Inhibitory profile of a LEDGF/p75 peptide against HIV-1 integrase: insight into integrase-DNA complex formation and catalysis

A lens epithelium-derived growth factor (LEDGF)/p75 peptide was evaluated for human immunodeficiency virus type 1 integrase (IN) inhibitory activity. The LEDGF/p75 peptide modestly inhibited IN catalysis and was dependent on IN-DNA assembly. The peptide was also effective at disrupting LEDGF/p75-IN complex formation. We next investigated the activity of the LEDGF/p75 peptide on IN mutant proteins that are unable to catalyze the DNA strand transfer reaction. The LEDGF/p75 peptide displayed an increased potency on these IN proteins, from 5-fold to greater than 10-fold, indicating the IN multimeric state greatly influences the peptide inhibitory effects. These results shed light on IN-DNA multimeric formation, and how this process influences the LEDGF/p75-IN interaction.     

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.     

Structural and Dynamical Properties of a Full-length HIV-1 Integrase: Molecular Dynamics Simulations

Abstract

The structural and dynamical properties of the complete full-length structure of HIV-1 integrase were investigated using Molecular Dynamics approach. Simulations were carried out for the three systems, core domain only (CORE), full-length structure without (FULL) and with a Mg²⁺ (FULL+ION) in its active site, aimed to investigate the difference in the molecular properties of the full-length models due to their different construction procedures as well as the effects of the two ends, C- and N-terminal, on those properties in the core domain. The full-length structure was prepared from the two experimental structures of two-domain fragment. The following properties were observed to differ significantly from the previous reports: (i) relative topology formed by an angle between the three domains; (ii) the cavity size defined by the catalytic triad, Asp64, Asp116, and Glul52; (iii) distances and solvation of the Mg²⁺; and (iv) conformation of the catalytic residues. In addition, the presence of the two terminal domains decreases the mobility of the central core domain significantly.     

Development of tricyclic hydroxy-1H-pyrrolopyridine-trione containing HIV-1 integrase inhibitors

New tricyclic HIV-1 integrase (IN) inhibitors were prepared that combined structural features of bicyclic pyrimidinones with recently disclosed 4,5-dihydroxy-1H-isoindole-1,3(2H)-diones. This combination resulted in the introduction of a nitrogen into the aryl ring and the addition of a fused third ring to our previously described inhibitors. The resulting analogues showed low micromolar inhibitory potency in in vitro HIV-1 integrase assays, with good selectivity for strand transfer relative to 3′-processing.     

Enzymatic capability of HIS-tagged HIV-1 integrase using oligonucleotide disintegration substrates

Disintegration, wherein a half-site integration substrate is resolved into separate viral and host DNA components via DNA strand transfer, is one of three well-established in vitro activities of HIV-1 integrase. The role of disintegration in the HIV-1 replicative cycle, however, remains a mystery. In this report, we describe the expression inEscherichia coli and purification of HIV-1 integrase as a fusion protein containing a 6×His tag at its amino terminus. Integrase resolved dumbbell and Y-substrates optimally at pH 6.8–7.2 in the presence of 2 mM MnCl₂. Substrate requirements for intramolecular disintegration included a 10 base pair viral U5 LTR arm and a CA dinucleotide located at the 3 end of the LTR. Disintegration was not sensitive to changes in the host DNA portion of the substrate. A dumbbell substrate with a 5 oligo-dA tail also underwent disintegration. The released LTR arm with an oligo-dA tail was utilized as a template primer by several DNA polymerases indicating that disintegration occurred via nucleophilic attack on the phosphodiester bond located immediately adjacent to the CA dinucleotide at the 3 end of the LTR. Coupled disintegration-DNA polymerase reactions provided a highly efficient and sensitive means of detecting disintegration activity. Integrase also catalyzed an apparently concerted disintegration-5-end joining reaction in which an LTR arm was transferred from one dumbbell substrate molecule to another.     

Novel furocoumarins as potential HIV-1 integrase inhibitors

A series of seven novel, rationally designed N-substituted 3-{3,5-dimethylfuro[3,2-g]coumarin-6-yl}propanamides have been prepared as potential HIV-1 integrase (IN) inhibitors via a five-step pathway commencing with resorcinol and diethyl 2-acetylglutarate, and the HIV-1 IN inhibition potential of these compounds has been examined relative to raltegravir, a known HIV-1 IN inhibitor.     

Cellular protein TTRAP interacts with HIV-1 integrase to facilitate viral integration

TTRAP is a PML-NB protein that is involved in the NF-κB signaling pathway. TTRAP was recently identified by yeast two-hybrid analysis as a HIV-1 integrase (HIV-1 IN) interacting protein. This interaction was verified by co-immunoprecipitation, GST pull-down, and intracellular imaging, and deletion assays suggested that the N-terminal 180 residues of TTRAP are responsible for the interaction. In stable TTRAP knock-down cell lines, the integration of viral vectors decreased significantly compared with non-silenced cell lines. Conversely, overexpression of TTRAP by transient transfection increased the percentage of integration events. This is the first time that TTRAP has been shown to interact with HIV-1 IN and facilitate lentiviral vector integration. These findings reveal a new function of TTRAP and expand our understanding of the cellular response to HIV infection. The interaction between TTRAP and HIV-1 IN may be useful in designing new anti-viral strategies as well as for improving the efficiency of lentiviral-vector-mediated gene delivery.     

4-Hydroxy-5-pyrrolinone-3-carboxamide HIV-1 integrase inhibitors

The viral enzyme integrase is essential for the replication of HIV-1 and, after the discovery of Isentress™, represents a validated target for anti-retroviral therapy. Incorporation of the dihydroxycarbonyl pharmacophore into a pyrrolinone scaffold led to the discovery of 5-pyrrolinone-3-carboxamides as a structurally diverse class of HIV-1 integrase inhibitors.     

Active site binding modes of the beta-diketoacids: a multi-active site approach in HIV-1 integrase inhibitor design

Predicting a bioactive conformation of a ligand is of paramount importance in rational drug design. The task becomes very difficult when the receptor site possesses a region with unusual conformational flexibility. Significant conformational differences are present in the active site regions in the available crystal structures of the core domains of HIV-1 integrase (IN). Among all reported IN inhibitors, the β-diketoacid class of compounds has proved to be of most promise and indeed S-1360 was the first IN inhibitor to enter clinical studies. With an aim to predict the bioactive (active site bound) conformation of S-1360, we performed extensive docking studies using three different reported crystal structures where the active site or partial active site region was resolved. For comparison we extended our studies to include 5CITEP (the first compound cocrystallized with IN core domain) and a bis-diketoacid (BDKA). We found that the conformation of S-1360 when bound in one of the active sites matches that of the experimentally observed results of IN escape mutants resistant to S-1360. Therefore, we propose that this active site conformation is the biologically relevant conformation and can be used for the future structure-based drug design studies selectively targeting IN.     

Discovery of small-molecule HIV-1 fusion and integrase inhibitors oleuropein and hydroxytyrosol: part II. integrase inhibition

We report molecular modeling and functional confirmation of Ole and HT binding to HIV-1 integrase. Docking simulations identified two binding regions for Ole within the integrase active site. Region I encompasses the conserved D64-D116-E152 motif, while region II involves the flexible loop region formed by amino acid residues 140-149. HT, on the other hand, binds to region II. Both Ole and HT exhibit favorable interactions with important amino acid residues through strong H-bonding and van der Waals contacts, predicting integrase inhibition. To test and confirm modeling predictions, we examined the effect of Ole and HT on HIV-1 integrase activities including 3'-processing, strand transfer, and disintegration. Ole and HT exhibit dose-dependent inhibition on all three activities, with EC(50)s in the nanomolar range. These studies demonstrate that molecular modeling of target-ligand interaction coupled with structural-activity analysis should facilitate the design and identification of innovative integrase inhibitors and other therapeutics.     

Development of a high-throughput assay for the HIV-1 integrase disintegration reaction

Both HIV-1 integrase (IN) and the central catalytic domain of IN (IN-CCD) catalyze the disintegration reaction in vitro. In this study, IN and IN-CCD proteins were expressed and purified, and a high-throughput format enzyme-linked immunosorbent assay (ELISA) was developed for the disintegration reaction. IN exhibited a marked preference for Mn²⁺ over Mg²⁺ as the divalent cation cofactor in disintegration. Baicalein, a known IN inhibitor, was found to be an IN-CCD inhibitor. The assay is sensitive and specific for the study of disintegration reaction as well as for the in vitro identification of antiviral drugs targeting IN, especially targeting IN-CCD.     

HIV-1 integrase blocks infection of bacteria by single-stranded DNA and RNA bacteriophages

Expression of human immunodeficiency virus-1 integrase in Escherichia coli, at levels that had no effect on bacterial cell growth, blocked plaque formation by bacteriophages having single-stranded genomic DNA (M13) or RNA (R17, Q, PRR1). Plaque formation by phages having double-stranded genomic DNA (T4, PR4) was unaffected. Integrase also inhibited infection by the phagemid M13KO7, but it had no effect on production of phage once infection by M13KO7 was established. This result indicated that integrase affects an early stage in infection. Integrase also inhibited phage production following transfection by either single-stranded or double-stranded (replicative form) M13 DNA, it blocked M13 DNA replication, as assayed by incorporation of radioactive nucleotides into DNA, and it failed to affect bacterial pilus function. These data suggest that integrase interacts in vivo with phage nucleic acid, a conclusion supported by studies in which integrase was shown to have a DNA-binding activity in its C-terminal portion. This portion of integrase was both necessary and sufficient for interference of plaque formation by M13 in the present study. Expression of the N-terminal portion of integrase at the same level as intact integrase had little effect on phage growth, indicating that expression of foreign protein in general was not responsible for the inhibitory effect. The simple bacteriophage assay described is potentially useful for identifying integrase mutants that lack single-stranded DNA binding activity.     

Design of inhibitors of the HIV-1 integrase core domain using virtual screening

Acquired immunodeficiency syndrome (AIDS) is a disease of the human immune system caused by the human immunodeficiencyvirus (HIV). The integrase (IN) enzyme of HIV interacts with several cellular and viral proteins during the integration process.Thus, it represents an appropriate target for antiretroviral drugs (ARVs). We performed virtual screening of database compoundsand designed analogues using Elvitegravir (EVG) as a standard compound. The 378 screened compounds were retrieved fromZINC, ChemSpider, PubChem, and ChemBank Chemical Databases based on chemical similarity and literature searches related tothe structure of EVG. The Physiochemical properties, Bioactivity, Toxicity and Absorption, Distribution, Metabolism and Excretionof Molecules (ADME) of these compounds were predicted and docking Experiments were conducted using Molegro VirtualDocker software. The docking and ADME suggested very significant results in regard to EVG. The MolDock and Rerank scoreswere used to analyze the results. The compounds ZINC26507991 (-84.22), Analogue 9 (-68.49), ZINC20731658 (-66.79),ZINC00210363 (-43.44) showed better binding orientation with IN receptor model with respect to EVG (182.52). The ZINC26507991has showed significant ADME result.     

Crystal structure of the HIV-1 integrase core domain in complex with sucrose reveals details of an allosteric inhibitory binding site

HIV integrase (IN) is an essential enzyme in HIV replication and an important target for drug design. IN has been shown to interact with a number of cellular and viral proteins during the integration process. Disruption of these important interactions could provide a mechanism for allosteric inhibition of IN. We present the highest resolution crystal structure of the IN core domain to date. We also present a crystal structure of the IN core domain in complex with sucrose which is bound at the dimer interface in a region that has previously been reported to bind integrase inhibitors.

Structured summary

MINT-7713125: IN (uniprotkb:P04585) and IN (uniprotkb:P04585) bind (MI:0407) by X-ray crystallography (MI:0114)     

HIV-1整合酶核心区野生型和F185K突变型活性和溶解性的比较及分子动力学模拟分析

表达纯化了野生型(WT)及F185K突变型HIV-1整合酶核心区蛋白(IN_C),并对二者的溶解性和活性进行了比较．实验结果表明：F185K 突变后IN_C溶解性显著提高,活性有一定程度降低．对WT和F185K IN_C体系进行了1800 ps的分子动力学模拟．模拟结果表明：F185K IN_C功能loop区柔性和蛋白质整体运动性降低,使蛋白质活性降低,F185K突变后盐桥网络的变化驱动了IN_C局部构象改变,引起IN_C表面的部分疏水残基被包埋,亲水残基暴露,相对亲水溶剂可接近面积增大,同时,突变后IN_C与水之间形成氢键的数量增加,与水之间作用加强,以上变化使IN_C溶解性提高．分子动力学模拟与实验结果相吻合．为理解蛋白质溶解性和对蛋白质进行可溶性改造提供了一定的理论依据．     

病毒末端DNA与不同长度HIV-1整合酶四聚体的对接研究(英文)

整合酶(integrase,IN)是HIV病毒复制周期中的一个重要酶,一般认为,IN是以四聚体形式发挥其生物活性。本文用DOT软件包研究了IN四聚体与8个不同长度病毒末端DNA的结合模式,以及病毒DNA长度对二者识别的影响。结果表明,IN有3个DNA结合区域,其中两个是病毒DNA结合区域,另外一个是宿主DNA结合区域。模拟结果与实验数据吻合较好,本研究为基于整合酶结构的药物研发及整合酶整合机理的研究提供了良好的基础。     

Synthesis and evaluation of substituted 4-arylimino-3-hydroxybutanoic acids as potential HIV-1 integrase inhibitors

A series of readily accessible 4-arylimino-3-hydroxybutanoic acids have been prepared and evaluated as potential HIV-1 Integrase inhibitors. None of the ligands exhibited significant toxicity against human embryonic kidney (HEK 293) cells, while five of them showed activity against HIV-1 integrase – the most active (6c) with an IC₅₀ value of 3.5?μM. In silico docking studies indicate the capacity of ligand 6c to interact with several amino acid residues and the two Mg²⁺ cations in the HIV-1 integrase receptor cavity.     

用分子模拟方法研究HIV-1整合酶突变体的耐药性机理

二酮酸类化合物(DKAs)是目前最有前景的HIV-1整合酶(integrase, IN)抑制剂．为了解DKAs引起的多种耐药株共有的耐药性机理,选择3种S-1360引起的IN耐药突变体,用分子对接和分子动力学模拟,研究了野生型和突变型IN与S-1360的结合模式,基于该结合模式探讨了3种耐药突变体所共有的耐药性机理．结果表明：在突变体中,S-1360结合到耐药突变IN核心区中的位置靠近功能loop 3区却远离与 DNA结合的关键残基,结合位置不同导致S-1360的抑制作用部分丧失;残基138到166区域的柔性对IN发挥生物学功能很重要,S-1360能与DNA结合的关键残基N155及K159形成氢键,这2个氢键作用降低了该区域的柔性,突变体中无类似氢键,因而该区域柔性增高;在突变体中,S-1360的苯环远离病毒DNA结合区,不能阻止病毒DNA末端暴露给宿主DNA;T66I突变导致残基Ⅰ的长侧链占据IN的活性口袋,阻止抑制剂以与野生型中相同的方式结合到活性中心,这均是产生抗药性的重要原因．这些模拟结果与实验结果吻合,可为抗IN的抑制剂设计和改造提供帮助．     

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.