Pyridoxine hydroxamic acids as novel HIV-integrase inhibitors.

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.

Biochemical properties of the xenotropic murine leukemia virus-related virus integrase.

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 âˆ¼ 30 nM) whereas dolutegravir and elvitegravir were less effective (IC50 âˆ¼ 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.

Impact of the Ku complex on HIV-1 expression and latency.

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.

A protein ballet around the viral genome orchestrated by HIV-1 reverse transcriptase leads to an architectural switch: from nucleocapsid-condensed RNA to Vpr-bridged DNA.

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.

In Silico and In Vitro Comparison of HIV-1 Subtypes B and CRF02_AG Integrases Susceptibility to Integrase Strand Transfer Inhibitors.

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.

3' self-inactivating long terminal repeat inserts for the modulation of transgene expression from lentiviral vectors.

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.

Long-lasting persistence of integrase resistance mutations in HIV-2-infected patients after raltegravir withdrawal.

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.

G140S/Q148R and N155H mutations render HIV-2 Integrase resistant to raltegravir whereas Y143C does not.

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.

Extracellular ATP acts on P2Y2 purinergic receptors to facilitate HIV-1 infection.

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.

Ethyl malonate amides: a diketo acid offspring fragment for HIV integrase inhibition.

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.

Synthesis, biological evaluation and molecular modeling studies of quinolonyl diketo acid derivatives: new structural insight into the HIV-1 integrase inhibition.

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.

Hot spots of integrase genotypic changes leading to HIV-2 resistance to raltegravir.

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.

Structure-Activity Relationship Studies of HIV-1 Integrase Oligonucleotide Inhibitors.

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.

Chemistry and structure-activity relationship of the styrylquinoline-type HIV integrase inhibitors.

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.

New 2-arylnaphthalenediols and triol inhibitors of HIV-1 integrase--discovery of a new polyhydroxylated antiviral agent.

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.

Resistance to HIV-1 integrase inhibitors: A structural perspective.

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.

A cooperative and specific DNA-binding mode of HIV-1 integrase depends on the nature of the metallic cofactor and involves the zinc-containing N-terminal domain.

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.

Impact of Y143 HIV-1 integrase mutations on resistance to raltegravir in vitro and in vivo.

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.

Raltegravir: molecular basis of its mechanism of action.

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.

Synthesis and anti-HIV-1 integrase activity of modified dinucleotides.

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.