HIV-1 integrase. In this article, we have found that the dependence of the integrase catalytic activity on the concentration of oligonucleotides has a bell-shaped pattern. The modulation of HIV-1 integrase activity correlated with the oligonucleotide length and was not associated with specific sequences. Moreover, a similar mode of the oligonucleotide action was found for integrase from the prototype foamy virus. This dual effect of the oligonucleotide and their conjugates with eosin might be explained by their binding with retroviral integrase in two different sites; the oligodeoxynucleotide binding in the first site results in integrase activation, whereas interactions with another one lead to inhibition of the enzyme activity. Eosin coupling to oligonucleotides did not change the mode of their action but enhanced their affinity to both binding sites. The affinity increase was found to be much more important for the site responsible for the integrase inhibition, thus explaining the high inhibitory potency of oligonucleotide-eosin conjugates.     

Substrate features important for recognition and catalysis by human immunodeficiency virus type 1 integrase identified by using novel DNA substrates.

The integrase encoded by human immunodeficiency virus type 1 (HIV-1) is required for integration of viral DNA into the host cell chromosome. In vitro, integrase mediates a concerted cleavage-ligation reaction (strand transfer) that results in covalent attachment of viral DNA to target DNA. With a substrate that mimics the strand transfer product, integrase carries out disintegration, the reverse of the strand transfer reaction, resolving this integration intermediate into its viral and target DNA parts. We used a set of disintegration substrates to study the catalytic mechanism of HIV-1 integrase and the interaction between the protein and the viral and target DNA sequence. One substrate termed dumbbell consists of a single oligonucleotide that can fold to form a structure that mimics the integration intermediate. Kinetic analysis using the dumbbell substrate showed that integrase turned over, establishing that HIV-1 integrase is an enzyme. Analysis of the disintegration activity on the dumbbell substrate and its derivatives showed that both the viral and target DNA parts of the molecule were required for integrase recognition. Integrase recognized target DNA asymmetrically: the target DNA upstream of the viral DNA joining site played a much more important role than the downstream target DNA in protein-DNA interaction. The site of transesterification was determined by both the DNA sequence of the viral DNA end and the structure of the branched substrate. Using a series of disintegration substrates with various base modifications, we found that integrase had relaxed structural specificity for the hydroxyl group used in transesterification and could tolerate distortion of the double-helical structure of these DNA substrates.     

Irreversible inhibition of human immunodeficiency virus type 1 integrase by dicaffeoylquinic acids

Human immunodeficiency virus type 1 (HIV-1) and other retroviruses require integration of a double-stranded DNA copy of the RNA genome into the host cell chromosome for productive infection. The viral enzyme, integrase, catalyzes the integration of retroviral DNA and represents an attractive target for developing antiretroviral agents. We identified several derivatives of dicaffeoylquinic acids (DCQAs) that inhibit HIV-1 replication in tissue culture and catalytic activities of HIV-1 integrase in vitro. The specific step at which DCQAs inhibit the integration in vitro and the mechanism of inhibition were examined in the present study. Titration experiments with different concentrations of HIV-1 integrase or DNA substrate found that the effect of DCQAs was exerted on the enzyme and not the DNA. In addition to HIV-1, DCQAs also inhibited the in vitro activities of MLV integrase and truncated variants of feline immunodeficiency virus integrase, suggesting that these compounds interacted with the central core domain of integrase. The inhibition on retroviral integrases was relatively specific, and DCQAs had no effect on several other DNA-modifying enzymes and phosphoryltransferases. Kinetic analysis and dialysis experiments showed that the inhibition of integrase by DCQAs was irreversible. The inhibition did not require the presence of a divalent cation and was unaffected by preassembling integrase onto viral DNA. The results suggest that the irreversible inhibition by DCQAs on integrase is directed toward conserved amino acid residues in the central core domain during catalysis.     

Crystal structures of catalytic core domain of BIV integrase: implications for the interaction between integrase and target DNA

Integrase plays a critical role in the recombination of viral DNA into the host genome. Therefore, over the past decade, it has been a hot target of drug design in the fight against type 1 human immunodeficiency virus (HIV-1). Bovine immunodeficiency virus (BIV) integrase has the same function as HIV-1 integrase. We have determined crystal structures of the BIV integrase catalytic core domain (CCD) in two different crystal forms at a resolution of 2.45? and 2.2?, respectively. In crystal form I, BIV integrase CCD forms a back-to-back dimer, in which the two active sites are on opposite sides. This has also been seen in many of the CCD structures of HIV-1 integrase that were determined previously. However, in crystal form II, BIV integrase CCD forms a novel face-to-face dimer in which the two active sites are close to each other. Strikingly, the distance separating the two active sites is approximately 20 ?, a distance that perfectly matches a 5-base pair interval. Based on these data, we propose a model for the interaction of integrase with its target DNA, which is also supported by many published biochemical data. Our results provide important clues for designing new inhibitors against HIV-1.     

Structural and Functional Role of INI1 and LEDGF in the HIV-1 Preintegration Complex

Integration of the HIV-1 cDNA into the human genome is catalyzed by the viral integrase (IN) protein. Several studies have shown the importance of cellular cofactors that interact with integrase and affect viral integration and infectivity. In this study, we produced a stable complex between HIV-1 integrase, viral U5 DNA, the cellular cofactor LEDGF/p75 and the integrase binding domain of INI1 (INI1-IBD), a subunit of the SWI/SNF chromatin remodeling factor. The stoichiometry of the IN/LEDGF/INI1-IBD/DNA complex components was found to be 4/2/2/2 by mass spectrometry and Fluorescence Correlation Spectroscopy. Functional assays showed that INI1-IBD inhibits the 3′ processing reaction but does not interfere with specific viral DNA binding. Integration assays demonstrate that INI1-IBD decreases the amount of integration events but inhibits by-product formation such as donor/donor or linear full site integration molecules. Cryo-electron microscopy locates INI1-IBD within the cellular DNA binding site of the IN/LEDGF complex, constraining the highly flexible integrase in a stable conformation. Taken together, our results suggest that INI1 could stabilize the PIC in the host cell, by maintaining integrase in a stable constrained conformation which prevents non-specific interactions and auto integration on the route to its integration site within nucleosomes, while LEDGF organizes and stabilizes an active integrase tetramer suitable for specific vDNA integration. Moreover, our results provide the basis for a novel type of integrase inhibitor (conformational inhibitor) representing a potential new strategy for use in human therapy.     

Insights from atomic-resolution X-ray structures of chemically synthesized HIV-1 protease in complex with inhibitors

The human immunodeficiency virus 1 (HIV-1) protease (PR) is an aspartyl protease essential for HIV-1 viral infectivity. HIV-1 PR has one catalytic site formed by the homodimeric enzyme. We chemically synthesized fully active HIV-1 PR using modern ligation methods. When complexed with the classic substrate-derived inhibitors JG-365 and MVT-101, the synthetic HIV-1 PR formed crystals that diffracted to 1.04- and 1.2-A resolution, respectively. These atomic-resolution structures revealed additional structural details of the HIV-1 PR's interactions with its active site ligands. Heptapeptide inhibitor JG-365, which has a hydroxyethylamine moiety in place of the scissile bond, binds in two equivalent antiparallel orientations within the catalytic groove, whereas the reduced isostere hexapeptide MVT-101 binds in a single orientation. When JG-365 was converted into the natural peptide substrate for molecular dynamic simulations, we found putative catalytically competent reactant states for both lytic water and direct nucleophilic attack mechanisms. Moreover, free energy perturbation calculations indicated that the insertion of catalytic water into the catalytic site is an energetically favorable process.     

Thalassiolins A-C: new marine-derived inhibitors of HIV cDNA integrase

Human immunodeficiency virus (HIV) replication requires integration of viral cDNA into the host genome, a process mediated by the viral enzyme integrase. We describe a new series of HIV integrase inhibitors, thalassiolins A-C (1-3), isolated from the Caribbean sea grass Thalassia testudinum. The thalassiolins are distinguished from other flavones previously studied by the substitution of a sulfated beta-D-glucose at the 7-position, a substituent that imparts increased potency against integrase in biochemical assays. The most active of these molecules, thalassiolin A (1), displays in vitro inhibition of the integrase catalyzed strand transfer reaction (IC50=0.4 microM) and an antiviral IC50 of 30 microM. Molecular modeling studies indicate a favorable binding mode is probable at the catalytic core domain of HIV-1 integrase.