HIV-1 Integrase Core Domain Research Articles

Cyclic Hexapeptide Mimics of the LEDGF Integrase Recognition Loop in Complex with HIV-1 Integrase.

The p75 splice variant of lens epithelium-derived growth factor (LEDGF) is a 75 kDa protein, which is recruited by the human immunodeficiency virus (HIV) to tether the pre-integration complex to the host chromatin and promote integration of proviral DNA into the host genome. We designed a series of small cyclic peptides that are structural mimics of the LEDGF binding domain, which interact with integrase as potential binding inhibitors. Herein we present the X-ray crystal structures, NMR studies, SPR analysis, and conformational studies of four cyclic peptides bound to the HIV-1 integrase core domain. Although the X-ray studies show that the peptides closely mimic the LEDGF binding loop, the measured affinities of the peptides are in the low millimolar range. Computational analysis using conformational searching and free energy calculations suggest that the low affinity of the peptides is due to mismatch between the low-energy solution and bound conformations.

Docking and Molecular Dynamics Calculations of Some Previously Studied and newly Designed Ligands to Catalytic Core Domain of HIV-1 Integrase and an Investigation to Effects of Conformational Changes of Protein on Docking Results

Nowadays, AIDS still remains as a worldwide pandemic and continues to cause many death which arise from HIV-1 virus. For nearly 35 years, drugs that target various steps of virus life cycle have been developed. HIV-1 integrase is the one of these steps which is essential for virus life cycle. Computer aided drug design is being used in many drug design studies as also used in development of the first HIV-1 integrase inhibitor Raltegravir. In this study 3 ligands which are used as HIV-1 integrase inhibitors and 4 newly designed ligands were docked to catalytic core domain of HIV-1 integrase. Each of ligands docked to three different conformations of protein. Prepared complexes (21 item) were carried out by 50 ns MD simulations and results were analyzed. Finally, the binding free energies of ligands were calculated. Hereunder, it was determined that designed ligands L01 and L03 gave favorable results. The questions about the ligands which have low docking scores in a conformation of protein could give better scores in another conformation of protein and if the MD simulations carry the different oriented and different localized ligands in same position at the end of simulation were answered.

Inhibition of the Replication of Clinical Drug-Resistant HIV-1 Strains by Small Molecule Integrase Inhibitors M522 and M532

Objective: Integrase (IN) is an enzyme essential for HIV-1 replication that has been a target of antiretroviral drug therapy. Since emerging HIV-1 variants have frequently become clinically resistant to antiretroviral agents, it is necessary to develop alternative IN inhibitors. Methods: We tested IN inhibitors, M522 and M532, against clinically resistant HIV-1 strains to antiretroviral drugs (AZT, non-nucleoside reverse transcriptase inhibitors, IN drug raltegravir, protease inhibitors); wild-type and clinical isolate from HIV-infected patients. We performed disintegration studies to show the interaction of M522 and M532 with the catalytic core domain of HIV-1 IN and time-of-drug-addition experiments to determine the inhibition step of viral replication. We tested selection of HIV-1 with M522 and M532 to examine the emergence of new drug resistant virus. CD4+ cell count was calculated for several groups of cells infected with HIV, cells treated and non-treated with M522 and M532 to evaluate their protective effect. Results: M522 and M532 inhibited the replication of HIV-1 strains (wild-type; drug-resistant; clinical isolate from infected patients; and laboratory strains) with high potency. These inhibitors interacted with the catalytic core domain of HIV-1 IN and blocked its activity, prevented viral integration. M522 and M532 interfered with the viral replication precisely at the integration step. HIV-1 virus did not develop resistance to M522 and M532 for 20 viral passages (160 days). These IN inhibitors protected the infected cells from cytopathic effects and the CD4+ cell counts of these cells treated with M522 and M532 were found to be identical to those of the uninfected cells. Conclusion: M522 and M532 are potent against clinical isolate from HIV-infected patients, wild-type and clinically resistant strains especially the relevant raltegravir-resistant virus. Development of M522 and M532 as new mutationinsensitive drugs aiming for the protection of CD4+ T-cells during HIV infection in the clinical trials is in progress.

Preclinical and Clinical Profile of HIV-1 Integrase Strand-transfer Inhibitor GS-9224 Compared to its Parent Compound GS-9160

In an effort to optimize the pharmacokinetic profile of GS-9160, a potent (EC50=1.2 – 4 nM) antiviral tri-cyclic HIV-1 integrase (IN) strand transfer inhibitor (INSTI), various substitutions on the p-fluorobenzyl moiety of GS-9160 were explored. This effort led to the discovery of GS-9224, an analog containing a 5-chloro-2,4-di-fluorobenzyl moiety. GS-9224, like its predecessor GS-9160, has potent (EC50=1.3 – 9 nM) and selective antiviral activity against HIV-1 and acts as a bona fide integration inhibitor through elevation of 2-long terminal repeat (2-LTR) circles and decrease of integration junctions in HIV-1 infected cells, markers of failed viral integration. Viral resistance selections with GS-9224 yielded three mutations within the catalytic core domain of HIV-1 IN: G140S, L74M and Q148K. When tested against a panel of HIV-1 clones engineered to express INSTI resistance mutations, the profile of GS-9224 was comparable to GS-9160. GS-9224 exhibits improved hepatic microsome stability, better absorption potential and a lower in vitro intestinal efflux ratio compared to GS-9160. In addition, GS-9224 displayed higher dog plasma protein binding (~99%) than GS-9160 (~93%) suggesting a potential for protein restricted clearance. However, the improved metabolic stability, absorption potential and serum protein binding of GS-9224 compared to GS-9160 did not translate into an improved clinical pharmacokinetic profile. Results from a single-dose study in human healthy subjects revealed a fast systemic clearance of GS-9224 with a terminal half-life of ~ 1 h, resulting in a pharmacokinetic profile that would not support once-daily dosing in HIV-infected patients.

Real-time monitoring of disintegration activity of catalytic core domain of HIV-1 integrase using molecular beacon

HIV-1 integrase, an essential enzyme for retroviral replication, is a validated target for anti-HIV therapy development. The catalytic core domain of integrase (IN–CCD) is capable of catalyzing disintegration reaction. In this work, a hairpin-shaped disintegration substrate was designed and validated by enzyme-linked immunosorbent assay; a molecular beacon-based assay was developed for disintegration reaction of IN–CCD. Results showed that the disintegration substrate could be recognized and catalyzed by IN–CCD, and the disintegration reaction can be monitored according to the increase of fluorescent signal. The assay can be applied to real-time detection of disintegration with advantages of simplicity, high sensitivity, and excellent specificity.

Parallel Screening of Low Molecular Weight Fragment Libraries: Do Differences in Methodology Affect Hit Identification?

Fragment screening is becoming widely accepted as a technique to identify hit compounds for the development of novel lead compounds. In neighboring laboratories, we have recently, and independently, performed a fragment screening campaign on the HIV-1 integrase core domain (IN) using similar commercially purchased fragment libraries. The two campaigns used different screening methods for the preliminary identification of fragment hits; one used saturation transfer difference nuclear magnetic resonance spectroscopy (STD-NMR), and the other used surface plasmon resonance (SPR) spectroscopy. Both initial screens were followed by X-ray crystallography. Using the STD-NMR/X-ray approach, 15 IN/fragment complexes were identified, whereas the SPR/X-ray approach found 6 complexes. In this article, we compare the approaches that were taken by each group and the results obtained, and we look at what factors could potentially influence the final results. We find that despite using different approaches with little overlap of initial hits, both approaches identified binding sites on IN that provided a basis for fragment-based lead discovery and further lead development. Comparison of hits identified in the two studies highlights a key role for both the conditions under which fragment binding is measured and the criteria selected to classify hits.

Communications: Electron polarization critically stabilizes the Mg2+ complex in the catalytic core domain of HIV-1 integrase

In this paper, we present a detailed dynamics study of the catalytic core domain (CCD) of HIV-1 integrase using both polarized and nonpolarized force fields. The numerical results reveal the critical role of protein polarization in stabilizing Mg(2+) coordination complex in CCD. Specifically, when nonpolarized force field is used, a remarkable drift of the Mg(2+) complex away from its equilibrium position is observed, which causes the binding site blocked by the Mg(2+) complex. In contrast, when polarized force field is employed in MD simulation, HIV-1 integrase CCD structure is stabilized and both the position of the Mg(2+) complex and the binding site are well preserved. The detailed analysis shows the transition of alpha-helix to 3(10)-helix adjacent to the catalytic loop (residues 139-147), which correlates with the dislocation of the Mg(2+) complex. The current study demonstrates the importance of electronic polarization of protein in stabilizing the metal complex in the catalytic core domain of HIV-1 integrase.

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)

Study of the Conformational Dynamics of the Catalytic Loop of WT and G140A/G149A HIV-1 Integrase Core Domain Using Reversible Digitally Filtered Molecular Dynamics

The HIV-1 IN enzyme is one of three crucial virally encoded enzymes (HIV-1 IN, HIV-1 PR, and HIV-1 RT) involved in the life-cycle of the HIV-1 virus, making it an attractive target in the development of drugs against the AIDS virus. The structure and mechanism of the HIV-1 IN enzyme is the least understood of the three enzymes due to the lack of three-dimensional structural information. X-ray cystallographic studies have not yet been able to resolve the full-length structure, and studies have been mainly focused on the catalytic domain. This central domain possesses an important catalytic loop observed to overhang the active site, and experimental studies have shown that its dynamics affects the catalytic activity of mutant HIV-1 IN enzymes. In this study, the enhanced sampling technique, Reversible Digitally Filtered Molecular Dynamics (RDFMD), has been applied to the catalytic domain of the WT and G140A/G149A HIV-1 IN enzymes and has highlighted significant differences between the behavior of the catalytic loop which may explain the decrease of activity observed in experimental studies for this mutant.

Contribution of the C-terminal region within the catalytic core domain of HIV-1 integrase to yeast lethality, chromatin binding and viral replication

BackgroundHIV-1 integrase (IN) is a key viral enzymatic molecule required for the integration of the viral cDNA into the genome. Additionally, HIV-1 IN has been shown to play important roles in several other steps during the viral life cycle, including reverse transcription, nuclear import and chromatin targeting. Interestingly, previous studies have demonstrated that the expression of HIV-1 IN induces the lethal phenotype in some strains of Saccharomyces cerevisiae. In this study, we performed mutagenic analyses of the C-terminal region of the catalytic core domain of HIV-1 IN in order to delineate the critical amino acid(s) and/or motif(s) required for the induction of the lethal phenotype in the yeast strain HP16, and to further elucidate the molecular mechanism which causes this phenotype.ResultsOur study identified three HIV-1 IN mutants, V165A, A179P and KR186,7AA, located in the C-terminal region of the catalytic core domain of IN that do not induce the lethal phenotype in yeast. Chromatin binding assays in yeast and mammalian cells demonstrated that these IN mutants were impaired for the ability to bind chromatin. Additionally, we determined that while these IN mutants failed to interact with LEDGF/p75, they retained the ability to bind Integrase interactor 1. Furthermore, we observed that VSV-G-pseudotyped HIV-1 containing these IN mutants was unable to replicate in the C8166 T cell line and this defect was partially rescued by complementation with the catalytically inactive D64E IN mutant.ConclusionOverall, this study demonstrates that three mutations located in the C-terminal region of the catalytic core domain of HIV-1 IN inhibit the IN-induced lethal phenotype in yeast by inhibiting the binding of IN to the host chromatin. These results demonstrate that the C-terminal region of the catalytic core domain of HIV-1 IN is important for binding to host chromatin and is crucial for both viral replication and the promotion of the IN-induced lethal phenotype in yeast.

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 Å) 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.

Structure-activity relationships of HIV-1 integrase inhibitors--enzyme-ligand interactions.

HIV-1 integrase is an essential enzyme for retroviral replication. It is involved in the integration of HIV DNA into host chromosomal DNA and appears to have no functional equivalent in human cells. Therefore it is an attractive and rational target for selective anti-AIDS therapy. A great number of HIV-1 integrase inhibitors have been described in the last decade and numerous reviews have been published. The biochemical mechanism of HIV-1 DNA integration, the enzyme structure and the possible targets for drug intervention have been thoroughly analyzed. Structure-based drug design including both ligand-based (pharmacophore) and target-based (docking) methods has also been discussed. The recent report of the crystal structure of HIV-1 integrase core domain with an inhibitor has given a new boost leading in the last two years to the emergence of diketoacids (DKAs). To date, with the dicaffeoyltartaric acids they are the only two classes of molecules that meet the criteria necessary to be considered lead molecules in the search for clinically useful inhibitors of HIV-1 integrase. After a survey of the function and the structure of this enzyme and the different available assays for the identification of new IN inhibitors, structure-activity relationships of HIV-1 integrase inhibitors that are expected to interact with the active site (or in its vicinity) will be discussed with emphasis on their different proposed mechanisms of action.

Thalassiolins A—C: New Marine‐Derived Inhibitors of HIV cDNA Integrase.

AbstractFor Abstract see ChemInform Abstract in Full Text.

AutoDocking dinucleotides to the HIV-1 integrase core domain: exploring possible binding sites for viral and genomic DNA.

To understand the binding of both viral and human DNA to HIV-1 integrase, fully flexible dinucleotides were docked onto the core domain of integrase. AutoDocking did identify sites on integrase where favorable interactions with nucleotides can occur, and those sites were in agreement with recently published protein fingerprinting data. By analyzing the phosphates of the docked dinucleotides, we developed a model indicating where the viral cDNA and human DNA bind to the integrase core domain.

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 β- 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 (IC 50=0.4 μM) and an antiviral IC 50 of 30 μM. Molecular modeling studies indicate a favorable binding mode is probable at the catalytic core domain of HIV-1 integrase.

HIV-1 integrase: the next target for AIDS therapy?

HIV-1 is the aetiological agent of AIDS. Present treatment of AIDS uses a combination therapy with reverse transcriptase and protease inhibitors. Recently, the integrase (IN), the third enzyme of HIV-1 which is necessary for the integration process of proviral DNA into the host genome, has reached as a legitimate new drug target. Several families of inhibitors of the catalytic core domain of HIV-1 IN exhibiting submicromolar activities have now been identified. Our contribution in this field was related to the development of new polyhydroxylated styrylquinolines. The latter compounds have proved to be potent HIV-1 IN inhibitors, that block the replication of HIV-1 in cell culture, and are devoid of cytotoxicity. The crystal structure of the catalytically active core domain of a HIV-1 IN mutant has been determined. The active site region is identified by the position of two of the conserved carboxylate residues essential for catalysis, Asp 64 and Asp 116, which coordinate a Mg 2+ ion, whereas the third catalytic residue, Glu 152 does not participate in metal binding. However, a recent molecular dynamics simulation of the HIV-1 IN catalytic domain provides support to the hypothesis that a second metal ion is likely to be carried into the HIV-1 IN active site by the DNA substrate. The structure of a complex of the HIV-1 IN core domain with the inhibitor 5-CITEP has been recently reported. The inhibitor binds centrally in the active site of the IN and makes a number of close contacts with the protein, particularly with Lys 156, Lys 159 and Gln 148, amino acids which were identified to be near the active site of the enzyme, through site-directed mutagenis and photo-crosslinking experiments. The exact mechanism by which HIV-1 IN inhibitors block the catalytic activity of the protein remains unknown. However, several putative pharmacophore components have been characterized. All these groups lie in a possible coordination to a divalent ion, supporting thus the hypothesis that the interaction causing the inhibition is mediated by one or two cations. Finally, among the HIV-1 IN inhibitors, three classes have proved to exhibit significant antiviral activities. Thus, it seems likely that the efficient use of HIV-1 IN as a target for rational design will become possible in the next future, possibly through the use of combination regimens including IN inhibitors.

Structure of the HIV-1 integrase catalytic domain complexed with an inhibitor: a platform for antiviral drug design.

HIV integrase, the enzyme that inserts the viral DNA into the host chromosome, has no mammalian counterpart, making it an attractive target for antiviral drug design. As one of the three enzymes produced by HIV, it can be expected that inhibitors of this enzyme will complement the therapeutic use of HIV protease and reverse transcriptase inhibitors. We have determined the structure of a complex of the HIV-1 integrase core domain with a novel inhibitor, 5ClTEP, 1-(5-chloroindol-3-yl)-3-hydroxy-3-(2H-tetrazol-5-yl)-pro penone, to 2.1-A resolution. The inhibitor binds centrally in the active site of the integrase and makes a number of close contacts with the protein. Only minor changes in the protein accompany inhibitor binding. This inhibitor complex will provide a platform for structure-based design of an additional class of inhibitors for antiviral therapy.

Three new structures of the core domain of HIV-1 integrase: an active site that binds magnesium.

HIV-1 integrase is an essential enzyme in the life cycle of the virus, responsible for catalyzing the insertion of the viral genome into the host cell chromosome; it provides an attractive target for antiviral drug design. The previously reported crystal structure of the HIV-1 integrase core domain revealed that this domain belongs to the superfamily of polynucleotidyltransferases. However, the position of the conserved catalytic carboxylic acids differed from those observed in other enzymes of the class, and attempts to crystallize in the presence of the cofactor, Mg2+, were unsuccessful. We report here three additional crystal structures of the core domain of HIV-1 integrase mutants, crystallized in the presence and absence of cacodylate, as well as complexed with Mg2+. These three crystal forms, containing between them seven independent core domain structures, demonstrate the unambiguous extension of the previously disordered helix alpha4 toward the amino terminus from residue M154 and show that the catalytic E152 points in the general direction of the two catalytic aspartates, D64 and D116. In the vicinity of the active site, the structure of the protein in the absence of cacodylate exhibits significant deviations from the previously reported structures. These differences can be attributed to the modification of C65 and C130 by cacodylate, which was an essential component of the original crystallization mixture. We also demonstrate that in the absence of cacodylate this protein will bind to Mg2+, and could provide a satisfactory platform for binding of inhibitors.

The crystal structure of the catalytic core domain of HIV-1 integrase in a new crystal form

The crystal structure of the catalytic core domain of HIV-1 integrase in a new crystal form

Structure of the bacteriophage Mu transposase core: A common structural motif for DNA transposition and retroviral integration

The crystal structure of the core domain of bacteriophage Mu transposase, MuA, has been determined at 2.4 Å resolution. The first of two subdomains contains the active site and, despite very limited sequence homology, exhibits a striking similarity to the core domain of HIV-1 integrase, which carries out a similar set of biochemical reactions. It also exhibits more limited similarity to other nucleases, RNase H and RuvC. The second, a β barrel, connects to the first subdomain through several contacts. Three independent determinations of the monomer structure from two crystal forms all show the active site held in a similar, apparently inactive configuration. The enzymatic activity of MuA is known to be activated by formation of a DNA-bound tetramer of the protein. We propose that the connections between the two subdomains may be involved in the cross-talk between the active site and the other domains of the transposase that controls the activity of the protein.