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A series of pyridoxine hydroxamic acid analog bearing a 5-aryl-spacers were synthesized. Evaluation of these novel HIV integrase complex inhibitors revealed compounds with high potency against wild-type HIV virus., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Xenotropic Murine Leukemia Virus-related Virus (XMRV) is a new gammaretrovirus generated by genetic recombination between two murine endogenous retroviruses, PreXMRV1 and PreXMRV2, during passaging of human prostate cancer xenografts in laboratory mice. XMRV is representative of an early founder virus that jumps species from mouse to human cell lines. Relatively little information is available concerning the XMRV integrase (IN), an enzyme that catalyzes a key stage in the retroviral cycle, and whose sequence is conserved among replication competent retroviruses emerging from recombination between the murine endogenous PreXMRV-1 and PreXMRV-2 genomes. Previous studies have shown that IN inhibitors efficiently block XMRV multiplication in cells. We thus aimed at characterizing the biochemical properties and sensitivity of the XMRV IN to the raltegravir, dolutegravir, 118-D-24 and elvitegravir inhibitors in vitro. We report for the first time the purification and enzymatic characterization of recombinant XMRV IN. This IN, produced in Escherichia coli and purified under native conditions, is optimally active over a pH range of 7-8.5, in the presence of Mg(2+) (15 mM and 30 mM for 3'-processing and strand transfer, respectively) and is poorly sensitive to the addition of dithiothreitol. Raltegravir was shown to be a very potent inhibitor (IC50 ∼ 30 nM) whereas dolutegravir and elvitegravir were less effective (IC50 ∼ 230 nM and 650 nM, respectively). The 118-D-24 drug had no impact on XMRV IN activity. Interestingly, the substrate specificity of XMRV IN seems to be less marked compared to HIV-1 IN since XMRV IN is able to process various donor substrates that share little homology. Finally, our analysis revealed some original properties of the XMRV IN such as its relatively low sequence specificity., (Copyright © 2014 Elsevier B.V. and Société française de biochimie et biologie Moléculaire (SFBBM). All rights reserved.)

Ku, a cellular complex required for human cell survival and involved in double strand break DNA repair and multiple other cellular processes, may modulate retroviral multiplication, although the precise mechanism through which it acts is still controversial. Recently, Ku was identified as a possible anti-human immunodeficiency virus type 1 (HIV-1) target in human cells, in two global approaches. Here we investigated the role of Ku on the HIV-1 replication cycle by analyzing the expression level of a panel of non-replicative lentiviral vectors expressing the green fluorescent protein in human colorectal carcinoma HCT 116 cells, stably or transiently depleted of Ku. We found that in this cellular model the depletion of Ku did not affect the efficiency of (pre-)integrative steps but decreased the early HIV-1 expression by acting at the transcriptional level. This negative effect was specific of the HIV-1 promoter, required the obligatory step of viral DNA integration and was reversed by transient depletion of p53. We also provided evidence on a direct binding of Ku to HIV-1 LTR in transduced cells. Ku not only promotes the early transcription from the HIV-1 promoter, but also limits the constitution of viral latency. Moreover, in the presence of a normal level of Ku, HIV-1 expression was gradually lost over time, likely due to the counter-selection of HIV-1-expressing cells. On the contrary, the reactivation of transgene expression from HIV-1 by means of trichostatin A- or tumor necrosis factor α-administration was enhanced under condition of Ku haplodepletion, suggesting a phenomenon of provirus latency. These observations plead in favor of the hypothesis that Ku has an impact on HIV-1 expression and latency at early- and mid-time after integration.

HIV-1 reverse transcription is achieved in the newly infected cell before viral DNA (vDNA) nuclear import. Reverse transcriptase (RT) has previously been shown to function as a molecular motor, dismantling the nucleocapsid complex that binds the viral genome as soon as plus-strand DNA synthesis initiates. We first propose a detailed model of this dismantling in close relationship with the sequential conversion from RNA to double-stranded (ds) DNA, focusing on the nucleocapsid protein (NCp7). The HIV-1 DNA-containing pre-integration complex (PIC) resulting from completion of reverse transcription is translocated through the nuclear pore. The PIC nucleoprotein architecture is poorly understood but contains at least two HIV-1 proteins initially from the virion core, namely integrase (IN) and the viral protein r (Vpr). We next present a set of electron micrographs supporting that Vpr behaves as a DNA architectural protein, initiating multiple DNA bridges over more than 500 base pairs (bp). These complexes are shown to interact with NCp7 bound to single-stranded nucleic acid regions that are thought to maintain IN binding during dsDNA synthesis, concurrently with nucleocapsid complex dismantling. This unexpected binding of Vpr conveniently leads to a compacted but filamentous folding of the vDNA that should favor its nuclear import. Finally, nucleocapsid-like aggregates engaged in dsDNA synthesis appear to efficiently bind to F-actin filaments, a property that may be involved in targeting complexes to the nuclear envelope. More generally, this article highlights unique possibilities offered by in vitro reconstitution approaches combined with macromolecular imaging to gain insights into the mechanisms that alter the nucleoprotein architecture of the HIV-1 genome, ultimately enabling its insertion into the nuclear chromatin., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Gene transfer for research or gene therapy requires the design of vectors that allow for adequate and safe transgene expression. Current methods to modulate the safety and expression profile of retroviral vectors can involve the insertion of insulators or scaffold/matrix-attachment regions in self-inactivating long terminal repeats (SIN-LTRs). Here, we generated a set of lentiviral vectors (with internal CMV or PGK promoter) in which we inserted (at the level of SIN-LTRs) sequences of avian (i.e., chicken hypersensitive site-4, cHS4), human (i.e., putative insulator and desert sequence), or bacterial origin. We characterized them with respect to viral titer, integration, transduction efficiency and transgene expression levels, in both integrase-proficient and -deficient contexts. We found that the cHS4 insulator enhanced transgene expression by a factor of 1.5 only when cloned in the antisense orientation. On the other hand, cHS4 in the sense orientation as well as all other inserts decreased transgene expression. This attenuation phenomenon persisted over long periods of time and did not correspond to extinction or variegation. Decreased transgene expression was associated with lower mRNA levels, yet RNA stability was not affected. Insertions within the SIN-LTRs may negatively affect transgene transcription in a direct fashion through topological rearrangements. The lentiviral vectors that we generated constitute valuable genetic tools for manipulating the level of transgene expression. Moreover, this study demonstrates that SIN-LTR inserts can decrease transgene expression, a phenomenon that might be overcome by modifying insert orientation, thereby highlighting the importance of careful vector design for gene therapy.

Most antiretroviral medical treatments were developed and tested principally on HIV-1 B nonrecombinant strain, which represents less than 10% of the worldwide HIV-1-infected population. HIV-1 circulating recombinant form CRF02_AG is prevalent in West Africa and is becoming more frequent in other countries. Previous studies suggested that the HIV-1 polymorphisms might be associated to variable susceptibility to antiretrovirals. This study is pointed to compare the susceptibility to integrase (IN) inhibitors of HIV-1 subtype CRF02_AG IN respectively to HIV-1 B. Structural models of B and CRF02_AG HIV-1 INs as unbound enzymes and in complex with the DNA substrate were built by homology modeling. IN inhibitors-raltegravir (RAL), elvitegravir (ELV) and L731,988-were docked onto the models, and their binding affinity for both HIV-1 B and CRF02_AG INs was compared. CRF02_AG INs were cloned and expressed from plasma of integrase strand transfer inhibitor (INSTI)-naïve infected patients. Our in silico and in vitro studies showed that the sequence variations between the INs of CRF02_AG and B strains did not lead to any notable difference in the structural features of the enzyme and did not impact the susceptibility to the IN inhibitors. The binding modes and affinities of INSTI inhibitors to B and CRF02_AG INs were found to be similar. Although previous studies suggested that several naturally occurring variations of CRF02_AG IN might alter either IN/vDNA interactions or INSTIs binding, our study demonstrate that these variations do affect neither IN activity nor its susceptibility to INSTIs.

Extracellular adenosine triphosphate (ATP) can activate purinergic receptors of the plasma membrane and modulate multiple cellular functions. We report that ATP is released from HIV-1 target cells through pannexin-1 channels upon interaction between the HIV-1 envelope protein and specific target cell receptors. Extracellular ATP then acts on purinergic receptors, including P2Y2, to activate proline-rich tyrosine kinase 2 (Pyk2) kinase and transient plasma membrane depolarization, which in turn stimulate fusion between Env-expressing membranes and membranes containing CD4 plus appropriate chemokine co-receptors. Inhibition of any of the constituents of this cascade (pannexin-1, ATP, P2Y2, and Pyk2) impairs the replication of HIV-1 mutant viruses that are resistant to conventional antiretroviral agents. Altogether, our results reveal a novel signaling pathway involved in the early steps of HIV-1 infection that may be targeted with new therapeutic approaches., (© 2011 Séror et al.)

Background: HIV-2 is endemic in West Africa and has spread throughout Europe. However, the alternatives for HIV-2-infected patients are more limited than for HIV-1. Raltegravir, an integrase inhibitor, is active against wild-type HIV-2, with a susceptibility to this drug similar to that of HIV-1, and is therefore a promising option for use in the treatment of HIV-2-infected patients. Recent studies have shown that HIV-2 resistance to raltegravir involves one of three resistance mutations, N155H, Q148R/H and Y143C, previously identified as resistance determinants in the HIV-1 integrase coding sequence. The resistance of HIV-1 IN has been confirmed in vitro for mutated enzymes harboring these mutations, but no such confirmation has yet been obtained for HIV-2., Results: The integrase coding sequence was amplified from plasma samples collected from ten patients infected with HIV-2 viruses, of whom three RAL-naïve and seven on RAL-based treatment at the time of virological failure. The genomes of the resistant strains were cloned and three patterns involving N155H, G140S/Q148R or Y143C mutations were identified. Study of the susceptibility of integrases, either amplified from clinical isolates or obtained by mutagenesis demonstrated that mutations at positions 155 and 148 render the integrase resistant to RAL. The G140S mutation conferred little resistance, but compensated for the catalytic defect due to the Q148R mutation. Conversely, Y143C alone did not confer resistance to RAL unless E92Q is also present. Furthermore, the introduction of the Y143C mutation into the N155H resistant background decreased the resistance level of enzymes containing the N155H mutation., Conclusion: This study confirms that HIV-2 resistance to RAL is due to the N155H, G140S/Q148R or E92Q/Y143C mutations. The N155H and G140S/Q148R mutations make similar contributions to resistance in both HIV-1 and HIV-2, but Y143C is not sufficient to account for the resistance of HIV-2 genomes harboring this mutation. For Y143C to confer resistance in vitro, it must be accompanied by E92Q, which therefore plays a more important role in the HIV-2 context than in the HIV-1 context. Finally, the Y143C mutation counteracts the resistance conferred by the N155H mutation, probably accounting for the lack of detection of these mutations together in a single genome.

While searching for new HIV integrase inhibitors we discovered that some ethyl malonate amides (EMA) are active against this enzyme. Surprisingly, the main function can only very rarely be found among the reported drug candidates. We synthesised a series of compounds in order to establish and analyse the structure-activity relationship. The similarity to the important classes of HIV integrase inhibitors as well as the synthetic availability of the different targets including this pharmacophore makes EMA compounds an interesting object of investigations., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

New quinolonyl diketo acid compounds bearing various substituents at position 6 of the quinolone scaffold were designed and synthesized as potential HIV-1 integrase inhibitors. These new compounds were evaluated for their antiviral and anti-integrase activity and showed inhibitory potency similar to that of 6-bromide analog 2. Molecular modeling and docking studies were performed to rationalize these data and to provide a detailed understanding of the mechanism of inhibition for this class of compounds., (Copyright © 2011 Elsevier Masson SAS. All rights reserved.)

Integration of human immunodeficiency virus type 1 DNA into an infected cell genome is one of the key steps of the viral replication cycle. Therefore, viral enzyme integrase, which realizes the integration, represents an attractive and validated target for the development of new antiviral drugs. In this paper, the anti-integrase activity of a series of conjugates of single-stranded oligonucleotides with hydrophobic molecules was tested, and the structure-activity relationships were also analyzed. Both oligonucleotide and hydrophobic parts of the conjugates influenced the inhibitory potency. Conjugates of 11-mer phosphorothioate oligonucleotides with 6-carboxy-4,7,2',4',5',7'-hexachlorofluorescein (HEX) were found to be the most efficient inhibitors (IC50 = 20 nM) and might be considered as lead compounds for further development of integrase inhibitors.

We studied seven heavily pretreated HIV-2-infected patients exhibiting a virological failure while receiving a salvage raltegravir-containing regimen. At the time of virological failure, different resistance genetic pathways were observed: T97A-Y143C, Q148K, Q148R, G140S-Q148R, E92Q-Y143R-N155H, and T97A-N155H. Thus, despite a 40% difference in integrase genes between HIV-1 and HIV-2, the genetic pathways leading to raltegravir resistance are similar.

Background: Little is known in HIV-2 infection about the kinetics of disappearance of raltegravir (RAL)-resistant virus after RAL withdrawal., Methods: RAL was interrupted in four highly antiretroviral-experienced HIV-2-infected patients exhibiting a virological failure when receiving RAL. Integrase gene was sequenced from plasma samples collected at the time of RAL failure and at further time points following RAL withdrawal., Results: At the time of RAL withdrawal, virus exhibited different integrase resistance pathways: G140S/Q148R, E92Q/N155H, T97A/N155H and T97A/Y143C. In patient 1, the G140S/Q148R double-mutant was still detected at month (M)7 and at M11 after stopping RAL, but was no longer detected at M15. Regarding patient 2, the double-mutant E92Q/N155H was still present at M2 and M8 after stopping RAL, and was no longer detected at M12. In patient 3, RAL-resistant virus with T97A/N155H mutations were still present 1 month after stopping RAL, and were no longer detected at M14. Regarding patient 4, the mutant T97A/Y143C was still detected at M1 and M3 following RAL withdrawal. At M18 after RAL stop, integrase genotypic pattern evolved to T97A/Y143G., Conclusions: Persistence of HIV-2 RAL-resistant mutants was observed in all the key genetic RAL resistance pathways. These findings have clinical implications especially in HIV-2-infected patients for whom therapeutic arsenal is limited compared to HIV-1, since the persistence of resistant mutants might compromise the possible efficacy of upcoming second-generation integrase inhibitors., (© 2011 International Medical Press)

Strand-transfer inhibitors, of which raltegravir, elvitegravir and S/GSK1349572, is a new class of antiretrovirals that inhibit HIV integrase-catalyzed insertion of the HIV-1 genome into cell chromosomes. The results of clinical trials were very encouraging regarding their viral efficiency and tolerance. However resistance mutations were identified in patients failing to respond to treatment with these inhibitors, involving primary mutations as well as numerous secondary mutations. This review focuses on recent advanced computational studies that have highlighted the contribution of those residues subject to primary mutations and the role of conformational flexibility of the enzyme in binding to strand-transfer inhibitors., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

A series of 13 hydroxylated 2-arylnaphthalenes have been synthesized and evaluated as HIV-1 integrase inhibitors. 7-(3,4,5-trihydroxyphenyl)naphthalene-1,2,3-triol 1c revealed chemical instability upon storage, leading to the isolation of a dimer 5c which was also tested. In the 2-arylnaphthalene series, all compounds were active against HIV-1 IN with IC50's within the 1-10 microM range, except for 1c and 5c which displayed submicromolar activity. Antiviral activity against HIV-1 replication was measured on 1b-c and 5c. Amongst the tested molecules, only 5c was found to present antiviral properties with a low cytotoxicity on two different cell lines., (Copyright (c) 2010 Elsevier Ltd. All rights reserved.)

HIV-1 integrase catalyzes the insertion of the viral genome into chromosomal DNA. We characterized the structural determinants of the 3'-processing reaction specificity--the first reaction of the integration process--at the DNA-binding level. We found that the integrase N-terminal domain, containing a pseudo zinc-finger motif, plays a key role, at least indirectly, in the formation of specific integrase-DNA contacts. This motif mediates a cooperative DNA binding of integrase that occurs only with the cognate/viral DNA sequence and the physiologically relevant Mg(2+) cofactor. The DNA-binding was essentially non-cooperative with Mn(2+) or using non-specific/random sequences, regardless of the metallic cofactor. 2,2'-Dithiobisbenzamide-1 induced zinc ejection from integrase by covalently targeting the zinc-finger motif, and significantly decreased the Hill coefficient of the Mg(2+)-mediated integrase-DNA interaction, without affecting the overall affinity. Concomitantly, 2,2'-dithiobisbenzamide-1 severely impaired 3'-processing (IC(50) = 11-15 nM), suggesting that zinc ejection primarily perturbs the nature of the active integrase oligomer. A less specific and weaker catalytic effect of 2,2'-dithiobisbenzamide-1 is mediated by Cys 56 in the catalytic core and, notably, accounts for the weaker inhibition of the non-cooperative Mn(2+)-dependent 3'-processing. Our data show that the cooperative DNA-binding mode is strongly related to the sequence-specific DNA-binding, and depends on the simultaneous presence of the Mg(2+) cofactor and the zinc effector.

In spite of significant progress in anti-HIV-1 therapy, current antiviral chemo-therapy still suffers from deleterious side effects and emerging drug resistance. Therefore, the development of novel antiviral drugs remains a crucial issue for the fight against AIDS. HIV-1 integrase is a key enzyme in the replication cycle of the retrovirus since it catalyzes the integration of the reverse transcribed viral DNA into the chromosomal DNA. Efforts to develop anti-integrase drugs started during the early nineties, culminating with the recent approval of Raltegravir. The discovery and the development of the styrylquinoline inhibitor class was an important step in the overall process. In this review we have described the key synthetic issues and the structure-activity relationship of this family of integrase inhibitors. Crystallographic and docking studies that shed light on their mechanism of action are also examined.

Integrase (IN), the HIV-1 enzyme responsible for the integration of the viral genome into the chromosomes of infected cells, is the target of the recently approved antiviral raltegravir (RAL). Despite this drug's activity against viruses resistant to other antiretrovirals, failures of raltegravir therapy were observed, in association with the emergence of resistance due to mutations in the integrase coding region. Two pathways involving primary mutations on residues N155 and Q148 have been characterized. It was suggested that mutations at residue Y143 might constitute a third primary pathway for resistance. The aims of this study were to investigate the susceptibility of HIV-1 Y143R/C mutants to raltegravir and to determine the effects of these mutations on the IN-mediated reactions. Our observations demonstrate that Y143R/C mutants are strongly impaired for both of these activities in vitro. However, Y143R/C activity can be kinetically restored, thereby reproducing the effect of the secondary G140S mutation that rescues the defect associated with the Q148R/H mutants. A molecular modeling study confirmed that Y143R/C mutations play a role similar to that determined for Q148R/H mutations. In the viral replicative context, this defect leads to a partial block of integration responsible for a weak replicative capacity. Nevertheless, the Y143 mutant presented a high level of resistance to raltegravir. Furthermore, the 50% effective concentration (EC(50)) determined for Y143R/C mutants was significantly higher than that obtained with G140S/Q148R mutants. Altogether our results not only show that the mutation at position Y143 is one of the mechanisms conferring resistance to RAL but also explain the delayed emergence of this mutation.

The synthesis of a series of thirty-eight new modified dinucleotides and dinucleotide conjugate analogues of d-(5')ApC(3') is described. The inhibitory activity of these compounds toward HIV-1 integrase was examined in enzymatic assays using the natural dinucleotide as a reference. Among the compounds, a perylene-dinucleotide conjugate has shown a two micromolar anti-integrase activity due to the presence of both the intercalator and the dinucleotide.

Integration of the HIV-1 viral DNA generated by reverse transcription of the RNA genome into the host cell chromosomes is a key step of viral replication, catalyzed by the viral integrase. In October 2007, the first integrase inhibitor, raltegravir, was approved for clinical use under the name of Isentress superset. The results of the various clinical trials that have evaluated raltegravir have been very encouraging with regard to the immunological and virological efficacy and tolerance. However, as observed for other anti-retrovirals, specific resistance mutations have been identified in patients failing to respond to treatment with raltegravir. Although knowledge of the integrase structural biology remains fragmentary, the structures and modeling data available might provide relevant clues on the origin of the emergence of these resistance mutations. In this review, we describe the mechanism of action of this drug and the main data relating to its use in vivo, together with recent structural data important to our understanding of the origin of viral resistance.

Virologic failure during treatment with raltegravir, the first effective drug targeting HIV integrase, is associated with two exclusive pathways involving either Q148H/R/K, G140S/A or N155H mutations. We carried out a detailed analysis of the molecular and structural effects of these mutations. We observed no topological change in the integrase core domain, with conservation of a newly identified Omega-shaped hairpin containing the Q148 residue, in particular. In contrast, the mutations greatly altered the specificity of DNA recognition by integrase. The native residues displayed a clear preference for adenine, whereas the mutant residues strongly favored pyrimidines. Raltegravir may bind to N155 and/or Q148 residues as an adenine bioisoster. This may account for the selected mutations impairing raltegravir binding while allowing alternative DNA recognition by integrase. This study opens up new opportunities for the design of integrase inhibitors active against raltegravir-resistant viruses.

Ethyl [6-bromo-1-(4-fluorophenylmethyl)-4(1H)-quinolinon-3-yl]-4-hydroxy-2-oxo-3-butenoate 1 and [6-bromo-1-(4-fluorophenylmethyl)-4(1H)-quinolinon-3-yl)]-4-hydroxy-2-oxo-3-butenoïc acid 2 were synthesized as potential HIV-1 integrase inhibitors and evaluated for their enzymatic and antiviral activity, acidic compound 2 being more potent than ester compound 1. X-ray diffraction analyses and theoretical calculations show that the diketoacid chain of compound 2 is preferentially coplanar with the quinolinone ring (dihedral angle of 0-30 degrees ). Docking studies suggest binding modes in agreement with structure-activity relationships.

Integration of the human immunodeficiency virus (HIV-1) cDNA into the human genome is catalysed by integrase. Several studies have shown the importance of the interaction of cellular cofactors with integrase for viral integration and infectivity. In this study, we produced a stable and functional complex between the wild-type full-length integrase (IN) and the cellular cofactor LEDGF/p75 that shows enhanced in vitro integration activity compared with the integrase alone. Mass spectrometry analysis and the fitting of known atomic structures in cryo negatively stain electron microscopy (EM) maps revealed that the functional unit comprises two asymmetric integrase dimers and two LEDGF/p75 molecules. In the presence of DNA, EM revealed the DNA-binding sites and indicated that, in each asymmetric dimer, one integrase molecule performs the catalytic reaction, whereas the other one positions the viral DNA in the active site of the opposite dimer. The positions of the target and viral DNAs for the 3' processing and integration reaction shed light on the integration mechanism, a process with wide implications for the understanding of viral-induced pathologies.

Objectives: Raltegravir is the first approved inhibitor of HIV-1 integrase (IN). In most patients, raltegravir failure is associated with mutations in the IN gene, through two different genetic pathways: 155 (N155H) or 148 (Q148K/R/H). The objective of this study was to characterize the dynamics of HIV-1 quasispecies variant populations in patients who failed to respond to raltegravir treatment., Patients and Methods: Bulk genotyping and clonal analysis were performed during the follow-up of 10 patients who failed to respond to raltegravir treatment., Results: Treatment failed through the 155 pathway in six patients and through the 148 pathway in two patients; two further patients switched from the 155 to the 148 pathway. In the two patients switching from the 155 to the 148 pathway, clonal analysis showed that Q148R/H and N155H mutations were present on different strands, suggesting that these two pathways are independent. This was consistent with our finding that each genetic profile was associated with different secondary mutations. We observed a greater variability among quasispecies associated with the 155 pathway, and IC(50) determinations showed that the fold resistance to raltegravir, relative to wild-type, was 10 for the N155H mutant and 50 for the G140S+Q148H mutant., Conclusions: Clonal analysis strongly suggests that the two main genetic pathways, 155 and 148, involved in the development of resistance to raltegravir are independent and exclusive. Moreover, the switch of the resistance profile from 155 to 148 may be related to the higher level of resistance to raltegravir conferred by the 148 pathway and also to the higher instability of the 155 pathway.

Raltegravir (MK-0518) is the first integrase (IN) inhibitor to be approved by the US FDA and is currently used in clinical treatment of viruses resistant to other antiretroviral compounds. Virological failure of Raltegravir treatment is associated with mutations in the IN gene following two main distinct genetic pathways involving either the N155 or Q148 residue. Importantly, in most cases, an additional mutation at the position G140 is associated with the Q148 pathway. Here, we investigated the viral DNA kinetics for mutants identified in Raltegravir-resistant patients. We found that (i) integration is impaired for Q148H when compared with the wild-type, G140S and G140S/Q148H mutants; and (ii) the N155H and G140S mutations confer lower levels of resistance than the Q148H mutation. We also characterized the corresponding recombinant INs properties. Enzymatic performances closely parallel ex vivo studies. The Q148H mutation 'freezes' IN into a catalytically inactive state. By contrast, the conformational transition converting the inactive form into an active form is rescued by the G140S/Q148H double mutation. In conclusion, the Q148H mutation is responsible for resistance to Raltegravir whereas the G140S mutation increases viral fitness in the G140S/Q148H context. Altogether, these results account for the predominance of G140S/Q148H mutants in clinical trials using Raltegravir.

Background: HIV type-1 (HIV-1) integrase (IN) inhibitor resistance is the consequence of mutations that are selected in the viral IN gene targeted by antiretroviral drugs, such as raltegravir (RAL) and elvitegravir (EVG). The genetic barrier, defined as the number of viral mutations required to overcome the drug-selective pressure, is one of the important factors in the development of drug resistance. The genetic barrier for IN inhibitor resistance was compared between HIV-1 subtype B and HIV-1 subtype CRF02_AG, which is highly prevalent in West Africa and becoming more frequent in developed countries., Methods: IN nucleotide sequences from 73 HIV-1 subtype B and 77 HIV-1 subtype CRF02_AG antiretroviral-naive patients were examined at 19 IN amino acid positions implicated in RAL and EVG resistance., Results: The majority (14/19) of the studied positions showed a high degree of conservation of the predominant codon sequences leading to a similar genetic barrier between subtypes B and CRF02_AG. Nevertheless, at positions 140 and 151, the variability between subtypes affected the genetic barrier for the mutations G140C, G140S and V1511 with a higher genetic barrier being calculated for subtype CRF02_AG., Conclusions: The major IN mutations E92Q, Q148K/R/H, N155H and E157Q (implicated in the resistance of IN inhibitors RAL and EVG) are highly conserved between subtypes B and CRF02_AG and display a similar genetic barrier. However, subtype CRFO2_AG showed a higher genetic barrier to acquire mutations 6140S, 6140C and V1511 as compared with subtype B, which could play a role in the resistance to RAL and/or EV6.

We report herein the synthesis of a series of 19 2-hydroxyisoquinoline-1,3(2H,4H)-dione derivatives variously substituted at position 7 aimed at inhibiting selectively two-metal ion catalytic active sites. The compounds were tested against HIV-1 reverse transcriptase (RT) polymerase, HIV-1 RT ribonuclease H (RNase H), and HIV-1 integrase (IN). Most compounds displayed poor inhibition of RT polymerase even at 50 microM. The majority of the synthesized compounds inhibited RNase H and IN at micromolar concentrations, and some of them were weakly selective for IN. Surprisingly, two new hits were discovered, which displayed a high selectivity for IN with submicromolar IC50 values. These enzymatic inhibitory properties may be related to the metal binding abilities of the compounds. Physicochemical studies were consistent with a 1/1 stoichiometry of the magnesium complexes in solution, and the metal complexation was strictly dependent on the enolization abilities of the compounds. Unfortunately, all tested compounds exhibited high cellular cytotoxicity in cell culture which limits their applications as antiviral agents.

Integration of retroviral DNA is an obligatory step of retrovirus replication because proviral DNA is the template for productive infection. Integrase, a retroviral enzyme, catalyses integration. The process of integration can be divided into two sequential reactions. The first one, named 3'-processing, corresponds to a specific endonucleolytic reaction which prepares the viral DNA extremities to be competent for the subsequent covalent insertion, named strand transfer, into the host cell genome by a trans-esterification reaction. Recently, a novel specific activity of the full length integrase was reported, in vitro, by our group for two retroviral integrases (HIV-1 and PFV-1). This activity of internal cleavage occurs at a specific palindromic sequence mimicking the LTR-LTR junction described into the 2-LTR circles which are peculiar viral DNA forms found during viral infection. Moreover, recent studies demonstrated the existence of a weak palindromic consensus found at the integration sites. Taken together, these data underline the propensity of retroviral integrases for binding symmetrical sequences and give perspectives for targeting specific sequences used for gene therapy.

Integration catalyzed by integrase (IN) is a key process in the retrovirus life cycle. Many biochemical or structural human immunodeficiency virus, type 1 (HIV-1) IN studies have been severely impeded by its propensity to aggregate. We characterized a retroviral IN (primate foamy virus (PFV-1)) that displays a solubility profile different from that of HIV-1 IN. Using various techniques, including fluorescence correlation spectroscopy, time-resolved fluorescence anisotropy, and size exclusion chromatography, we identified a monomer-dimer equilibrium for the protein alone, with a half-transition concentration of 20-30 mum. We performed specific enzymatic labeling of PFV-1 IN and measured the fluorescence resonance energy transfer between carboxytetramethylrhodamine-labeled IN and fluorescein-labeled DNA substrates. FRET and fluorescence anisotropy highlight the preferential binding of PFV-1 IN to the 3'-end processing site. Sequence-specific DNA binding was not observed with HIV-1 IN, suggesting that the intrinsic ability of retroviral INs to bind preferentially to the processing site is highly underestimated in the presence of aggregates. IN is in a dimeric state for 3'-processing on short DNA substrates, whereas IN polymerization, mediated by nonspecific contacts at internal DNA positions, occurs on longer DNAs. Additionally, aggregation, mediated by nonspecific IN-IN interactions, occurs preferentially with short DNAs at high IN/DNA ratios. The presence of either higher order complex is detrimental for specific activity. Ionic strength favors catalytically competent over higher order complexes by selectively disrupting nonspecific IN-IN interactions. This counteracting effect was not observed with polymerization. The synergic effect on the selection of specific/competent complexes, obtained by using short DNA substrates under high salt conditions, may have important implications for further structural studies in IN.DNA complexes.

A successful synthesis of fukiic acid is described in 7% overall yield (6 steps from veratraldehyde). rac-Fukiic acid was found to be a potent inhibitor of HIV-1 integrase but did not reveal any antiviral activity in the MT-4 cells assay.

An efficient synthesis of the acid part of salvianolic acid E 2 is described. Compound 2 was obtained from vanillin in 10 steps and 21% overall yield. During the synthesis of 2 an unexpected 5-oxo-4b,9b-dihydroindano[1,2-b]benzofuran rac-12 was isolated. Both compounds together with the acid part of salvianolic acid D were active as HIV-1 integrase inhibitors at the submicromolar level. But they did not inhibit the replication of the virus on MT-4 cells.

A series of thirteen 4,5-diaryl-3-hydroxy-2(5H)-furanones were synthesized. They were evaluated for their antioxidant potencies and inhibitory properties of 5-lipoxygenase, cyclooxygenases, HIV-1 integrase and PC3 cell proliferation. New hits were discovered either in the anti-proliferation test or in the HIV anti-integrase test.

HIV-1 integrase is one of the three essential enzyme required for viral replication and has a great potential as a novel target for anti-HIV drugs. The sequence variability of the entire integrase (IN) was examined in HIV-1 subtype B and CRF02-AG antiretroviral naïve infected patients for the presence of naturally occurring polymorphisms IN gene sequences and protein structures from both subtypes were compared. The phylogenetic analysis showed a total concordance between the 3 pol gene sequences for patients identified as subtype B whereas 3% of patients identified as CRF02-AG showed a mixture of subtypes. The analysis of IN aa sequences showed that 13 positions (K/R14, V/I31, L/I101, T/V112, T/A124, T/A125, G/N134, I/V135, K/T136, V/I201, T/S206, L/I234, and S/G283) differed between subtypes B and CRF02-AG. As observed in the 3D model of the preintegration complex, these differences may impact the functional property of IN. The fact that most variations were grouped suggests that some of them are linked together through compensatory mechanisms. This comparison allowed us to identify several variations of amino acids in HIV-1 IN subtype CRF02-AG that could have a putative impact on anti-integrase sensitivity. In particular, the region formed by Thr125, Thr124, Val31 contains at least one residue, T125, which variation has been involved in eliciting resistance to the naphtyridine carboxamide L870,810 IN inhibitor. In conclusion, virological response to anti-integrase should be studied carefully, according to the subtype, in clinical trials.

Rosmarinic acid was reacted with nitrite ions under acidic conditions to give 6'-nitro- and 6',6''-dinitrorosmarinic acids according to the reaction time. Both compounds were active as HIV-1 integrase inhibitors at the submicromolar level. They also inhibited the viral replication in MT-4 cells with modest and similar selectivity indexes. The nitration of rosmarinic acid strongly improves the anti-integrase inhibition and the antiviral activity without increasing the cellular toxicity.

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.

Present studies were undertaken on the preparation of synthetic analogues of bis- or tetra-coumarins and their activity against HIV-1 integrase (HIV-1 IN). Among these coumarin analogues, compounds 14, 16 and 18 were found to be potent molecules against HIV-1 IN at IC50 values of 0.96, 0.58, and 0.49 microM, respectively. The results provided a tool for guiding the further design of more potent antiviral agents and for predicting the affinity of related compounds.

The heterodimer Ku70/80 Ku is the DNA-binding component of the DNA-PK complex required for the nonhomologous end-joining pathway. It participates in numerous nuclear processes, including telomere and chromatin structure maintenance, replication, and transcription. Ku interacts with retroviral preintegration complexes and is thought to interfere with the retroviral replication cycle, in particular the formation of 2-long terminal repeat (LTR) viral DNA circles, viral DNA integration, and transcription. We describe here the effect of Ku80 on both provirus integration and the resulting transgene expression in cells transduced with retroviral vectors. We found that transgene expression was systematically higher in Ku80-deficient xrs6 cells than in Ku80-expressing CHO cells. This higher expression was observed irrespective of the presence of the viral LTR and was also not related to the nature of the promoter. Real-time PCR monitoring of the early viral replicative steps demonstrated that the absence of Ku80 does not affect the efficiency of transduction. We analyzed the transgene distributions localization in nucleus by applying a three-dimensional reconstruction model to two-dimensional fluorescence in situ hybridization images. This indicated that the presence of Ku80 resulted in a bias toward the transgenes being located at the periphery of the nucleus associated with their being repressed; in the absence of this factor the transgenes tend to be randomly distributed and actively expressed. Therefore, although not strictly required for retroviral integration, Ku may be involved in targeting retroviral elements to chromatin domains prone to gene silencing.

Background: HIV-1 integrase (IN) catalyses the retroviral integration process, removing two nucleotides from each long terminal repeat and inserting the processed viral DNA into the target DNA. It is widely assumed that the strand transfer step has no sequence specificity. However, recently, it has been reported by several groups that integration sites display a preference for palindromic sequences, suggesting that a symmetry in the target DNA may stabilise the tetrameric organisation of IN in the synaptic complex., Methodology/principal Findings: We assessed the ability of several palindrome-containing sequences to organise tetrameric IN and investigated the ability of IN to catalyse DNA cleavage at internal positions. Only one palindromic sequence was successfully cleaved by IN. Interestingly, this symmetrical sequence corresponded to the 2-LTR junction of retroviral DNA circles-a palindrome similar but not identical to the consensus sequence found at integration sites. This reaction depended strictly on the cognate retroviral sequence of IN and required a full-length wild-type IN. Furthermore, the oligomeric state of IN responsible for this cleavage differed from that involved in the 3'-processing reaction. Palindromic cleavage strictly required the tetrameric form, whereas 3'-processing was efficiently catalysed by a dimer., Conclusions/significance: Our findings suggest that the restriction-like cleavage of palindromic sequences may be a general physiological activity of retroviral INs and that IN tetramerisation is strongly favoured by DNA symmetry, either at the target site for the concerted integration or when the DNA contains the 2-LTR junction in the case of the palindromic internal cleavage.

Catechol and beta-ketoenol are important pharmacophores of HIV-1 integrase (IN) inhibitors. We investigated their recognition of the divalent metals, Mg and Mn, and of hydrogen bond donors (HBD) and acceptors (HBA). We used data retrieved from the Cambridge Structural Database (CSD), applying a 3-D structure-based, in silico-driven approach. We found that both biophores were stabilized by intramolecular H-bonding (IHB), which was weak in catechols and very strong in beta-ketoenols. Catechols tended to recognize environmental HBD and HBA, demonstrating their ability to make use of both hydroxyl groups to form multiple, strong intermolecular H-bonds. In contrast, beta-ketoenols stabilized by strong IHB inefficiently formed intermolecular H-bonds. beta-Ketoenolate chelated both Mg and Mn ions much more efficiently than dioxolene, which was highly selective for Mn cations. The significant differences in the ability of these two pharmacophores to bind HBD and HBA and in their ability to chelate Mg and Mn have important consequences for competitive inhibitor binding and selectivity for metals and integrase DNA-binding sites.

Flavonoid compounds represent an important natural source of antiretrovirals for AIDS therapy due to their significant anti-HIV-1 activity and low toxicity. Here we propose a simple theoretical criterion to discriminate active from inactive flavonoids that is suitable for rapid in silico screening of flavonoid libraries, and selection and optimization of lead compounds with anti-HIV-1 activity.

A range of novel N-terminal lipid-functionalized peptide nucleic acid (PNA) monomers have been prepared and their abilities to inhibit HIV-1 reverse transcriptase and integrase have been examined.

We used comparative molecular surface analysis to design molecules for the synthesis as part of the search for new HIV-1 integrase inhibitors. We analyzed the virtual combinatorial library (VCL) constituted from various moieties of styrylquinoline and styrylquinazoline inhibitors. Since imines can be applied in a strategy of dynamic combinatorial chemistry (DCC), we also tested similar compounds in which the -C=N- or -N=C- linker connected the heteroaromatic and aromatic moieties. We then used principal component analysis (PCA) or self-organizing maps (SOM), namely, the Kohonen neural networks to obtain a clustering plot analyzing the diversity of the VCL formed. Previously synthesized compounds of known activity, used as molecular probes, were projected onto this plot, which provided a set of promising virtual drugs. Moreover, we further modified the above mentioned VCL to include the single bond linker -C-N- or -N-C-. This allowed increasing compound stability but expanded also the diversity between the available molecular probes and virtual targets. The application of the CoMSA with SOM indicated important differences between such compounds and active molecular probes. We synthesized such compounds to verify the computational predictions.

We previously reported that the stimulation of monocyte-derived macrophages (MDM) by plate-bound i.v. Igs inhibits HIV-1 replication. In this study, we show that IgG immune complexes also suppress HIV-1 replication in MDMs and that activating receptors for the Fc portion of IgG-FcgammaRI, FcgammaRIIA, and FcgammaRIII-are responsible for the inhibition. MDM stimulation through FcgammaRs induces activation signals and the secretion of HIV-1 modulatory cytokines, such as M-CSF, TNF-alpha, and macrophage-derived chemokine. However, none of these cytokines contribute to HIV-1 suppression. HIV-1 entry and postintegration steps of viral replication are not affected, whereas reduced levels of reverse transcription products and of integrated proviruses, as determined by real-time PCR analysis, account for the suppression of HIV-1 gene expression in FcgammaR-activated MDMs. We found that FcgammaR-dependent activation of MDMs also inhibits the replication of HIV-2, SIVmac, and SIVagm, suggesting a common control mechanism for primate immunodeficiency lentiviruses in activated macrophages.

To replicate, human immunodeficiency virus, type 1 (HIV-1) needs to integrate a cDNA copy of its RNA genome into a chromosome of the host cell, a step controlled by the viral integrase (IN) protein. Viral integration involves the participation of several cellular proteins. SNF5/Ini1, a subunit of the SWI/SNF chromatin remodeling complex, was the first cofactor identified to interact with IN. We report here that SNF5/Ini1 interferes with early steps of HIV-1 replication. Inhibition of SNF5/Ini1 expression by RNA interference increases HIV-1 replication. Using quantitative PCR, we show that both the 2-long terminal repeat circle and integrated DNA forms accumulate upon SNF5/Ini1 knock down. By yeast two-hybrid assay, we screened a library of HIV-1 IN random mutants obtained by PCR random mutagenesis using SNF5/Ini1 as prey. Two different mutants of interaction, IN E69G and IN K71R, were impaired for SNF5/Ini1 interaction. The E69G substitution completely abolished integrase catalytic activity, leading to a replication-defective virus. On the contrary, IN K71R retained in vitro integrase activity. K71R substitution stimulates viral replication and results in higher infectious titers. Taken together, these results suggest that, by interacting with IN, SNF5/Ini1 interferes with early steps of HIV-1 infection.

The 3'-processing of the extremities of viral DNA is the first of two reactions catalyzed by HIV-1 integrase (IN). High order IN multimers (tetramers) are required for complete integration, but it remains unclear which oligomer is responsible for the 3'-processing reaction. Moreover, IN tends to aggregate, and it is unknown whether the polymerization or aggregation of this enzyme on DNA is detrimental or beneficial for activity. We have developed a fluorescence assay based on anisotropy for monitoring release of the terminal dinucleotide product in real-time. Because the initial anisotropy value obtained after DNA binding and before catalysis depends on the fractional saturation of DNA sites and the size of IN.DNA complexes, this approach can be used to study the relationship between activity and binding/multimerization parameters in the same assay. By increasing the IN:DNA ratio, we found that the anisotropy increased but the 3'-processing activity displayed a characteristic bell-shaped behavior. The anisotropy values obtained in the first phase were predictive of subsequent activity and accounted for the number of complexes. Interestingly, activity peaked and then decreased in the second phase, whereas anisotropy continued to increase. Time-resolved fluorescence anisotropy studies showed that the most competent form for catalysis corresponds to a dimer bound to one viral DNA end, whereas higher order complexes such as aggregates predominate during the second phase when activity drops off. We conclude that a single IN dimer at each extremity of viral DNA molecules is required for 3'-processing, with a dimer of dimers responsible for the subsequent full integration.

Nineteen biscoumarins bearing free and modified hydroxyl substituents at benzoyloxyphenyl linker have been synthesized by multiple step synthesis. Among these biscoumarins, thirteen were found to be active molecules against HIV-1 integrase (HIV-1 IN). The structure-activity relationship of the nineteen compounds on HIV IN may be useful for the design of potent therapeutic agents.

Following the discovery of diketoacid-containing compounds as HIV-1 integrase (IN) inhibitors, a plethora of new molecules have been published leading to four drugs under clinical trial. In an attempt to rationally design new dimeric diketoacids (DKAs) targeting two divalent metal ions on the active site of IN, potent inhibitors against purified IN were found with varied selectivity for strand transfer. In this context, we designed and synthesized a new series of catechol-DKA hybrids. These compounds presented micromolar anti-integrase activities with moderate antiviral properties.

The specific activity of the human immunodeficiency virus, type 1 (HIV-1), integrase on the viral long terminal repeat requires the binding of the enzyme to certain sequences located in the U3 and U5 regions at the ends of viral DNA, but the determinants of this specific DNA-protein recognition are not yet completely understood. We synthesized DNA duplexes mimicking the U5 region and containing either 2'-modified nucleosides or 1,3-propanediol insertions and studied their interactions with HIV-1 integrase, using Mn2+ or Mg2+ ions as integrase cofactors. These DNA modifications had no strong effect on integrase binding to the substrate analogs but significantly affected 3'-end processing rate. The effects of nucleoside modifications at positions 5, 6, and especially 3 strongly depended on the cationic cofactor used. These effects were much more pronounced in the presence of Mg2+ than in the presence of Mn2+. Modifications of base pairs 7-9 affected 3'-end processing equally in the presence of both ions. Adenine from the 3rd bp is thought to form at least two hydrogen bonds with integrase that are crucial for specific DNA recognition. The complementary base, thymine, is not important for integrase activity. For other positions, our results suggest that integrase recognizes a fine structure of the sugar-phosphate backbone rather than heterocyclic bases. Integrase interactions with the unprocessed strand at positions 5-8 are more important than interactions with the processed strand for specific substrate recognition. Based on our results, we suggest a model for integrase interaction with the U5 substrate.