In Vitro DNA Tethering of HIV-1 Integrase by the Transcriptional Coactivator LEDGF/p75

Although LEDGF/p75 is believed to act as a cellular cofactor of lentiviral integration by tethering integrase (IN) to chromatin, there is no good in vitro model to analyze this functionality. We designed an AlphaScreen assay to study how LEDGF/p75 modulates the interaction of human immunodeficiency virus type 1 IN with DNA. IN bound with similar affinity to DNA mimicking the long terminal repeat or to random DNA. While LEDGF/p75 bound DNA strongly, a mutant of LEDGF/p75 with compromised nuclear localization signal (NLS)/AT hook interacted weakly, and the LEDGF/p75 PWWP domain did not interact, corroborating previous reports on the role of NLS and AT hooks in charge-dependent DNA binding. LEDGF/p75 stimulated IN binding to DNA 10-fold to 30-fold. Stimulation of IN-DNA binding required a direct interaction between IN and the C-terminus of LEDGF/p75. Addition of either the C-terminus of LEDGF/p75 (amino acids 325-530) or LEDGF/p75 mutated in the NLS/AT hooks interfered with IN binding to DNA. Our results are consistent with an in vitro model of LEDGF/p75-mediated tethering of IN to DNA. The inhibition of IN-DNA interaction by the LEDGF/p75 C-terminus may provide a novel strategy for the inhibition of HIV IN activity and may explain the potent inhibition of HIV replication observed after the overexpression of C-terminal fragments in cell culture.     

Discovery of novel inhibitors of LEDGF/p75-IN protein–protein interactions

Human lens epithelium-derived growth factor (LEDGF)/p75 plays an important role in the HIV life cycle by stimulating integrase (IN)-led viral DNA integration into cellular chromosomes. Mechanistic studies show the majority of IN inhibitors chelate magnesium ions in the catalytic active site, a region topologically distant from the LEDGF/p75 binding site. Compounds disrupting the formation of LEDGF/p75 and IN complexes serve as a novel mechanistic approach different from current antiretroviral therapies. We previously built pharmacophore models mimicking LEDGF/p75 residues and identified four classes of LEDGF/p75-IN inhibitors. Substructure and similarity searches yielded additional LEDGF/p75-IN inhibitors containing an acylhydrazone moiety. The most potent of the acylhydrazones inhibited LEDGF/p75-IN interaction with an IC₅₀ value of 400 nM. We explored structure–activity relationships (SAR) and identified new acylhydrazones, hydrazines, and diazenes as lead molecules for further optimization. Two lead LEDGF/p75-IN inhibitors showed antiviral activity.     

Identification of the LEDGF/p75 binding site in HIV-1 integrase

Lens epithelium-derived growth factor (LEDGF)/p75 is an important cellular co-factor for human immunodeficiency virus (HIV) replication. We originally identified LEDGF/p75 as a binding partner of integrase (IN) in human cells. The interaction has been mapped to the integrase-binding domain (IBD) of LEDGF/p75 located in the C-terminal part. We have subsequently shown that IN carrying the Q168A mutation remains enzymatically active but is impaired for interaction with LEDGF/p75. To map the integrase/LEDGF interface in more detail, we have now identified and characterized two regions within the enzyme involved in the interaction with LEDGF/p75. The first region centers around residues W131 and W132 while the second extends from I161 up to E170. For the different IN mutants the interaction with LEDGF/p75 and the enzymatic activities were determined. IN(W131A), IN(I161A), IN(R166A), IN(Q168A) and IN(E170A) are impaired for interaction with LEDGF/p75, but retain 3' processing and strand transfer activities. Due to impaired integration, an HIV-1 strain containing the W131A mutation in IN displays reduced replication capacity, whereas virus carrying IN(Q168A) is replication defective. Comparison of the wild-type IN-LEDGF/p75 co-crystal structure with that of the modelled structure of the IN(Q168A) and IN(W131A) mutant integrases corroborated our experimental data.     

Mechanism of action of the HIV-1 integrase inhibitory peptide LEDGF 361-370

The HIV-1 integrase protein (IN) mediates integration of the viral cDNA into the host genome and is a target for anti-HIV drugs. We have recently described a peptide derived from residues 361-370 of the IN cellular partner protein LEDGF/p75, which inhibited IN catalytic activity in vitro and HIV-1 replication in cells. Here we performed a comprehensive study of the LEDGF 361-370 mechanism of action in vitro, in cells and in vivo. Alanine scan, fluorescence anisotropy binding studies, homology modeling and NMR studies demonstrated that all residues in LEDGF 361-370 contribute to IN binding and inhibition. Kinetic studies in cells showed that LEDGF 361-370 specifically inhibited integration of viral cDNA. Thus, the full peptide was chosen for in vivo studies, in which it inhibited the production of HIV-1 RNA in mouse model. We conclude that the full LEDGF 361-370 peptide is a potent HIV-1 inhibitor and may be used for further development as an anti-HIV lead compound.     

Identification of small peptides inhibiting the integrase‐LEDGF/p75 interaction through targeting the cellular co‐factor

The integration of the viral DNA into the host genome is one of the essential steps in the HIV replication cycle. This process is mediated by the viral enzyme integrase (IN) and lens epithelium‐derived growth factor (LEDGF/p75). LEDGF/p75 has been identified as a crucial cellular co‐factor of integration that acts by tethering IN to the cellular chromatin. Recently, circular peptides were identified that bind to the C‐terminal domain of IN and disrupt the interaction with LEDGF/p75. Starting from the circular peptides, we identified a short peptidic sequence able to inhibit the LEDGF/p75‐IN interaction at low μM concentration through its binding to the IN binding site of LEDGF/p75. This discovery can lead to the synthesis of peptidomimetics with high anti‐HIV activity targeting the cellular co‐factor LEDGF/p75 and not the viral protein IN. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Small molecules targeting the interaction between HIV-1 integrase and LEDGF/p75 cofactor

The search of small molecules as protein–protein interaction inhibitors represents a new attractive strategy to develop anti-HIV-1 agents. We previously reported a computational study that led to the discovery of new inhibitors of the interaction between enzyme HIV-1 integrase (IN) and the nuclear protein lens epithelium growth factor LEDGF/p75.¹Herein, we describe new findings about the binding site of LEDGF/p75 on IN employing a different computational approach. In this way further structural requirements, helpful to disrupt LEDGF/p75-IN binding, have been identified. The main result of this work was the exploration of a relevant hydrophobic region. So we planned the introduction of suitable and simple chemical modifications on our previously reported ‘hit’ and the new synthesized compounds were subjected to biological tests.The results obtained demonstrate that the hydrophobic pocket could play a key role in improving inhibitory efficacy thus opening new suggestions to design active ligands.     

Insights into the interactions between HIV-1 integrase and human LEDGF/p75 by molecular dynamics simulation and free energy calculation

Human cellular protein LEDGF/p75 (lens epithelium-derived growth factor) is an important binding partner of human immunodeficiency virus type 1 (HIV-1) integrase (IN). Without LEDGF/p75, HIV-1 can not complete its life cycle. To study the detailed interactions between LEDGF/p75 and HIV-1 IN, and then obtain the hotspots at the binding interface, 13 ns molecular dynamics simulations were carried out here. One-hundred snapshots extracted from the last 4 ns trajectories were used for calculation of binding free energy and decomposition of the energy by residue. First, the structural changes and their dynamic interactions were investigated focused on the production stage. And then, the free energy was discussed. On the basis of the above results, it could be suggested that residues Gln168, Glu170, and Thr174 in chain A of IN, Thr125, and Trp131 in chain B of IN as well as Ile365, Asp366, Phe406, and Val408 in LEDGF/p75 were responsible for their binding. These results might be helpful for discovery and design of small molecules to interrupt the interaction between HIV-1 IN and LEDGF/p75.     

D77, one benzoic acid derivative, functions as a novel anti-HIV-1 inhibitor targeting the interaction between integrase and cellular LEDGF/p75

Integration of viral-DNA into host chromosome mediated by the viral protein HIV-1 integrase (IN) is an essential step in the HIV-1 life cycle. In this process, Lens epithelium-derived growth factor (LEDGF/p75) is discovered to function as a cellular co-factor for integration. Since LEDGF/p75 plays an important role in HIV integration, disruption of the LEDGF/p75 interaction with IN has provided a special interest for anti-HIV agent discovery. In this work, we reported that a benzoic acid derivative, 4-[(5-bromo-4-{[2,4-dioxo-3-(2-oxo-2-phenylethyl)-1,3-thiazolidin-5-ylidene]methyl}-2-ethoxyphenoxy)methyl]benzoic acid (D77) could potently inhibit the IN-LEDGF/p75 interaction and affect the HIV-1 IN nuclear distribution thus exhibiting antiretroviral activity. Molecular docking with site-directed mutagenesis analysis and surface plasmon resonance (SPR) binding assays has clarified possible binding mode of D77 against HIV-1 integrase. As the firstly discovered small molecular compound targeting HIV-1 integrase interaction with LEDGF/p75, D77 might supply useful structural information for further anti-HIV agent discovery.     

Structure-based virtual screening toward the discovery of novel inhibitors for impeding the protein-protein interaction between HIV-1 integrase and human lens epithelium-derived growth factor (LEDGF/p75)

HIV-1 integrase is a unique promising component of the viral replication cycle, catalyzing the integration of reverse transcribed viral cDNA into the host cell genome. Generally, IN activity requires both viral as well as a cellular co-factor in the processing replication cycle. Among them, the human lens epithelium-derived growth factor (LEDGF/p75) represented as promising cellular co-factor which supports the viral replication by tethering IN to the chromatin. Due to its major importance in the early steps of HIV replication, the interaction between IN and LEDGF/p75 has become a pleasing target for anti-HIV drug discovery. The present study involves the finding of novel inhibitor based on the information of dimeric CCD of IN in complex with known inhibitor, which were carried out by applying a structure-based virtual screening concept with molecular docking. Additionally, Free binding energy, ADME properties, PAINS analysis, Density Functional Theory, and Enrichment Calculations were performed on selected compounds for getting a best lead molecule. On the basis of these analyses, the current study proposes top 3 compounds: Enamine-Z742267384, Maybridge-HTS02400, and Specs-AE-848/37125099 with acceptable pharmacological properties and enhanced binding affinity to inhibit the interaction between IN and LEDGF/p75. Furthermore, Simulation studies were carried out on these molecules to expose their dynamics behavior and stability. We expect that the findings obtained here could be future therapeutic agents and may provide an outline for the experimental studies to stimulate the innovative strategy for research community.     

Study on the interactions between diketo-acid inhibitors and prototype foamy virus integrase-DNA complex via molecular docking and comparative molecular dynamics simulation methods

Human immunodeficiency virus type 1 (HIV-1) integrase (IN) is an important drug target for anti-acquired immune deficiency disease (AIDS) treatment and diketo-acid (DKA) inhibitors are potent and selective inhibitors of HIV-1 IN. Due to lack of three-dimensional structures including detail interactions between HIV-1 IN and its substrate viral DNA, the drug design and screening platform remains incompleteness and deficient. In addition, the action mechanism of DKA inhibitors with HIV-1 IN is not well understood. In view of the high homology between the structure of prototype foamy virus (PFV) IN and that of HIV-1 IN, we used PFV IN as a surrogate model for HIV-1 IN to investigate the inhibitory mechanism of raltegravir (RLV) and the binding modes with a series of DKA inhibitors. Firstly, molecular dynamics simulations of PFV IN, IN-RLV, IN-DNA, and IN-DNA-RLV systems were performed for 10?ns each. The interactions and inhibitory mechanism of RLV to PFV IN were explored through overall dynamics behaviors, catalytic loop conformation distribution, and hydrogen bond network analysis. The results show that the coordinated interactions of RLV with IN and viral DNA slightly reduce the flexibility of catalytic loop region of IN, and remarkably restrict the mobility of the CA end of viral DNA, which may lead to the partial loss of the inhibitory activity of IN. Then, we docked a series of DKA inhibitors into PFV IN-DNA receptor and obtained the IN-DNA-inhibitor complexes. The docking results between PFV IN-DNA and DKA inhibitors agree well with the corresponding complex of HIV-1 IN, which proves the dependability of PFV IN-DNA used for the anti-AIDS drug screening. Our study may help to make clear some theoretical questions and to design anti-AIDS drug based on the structure of IN.     

Structural studies and biological evaluation of T30695 variants modified with single chiral glycerol-T reveal the importance of LEDGF/p75 for the aptamer anti-HIV-integrase activities

Some G-quadruplex (GQ) forming aptamers, such as T30695, exhibit particularly promising properties among the potential anti-HIV drugs. T30695?G-quadruplex binds to HIV-1 integrase (IN) and inhibits its activity during 3′-end processing at nanomolar concentrations. Herein we report a study concerning six T30695-GQ variants, in which the R or S chiral glycerol T, singly replaced the thymine residues at the T30695?G-quadruplex loops. CD melting, EMSA and HMRS experiments provided information about the thermal stability and the stoichiometry of T30695-GQ variants, whereas CD and ¹H NMR studies were performed to evaluate the effects of the modifications on T30695-GQ topology. Furthermore, LEDGF/p75 dependent and independent integration assays were carried out to evaluate how T loop modifications impact T30695-GQ biological activities. The obtained results showed that LEDGF/p75 adversely affects the potencies of T30695 and its variants. The IN inhibitory activities of the modified aptamers also depended on the position and on the chirality (R or S) of glycerol T loop in the GQ, mostly regardless of the G-quadruplex stabilities. In view of our and literature data, we suggest that the allosteric modulation of IN tetramer conformations by LEDGF/p75 alters the interactions between the aptamers and the enzyme. Therefore, the new T30695 variants could be suitable tools in studies aimed to clarify the HIV-1 IN tetramers allostery and its role in the integration activity.     

Allosteric Inhibition of Human Immunodeficiency Virus Integrase: LATE BLOCK DURING VIRAL REPLICATION AND ABNORMAL MULTIMERIZATION INVOLVING SPECIFIC PROTEIN DOMAINS*

HIV-1 replication in the presence of antiviral agents results in evolution of drug-resistant variants, motivating the search for additional drug classes. Here we report studies of GSK1264, which was identified as a compound that disrupts the interaction between HIV-1 integrase (IN) and the cellular factor lens epithelium-derived growth factor (LEDGF)/p75. GSK1264 displayed potent antiviral activity and was found to bind at the site occupied by LEDGF/p75 on IN by x-ray crystallography. Assays of HIV replication in the presence of GSK1264 showed only modest inhibition of the early infection steps and little effect on integration targeting, which is guided by the LEDGF/p75·IN interaction. In contrast, inhibition of late replication steps was more potent. Particle production was normal, but particles showed reduced infectivity. GSK1264 promoted aggregation of IN and preformed LEDGF/p75·IN complexes, suggesting a mechanism of inhibition. LEDGF/p75 was not displaced from IN during aggregation, indicating trapping of LEDGF/p75 in aggregates. Aggregation assays with truncated IN variants revealed that a construct with catalytic and C-terminal domains of IN only formed an open polymer associated with efficient drug-induced aggregation. These data suggest that the allosteric inhibitors of IN are promising antiviral agents and provide new information on their mechanism of action.     

Cyclic peptide inhibitors of HIV-1 integrase derived from the LEDGF/p75 protein

Restricting linear peptides to their bioactive conformation is an attractive way of improving their stability and activity. We used a cyclic peptide library with conformational diversity for selecting an active and stable peptide that mimics the structure and activity of the HIV-1 integrase (IN) binding loop from its cellular cofactor LEDGF/p75 (residues 361-370). All peptides in the library had the same primary sequence, and differed only in their conformation. Library screening revealed that the ring size and linker structure had a huge effect on the conformation, binding and activity of the peptides. One of the cyclic peptides, c(MZ 4-1), was a potent and stable inhibitor of IN activity in vitro and in cells even after 8 days. The NMR structure of c(MZ 4-1) showed that it obtains a bioactive conformation that is similar to the parent site in LEDGF/p75.     

Design of HIV-1 integrase inhibitors targeting the catalytic domain as well as its interaction with LEDGF/p75: a scaffold hopping approach using salicylate and catechol groups

HIV-1 integrase (IN) is a validated therapeutic target for antiviral drug design. However, the emergence of viral strains resistant to clinically studied IN inhibitors demands the discovery of novel inhibitors that are structurally as well mechanistically different. Herein, we describe the design and discovery of novel IN inhibitors targeting the catalytic domain as well as its interaction with LEDGF/p75, which is essential for the HIV-1 integration as an IN cofactor. By merging the pharmacophores of salicylate and catechol, the 2,3-dihydroxybenzamide (5a) was identified as a new scaffold to inhibit the strand transfer reaction efficiently. Further structural modifications on the 2,3-dihydroxybenzamide scaffold revealed that the heteroaromatic functionality attached on the carboxamide portion and the piperidin-1-ylsulfonyl substituted at the phenyl ring are beneficial for the activity, resulting in a low micromolar IN inhibitor (5p, IC(50)=5 μM) with more than 40-fold selectivity for the strand transfer over the 3'-processing reaction. More significantly, this active scaffold remarkably inhibited the interaction between IN and LEDGF/p75 cofactor. The prototype example, N-(cyclohexylmethyl)-2,3-dihydroxy-5-(piperidin-1-ylsulfonyl) benzamide (5u) inhibited the IN-LEDGF/p75 interaction with an IC(50) value of 8 μM. Using molecular modeling, the mechanism of action was hypothesized to involve the chelation of the divalent metal ions inside the IN active site. Furthermore, the inhibitor of IN-LEDGF/p75 interaction was properly bound to the LEDGF/p75 binding site on IN. This work provides a new and efficient approach to evolve novel HIV-1 IN inhibitors from rational integration and optimization of previously reported inhibitors.