Studies towards the Design and Synthesis of Novel 1,5-Diaryl-1H-imidazole-4-carboxylic Acids and 1,5-Diaryl-1H-imidazole-4-carbohydrazides as Host LEDGF/p75 and HIV-1 Integrase Interaction Inhibitors.

Two targeted sets of novel 1,5-diaryl-1H-imidazole-4-carboxylic acids 10 and carbohydrazides 11 were designed and synthesized from their corresponding ester intermediates 17, which were prepared via cycloaddition of ethyl isocyanoacetate 16 and diarylimidoyl chlorides 15. Evaluation of these new target scaffolds in the AlphaScreenTM HIV-1 IN-LEDGF/p75 inhibition assay identified seventeen compounds exceeding the pre-defined 50% inhibitory threshold at 100 µM concentration. Further evaluation of these compounds in the HIV-1 IN strand transfer assay at 100 µM showed that none of the compounds (with the exception of 10a, 10l, and 11k, with marginal inhibitory percentages) were actively bound to the active site, indicating that they are selectively binding to the LEDGF/p75-binding pocket. In a cell-based HIV-1 antiviral assay, compounds 11a, 11b, 11g, and 11h exhibited moderate antiviral percentage inhibition of 33-45% with cytotoxicity (CC50) values of >200 µM, 158.4 µM, >200 µM, and 50.4 µM, respectively. The antiviral inhibitory activity displayed by 11h was attributed to its toxicity. Upon further validation of their ability to induce multimerization in a Western blot gel assay, compounds 11a, 11b, and 11h appeared to increase higher-order forms of IN.

Analyses of HIV-1 integrase sequences prior to South African national HIV-treatment program and available of integrase inhibitors in Cape Town, South Africa.

HIV-Integrase (IN) has proven to be a viable target for highly specific HIV-1 therapy. We aimed to characterize the HIV-1 IN gene in a South African context and identify resistance-associated mutations (RAMs) against available first and second generation Integrase strand-transfer inhibitors (InSTIs). We performed genetic analyses on 91 treatment-naïve HIV-1 infected patients, as well as 314 treatment-naive South African HIV-1 IN-sequences, downloaded from Los Alamos HIV Sequence Database. Genotypic analyses revealed the absence of major RAMs in the cohort collected before the broad availability of combination antiretroviral therapy (cART) and INSTI in South Africa, however, occurred at a rate of 2.85% (9/314) in database derived sequences. RAMs were present at IN-positions 66, 92, 143, 147 and 148, all of which may confer resistance to Raltegravir (RAL) and Elvitegravir (EVG), but are unlikely to affect second-generation Dolutegravir (DTG), except mutations in the Q148 pathway. Furthermore, protein modeling showed, naturally occurring polymorphisms impact the stability of the intasome-complex and therefore may contribute to an overall potency against InSTIs. Our data suggest the prevalence of InSTI RAMs, against InSTIs, is low in South Africa, but natural polymorphisms and subtype-specific differences may influence the effect of individual treatment regimens.

Quinoline-based Protein-protein Interaction Inhibitors of LEDGF/p75 and HIV Integrase: An In Silico Study.

The failure of the Integrase Strand Transfer Inhibitors (INSTIs) due to the mutations occurring at the catalytic site of HIV integrase (IN) has led to the design of allosteric integrase inhibitors (ALLINIs). Lens epithelium derived growth factor (LEDGF/p75) is the host cellular cofactor which helps chaining IN to the chromatin. The protein-protein interactions (PPIs) were observed at the allosteric site (LEDGF/p75 binding domain) between LEDGF/p75 of the host cell and IN of virus. In recent years, many small molecules such as CX04328, CHIBA-3053 and CHI-104 have been reported as LEDGF/p75-IN interaction inhibitors (LEDGINs). LEDGINs have emerged as promising therapeutics to halt the PPIs by binding at the interface of both the proteins. In the present work, we correlated the docking scores for the reported LEDGINs containing quinoline scaffold with the in vitro biological data. The hierarchal clustering method was used to divide the compounds into test and training set. The robustness of the generated model was validated by q2 and r2 for the predicted set of compounds. The generated model between the docking score and biological data was assessed to predict the activity of the hits (quinoline scaffold) obtained from virtual screening of LEDGINs providing their structureactivity relationships to aim for the generation of potent agents.

An effective HIV-1 integrase inhibitor screening platform: Rationality validation of drug screening, conformational mobility and molecular recognition analysis for PFV integrase complex with viral DNA.

As an important target for the development of novel anti-AIDS drugs, HIV-1 integrase (IN) has been widely concerned. However, the lack of a complete accurate crystal structure of HIV-1 IN greatly blocks the discovery of novel inhibitors. In this work, an effective HIV-1 IN inhibitor screening platform, namely PFV IN, was filtered from all species of INs. Next, the 40.8% similarity with HIV-1 IN, as well as the high efficiency of virtual screening and the good agreement between calculated binding free energies and experimental ones all proved PFV IN is a promising screening platform for HIV-1 IN inhibitors. Then, the molecular recognition mechanism of PFV IN by its substrate viral DNA and six naphthyridine derivatives (NRDs) inhibitors was investigated through molecular docking, molecular dynamics simulations and water-mediated interactions analyses. The functional partition of NRDs IN inhibitors could be divided into hydrophobic and hydrophilic ones, and the Mg2+ ions, water molecules and conserved DDE motif residues all interacted with the hydrophilic partition, while the bases in viral DNA and residues like Tyr212, Pro214 interacted with the hydrophobic one. Finally, the free energy landscape (FEL) and cluster analyses were performed to explore the molecular motion of PFV IN-DNA system. It is found that the association with NRDs inhibitors would obviously decrease the motion amplitude of PFV IN-DNA, which may be one of the most potential mechanisms of IN inhibitors. This work will provide a theoretical basis for the inhibitor design based on the structure of HIV-1 IN.

Discovery of a Novel HIV-1 Integrase/p75 Interacting Inhibitor by Docking Screening, Biochemical Assay, and in Vitro Studies.

Protein-protein interaction between lens epithelium-derived growth factor (LEDGF/p75) and HIV-1 integrase becomes an attractive target for anti-HIV drug development. The blockade of this interaction by small molecules could potentially inhibit HIV-1 replication. These small molecules are termed as LEDGINs; and several newly identified LEDGINs have been reported to significantly reduce HIV-1 replication. Through this project, we have finished the docking screening of the Maybridge database against the p75 binding site of HIV-1 integrase using both DOCK and Autodock Vina software. Finally, we have successfully identified a novel scaffold LEDGINs inhibitor DW-D-5. Its antiviral activities and anticatalytic activity of HIV-1 integrase are similar to other LEDGINs under development. We demonstrated that the combination of DW-D-5 and FDA approved anti-HIV drugs resulted in additive inhibitory effects on HIV-1 replication, indicating that DW-D-5 could be an important component of combination pills for clinic use in HIV treatment.

The evaluation of statins as potential inhibitors of the LEDGF/p75-HIV-1 integrase interaction.

Lovastatin was identified through virtual screening as a potential inhibitor of the LEDGF/p75-HIV-1 integrase interaction. In an AlphaScreen assay, lovastatin inhibited the purified recombinant protein-protein interaction (IC50 = 1.97 ± 0.45 µm) more effectively than seven other tested statins. None of the eight statins, however, yielded antiviral activity in vitro, while only pravastatin lactone yielded detectable inhibition of HIV-1 integrase strand transfer activity (31.65% at 100 µm). A correlation between lipophilicity and increased cellular toxicity of the statins was observed.

Evolution of a novel pathway leading to dolutegravir resistance in a patient harbouring N155H and multiclass drug resistance.

OBJECTIVES: Dolutegravir has been recently approved for treatment-naive and -experienced HIV-infected subjects, including integrase inhibitor (INI)-experienced patients. Dolutegravir is a second-generation INI that can overcome many prior raltegravir and elvitegravir failures. Here, we report the evolution of resistance to dolutegravir in a highly treatment-experienced patient harbouring the major N155H mutation consequent to raltegravir treatment failure. METHODS: Genotypic and phenotypic analyses were done on longitudinal samples to determine viral resistance to INIs. Integrase amino acid sequence interactions with raltegravir and dolutegravir were assessed by molecular modelling and docking simulations. RESULTS: Five mutations (A49P, L68FL, T97A, E138K and L234V) were implicated in emergent dolutegravir resistance, with a concomitant severe compromise in viral replicative capacity. Molecular modelling and docking simulations revealed that dolutegravir binding to integrase was affected by these acquired dolutegravir mutations. CONCLUSIONS: Our findings identify a novel mutational pathway involving integrase mutations A49P and L234V, leading to dolutegravir resistance in a patient with the N155H raltegravir mutation.

Discovery of MK-8970: an acetal carbonate prodrug of raltegravir with enhanced colonic absorption.

Developing new antiretroviral therapies for HIV-1 infection with potential for less frequent dosing represents an important goal within drug discovery. Herein, we present the discovery of ethyl (1-((4-((4-fluorobenzyl)carbamoyl)-1-methyl-2-(2-(5-methyl- 1,3,4-oxadiazole-2-carboxamido)propan-2-yl)-6-oxo-1,6-dihydropyrimidin-5-yl)oxy)ethyl) carbonate (MK-8970), a highly optimized prodrug of raltegravir (Isentress). Raltegravir is a small molecule HIV integrase strand-transfer inhibitor approved for the treatment of HIV infection with twice-daily administration. Two classes of prodrugs were designed to have enhanced colonic absorption, and derivatives were evaluated in pharmacokinetic studies, both in vitro and in vivo in different species, ultimately leading to the identification of MK-8970 as a suitable candidate for development as an HIV therapeutic with the potential to require less frequent administration while maintaining the favorable efficacy, tolerability, and minimal drug-drug interaction profile of raltegravir.

Improving the use of ranking in virtual screening against HIV-1 integrase with triangular numbers and including ligand profiling with antitargets.

A delicate balance exists between a drug molecule's toxicity and its activity. Indeed, efficacy, toxicity, and side effect problems are a common cause for the termination of drug candidate compounds and development projects. To address this, an antitarget interaction profile is built and combined with virtual screening and cross docking for new inhibitors of HIV-1 integrase, in order to consider possible off-target interactions as early as possible in a drug or hit discovery program. New ranking techniques using triangular numbers improve ranking information on the compounds and recovery of known inhibitors into the top compounds using different docking programs. This improved ranking arises from using consensus of ranks between docking programs and ligand efficiencies to derive a new rank, instead of using absolute score values, or average of ranks. The triangular number rerank also allowed the objective combination of results from several protein targets or screen conditions and several programs. Triangular number reranking conserves more information than other reranking methods such as average of scores or averages of ranks. In addition, the use of triangular numbers for reranking makes possible the use of thresholds with a justified leeway based on the number of available known inhibitors, so that the majority of the compounds above the threshold in ranks compare to the compounds that have known experimentally determined biological activity. The battery of anti- or off-targets can be tailored to specific molecular or drug design challenges. In silico filters can thus be deployed in successive stages, for prefiltering, activity profiling, and for further analysis and triaging of libraries of compounds.

Discovery of a novel HIV-1 integrase inhibitor from natural compounds through structure based virtual screening and cell imaging.

The interaction between HIV-1 integrase and LEDGF/P75 has been validated as a target for anti-HIV drug development. Based on the crystal structure of integrase in complex with LEDGF/P75, a library containing 80 thousand natural compounds was filtered with virtual screening. 11 hits were selected for cell based assays. One compound, 3-(1,3-benzothiazol-2-yl)-8-{[bis(2-hydroxyethyl)amino]methyl}-7-hydroxy-2H-chromen-2-one (D719) inhibited integrase nuclear translocation in cell imaging. The binding mode of D719 was analyzed with molecular simulation. The anti-HIV activity of D719 was assayed by measuring the p24 antigen production in acute infection. The structure characteristics of D719 may provide valuable information for integrase inhibitor design.

Lead screening for HIV-1 integrase (IN) inhibited by traditional Chinese medicine.

Human immunodeficiency virus causes the acquired immunodeficiency syndrome (AIDS) and becomes a serious world-wide problem because of this disease's rapid propagation and incurability. Integrase strand transfer inhibitors (INSTIs) supports HIV have rapid drug resistance for antitreatment. Screening the traditional Chinese medicine (TCM) database by simulating molecular docking and molecular dynamics may select molecular compounds to inhibit INSTIs against HIV drug resistance. (S)-cathinone and (1S,2S)-norpseudoephedrine are selected based on structure and ligand-based drugs are designed and then get higher bioactivity predicted score from SVM than Raltegravir and other TCM compounds. The molecular dynamics are helpful in the analysis and detection of protein-ligand interactions. According to the docking poses, hydrophobic interactions and hydrogen bond variations define the main regions of important amino acids in integrase. In addition to the detection of TCM compound efficacy, we suggest (1S,2S)-norpseudoephedrine is better than the others based on the analysis of interaction and the effect on the structural variation.

Traditional Chinese medicine application in HIV: an in silico study.

Viral infection by human immunodeficiency virus (HIV) requires integration of viral DNA with host DNA which involves the binding of HIV integrase (IN) with its co-factor lens epithelium-derived growth factor (LEDGF/p75). Since disrupted binding of IN with LEDGF/p75 inhibits proliferation of HIV, inhibition or denaturation of IN is a possible method for inhibiting HIV replication. D77 is a known drug with demonstrated inhibition against HIV by binding to IN. Herein, we utilized D77 as a control to screen for traditional Chinese medicine (TCM) compounds that exhibit similar atomic-level characteristics. 9-Hydroxy-(10E)-octadecenoic acid and Beauveriolide I were found to have higher Dock Scores to IN than D77 through virtual screening. Multiple linear regression (R2 = 0.9790) and support vector machine (R2 = 0.9114) models consistently predicted potential bioactivity of the TCM candidates against IN. The 40 ns molecular dynamics simulation showed that the TCM compounds fulfilled the drug-like criteria of forming stable complexes with IN. Atomic-level investigations revealed that 9-hydroxy-(10E)-octadecenoic acid bound to an important residue A:Lys173, and Beauveriolide I formed stable interactions with the core LEDGF binding site and with Asn256 of the IN binding site on LEDGF. The TCM candidates also initiated loss of α-helices that could affect the functionality of IN. Taken together, the ability of 9-hydroxy-(10E)-octadecenoic acid and Beauveriolide I to (1) form stable interactions affecting IN-LEDGF binding and (2) have predicted bioactivity against IN suggests that the TCM candidates might be potential starting structures for developing compounds that may disrupt IN-LEDGF binding. An animated interactive 3D complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:JBSD:40.

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 IC(50) value of 400nM. 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.

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.

Phage display-directed discovery of LEDGF/p75 binding cyclic peptide inhibitors of HIV replication.

The interaction between the human immunodeficiency virus (HIV) integrase (IN) and its cellular cofactor lens epithelium-derived growth factor (LEDGF/p75) is crucial for HIV replication. While recently discovered LEDGINs inhibit HIV-1 replication by occupying the LEDGF/p75 pocket in IN, it remained to be demonstrated whether LEDGF/p75 by itself can be targeted. By phage display we identified cyclic peptides (CPs) as the first LEDGF/p75 ligands that inhibit the LEDGF/p75-IN interaction. The CPs inhibit HIV replication in different cell lines without overt toxicity. In accord with the role of LEDGF/p75 in HIV integration and its inhibition by LEDGINs, CP64, and CP65 block HIV replication primarily by inhibiting the integration step. The CPs retained activity against HIV strains resistant to raltegravir or LEDGINs. Saturation transfer difference (STD) NMR showed residues in CP64 that strongly interact with LEDGF/p75 but not with HIV IN. Mutational analysis identified tryptophan as an important residue responsible for the activity of the peptides. Serial passaging of virus in the presence of CPs did not yield resistant strains. Our work provides proof-of-concept for direct targeting of LEDGF/p75 as novel therapeutic strategy and the CPs thereby serve as scaffold for future development of new HIV therapeutics.

Discovery of small molecule HIV-1 integrase dimerization inhibitors.

Human immunodeficiency virus-1 integrase (HIV-1 IN) inserts the viral DNA into host cell chromatin in a multistep process. This enzyme exists in equilibrium between monomeric, dimeric, tetrameric and high order oligomeric states. However, monomers of IN are not capable of supporting its catalytic functions and the active form has been shown to be at least a dimer. As a consequence, the development of inhibitors targeting IN dimerization constitutes a promising novel antiviral strategy. In this work, we successfully combined different computational techniques in order to identify small molecule inhibitors of IN dimerization. Additionally, a novel AlphaScreen-based IN dimerization assay was used to evaluate the inhibitory activities of the selected compounds. To the best of our knowledge, this study represents the first successful virtual screening and evaluation of small molecule HIV-1 IN dimerization inhibitors, which may serve as attractive hit compounds for the development of novel anti-HIV.

Virtual screening for natural product inhibitors of HIV-1 integrase.

Since there is no human homolog of this enzyme, HIV-1 integrase (IN) represents a rational and important target for treating HIV infection and preventing AIDS. The 3D structure of full-length HIV-1 IN, either separately or in complex with its inhibitors, has been lacking. Thus, scarce information about the interactions between the HIV-1 IN and its inhibitors can be referenced. To more rationally design potent HIV-1 IN inhibitors, we have previously constructed a model of the full-length HIV-1 IN tetramer and a model of the protein-viral DNA complex, as well as the pharmacophore model of HIV-1 IN strand transfer inhibitors (INSTIs). In this paper, the pharmacophore model of INSTIs was used as a 3D query to screen the Traditional Chinese Medicine Database (TCMD). The hit compounds were further filtered by Lipinski's Rule of Five and docking study to refine the retrieved hits. Finally, 9 suitable ligands with similar structures belonging to the thioglycosides were selected. Subsequent molecular dynamics simulation showed that these compounds had interactions with HIV-1 IN binding site and their possible function as IN inhibitors was discussed.

Immunoregulatory and anti-HIV-1 enzyme activities of antioxidant components from lotus (Nelumbo nucifera Gaertn.) rhizome.

In the present study, two antioxidant micromolecular components (L2f-2 and L2f-3) and an antioxidant macromolecular component LB2 were extracted from lotus (Nelumbo nucifera Gaertn.) rhizomes. MS, FTIR (Fourier-transform IR) spectroscopy and NMR were used to identify these compounds. L2f-2 was (+/-)-gallocatechin, L2f-3 was (-)-catechin and LB2 was a polysaccharide-protein complex with a molecular mass of 18.8 kDa. LB2 was identified as a polysaccharide sulfate containing α/ß-pyranose and α-furanose according to its FTIR spectrogram. It was composed of mannose, rhamnose, glucose, galactose and xylose with a molar ratio 2:8:7:8:1. The antioxidant components L2f-2, L2f-3 and LB2 strongly inhibited HIV-1 RT (reverse transcriptase) and IN (integrase). LB2 inhibited RT with an IC50 value of 33.7 µM. It also exhibited the highest HIV-1 3'-processing inhibitory activity with an IC50 value of 5.28 µM. Both L2f-2 and L2f-3 up-regulated the expression of IL-2 (interleukin-2) and down-regulated IL-10, while LB2 exhibited positive regulation on IL-2, IL-4 and IL-10. Moreover, L2f-3 and LB2 might inhibit HIV-1 directly by down-regulating TNFα (tumour necrosis factor α). These natural antioxidant components with antiviral and immunoregulatory activities could be potentially important for anti HIV-1 drug development and application to HIV-1 therapy.

Solution conformation and dynamics of the HIV-1 integrase core domain.

The human immunodeficiency virus type 1 (HIV-1) integrase (IN) is a critical enzyme involved in infection. It catalyzes two reactions to integrate the viral cDNA into the host genome, 3' processing and strand transfer, but the dynamic behavior of the active site during catalysis of these two processes remains poorly characterized. NMR spectroscopy can reveal important structural details about enzyme mechanisms, but to date the IN catalytic core domain has proven resistant to such an analysis. Here, we present the first NMR studies of a soluble variant of the catalytic core domain. The NMR chemical shifts are found to corroborate structures observed in crystals, and confirm prior studies suggesting that the alpha4 helix extends toward the active site. We also observe a dramatic improvement in NMR spectra with increasing MgCl(2) concentration. This improvement suggests a structural transition not only near the active site residues but also throughout the entire molecule as IN binds Mg(2+). In particular, the stability of the core domain is linked to the conformation of its C-terminal helix, which has implications for relative domain orientation in the full-length enzyme. (15)N relaxation experiments further show that, although conformationally flexible, the catalytic loop of IN is not fully disordered in the absence of DNA. Indeed, automated chemical shift-based modeling of the active site loop reveals several stable clusters that show striking similarity to a recent crystal structure of prototype foamy virus IN bound to DNA.

Mutations associated with failure of raltegravir treatment affect integrase sensitivity to the inhibitor in vitro.

Raltegravir (MK-0518) is a potent inhibitor of human immunodeficiency virus (HIV) integrase and is clinically effective against viruses resistant to other classes of antiretroviral agents. However, it can select mutations in the HIV integrase gene. Nine heavily pretreated patients who received salvage therapy including raltegravir and who subsequently developed virological failure under raltegravir therapy were studied. For each patient, the sequences of the integrase-coding region were determined and compared to that at the beginning of the treatment. Four different mutation profiles were identified in these nine patients: E92Q, G140S Q148H, N155H, and E157Q mutations. For four patients, each harboring a different profile, the wild-type and mutated integrases were produced, purified, and assayed in vitro. All the mutations identified altered the activities of integrase protein: both 3' processing and strand transfer activities were moderately affected in the E92Q mutant; strand transfer was markedly impaired in the N155H mutant; both activities were strongly impaired in the G140S Q148H mutant; and the E157Q mutant was almost completely inactive. The sensitivities of wild-type and mutant integrases to raltegravir were compared. The E92Q and G140S Q148H profiles were each associated with a 7- to 8-fold decrease in sensitivity, and the N155H mutant was more than 14-fold less sensitive to raltegravir. At least four genetic profiles (E92Q, G140S Q148H, N155H, and E157Q) can be associated with in vivo treatment failure and resistance to raltegravir. These mutations led to strong impairment of enzymes in vitro in the absence of raltegravir: strand transfer activity was affected, and in some cases 3' processing was also impaired.