Showing papers on "Integrase published in 2013"

Safety and Efficacy of Dolutegravir in Treatment-Experienced Subjects With Raltegravir-Resistant HIV Type 1 Infection: 24-Week Results of the VIKING Study

Abstract: Integrase inhibitors (INIs) represent a class of drugs for the treatment of human immunodeficiency virus (HIV)–infected individuals, blocking HIV genome integration into the host cell DNA [1]. They have been shown to be highly effective for the treatment of antiretroviral-naive and antiretroviral-experienced subjects, as demonstrated first with raltegravir (RAL) and more recently with elvitegravir (EVG) [2–6]. However, these first-generation INIs share common resistance pathways. In clinical studies of RAL, subjects with virologic failure and reduced RAL susceptibility typically harbored virus with 1 of 3 signature mutational pathways (ie, N155H, Q148H/K/R, or Y143C/H/R) in the integrase gene [7]. Continuing RAL treatment in these circumstances may lead to the addition of secondary mutations or pathway evolution; N155H may evolve to Y143 or Q148 pathways [4]. In addition, EVG does not appear to have activity against RAL-resistant isolates, and RAL does not appear to have activity against EVG-resistant isolates [8–10]. Therefore, there is a need for an INI with a high barrier to resistance and activity in subjects with human immunodeficiency virus type 1 (HIV-1) resistant to EVG and RAL. Dolutegravir (DTG) is a new HIV-1 INI that has demonstrated good efficacy and safety in treatment-naive, HIV-infected individuals [11]. In vitro studies demonstrate limited cross-resistance between DTG and RAL or EVG, with no or minimal impact on DTG fold-change against Q148 single mutants or against viruses with Y143 or N155 signature mutations regardless of RAL-associated secondary mutations [12, 13]. However, the DTG fold-change increased for Q148H/K/R as secondary RAL resistance–associated mutations increased. On the basis of these in vitro findings, this phase IIb pilot study was conducted to assess and demonstrate the activity of DTG in HIV-1–infected individuals with RAL-resistant viral isolates.

Update of the drug resistance mutations in HIV-1: March 2013.

Abstract: In this update, 2 integrase strand transfer inhibitors (InSTIs), elvitegra-vir and dolutegravir, have become available and were added to the fig-ure. Elvitegravir was approved by the US Food and Drug Administration (FDA) in August 2012 for HIV-1 treat-ment-naive patients as part of a fixed-dose combination of elvitegravir/ cobicistat/tenofovir/emtricitabine.

Allosteric integrase inhibitor potency is determined through the inhibition of HIV-1 particle maturation.

Abstract: Integration is essential for HIV-1 replication, and the viral integrase (IN) protein is an important therapeutic target. Allosteric IN inhibitors (ALLINIs) that engage the IN dimer interface at the binding site for the host protein lens epithelium-derived growth factor (LEDGF)/transcriptional coactivator p75 are an emerging class of small molecule antagonists. Consistent with the inhibition of a multivalent drug target, ALLINIs display steep antiviral dose–response curves ex vivo. ALLINIs multimerize IN protein and concordantly block its assembly with viral DNA in vitro, indicating that the disruption of two integration-associated functions, IN catalysis and the IN-LEDGF/p75 interaction, determines the multimode mechanism of ALLINI action. We now demonstrate that ALLINI potency is unexpectedly accounted for during the late phase of HIV-1 replication. The compounds promote virion IN multimerization and, reminiscent of class II IN mutations, block the formation of the electron-dense viral core and inhibit reverse transcription and integration in subsequently infected target cells. Mature virions are recalcitrant to ALLINI treatment, and compound potency during virus production is independent of the level of LEDGF/p75 expression. We conclude that cooperative multimerization of IN by ALLINIs together with the inability for LEDGF/p75 to effectively engage the virus during its egress from cells underscores the multimodal mechanism of ALLINI action. Our results highlight the versatile nature of allosteric inhibitors to primarily inhibit viral replication at a step that is distinct from the catalytic requirement for the target enzyme. The vulnerability of IN to small molecules during the late phase of HIV-1 replication unveils a pharmacological Achilles’ heel for exploitation in clinical ALLINI development.

BET proteins promote efficient murine leukemia virus integration at transcription start sites

Abstract: The selection of chromosomal targets for retroviral integration varies markedly, tracking with the genus of the retrovirus, suggestive of targeting by binding to cellular factors. γ-Retroviral murine leukemia virus (MLV) DNA integration into the host genome is favored at transcription start sites, but the underlying mechanism for this preference is unknown. Here, we have identified bromodomain and extraterminal domain (BET) proteins (Brd2, -3, -4) as cellular-binding partners of MLV integrase. We show that purified recombinant Brd4(1-720) binds with high affinity to MLV integrase and stimulates correct concerted integration in vitro. JQ-1, a small molecule that selectively inhibits interactions of BET proteins with modified histone sites impaired MLV but not HIV-1 integration in infected cells. Comparison of the distribution of BET protein-binding sites analyzed using ChIP-Seq data and MLV-integration sites revealed significant positive correlations. Antagonism of BET proteins, via JQ-1 treatment or RNA interference, reduced MLV-integration frequencies at transcription start sites. These findings elucidate the importance of BET proteins for MLV integration efficiency and targeting and provide a route to developing safer MLV-based vectors for human gene therapy.

Carbamoyl Pyridone HIV-1 Integrase Inhibitors 3. A Diastereomeric Approach to Chiral Nonracemic Tricyclic Ring Systems and the Discovery of Dolutegravir (S/GSK1349572) and (S/GSK1265744)

Abstract: We report herein the discovery of the human immunodeficiency virus type-1 (HIV-1) integrase inhibitors dolutegravir (S/GSK1349572) (3) and S/GSK1265744 (4). These drugs stem from a series of carbamoyl pyridone analogues designed using a two-metal chelation model of the integrase catalytic active site. Structure-activity studies evolved a tricyclic series of carbamoyl pyridines that demonstrated properties indicative of once-daily dosing and superior potency against resistant viral strains. An inherent hemiaminal ring fusion stereocenter within the tricyclic carbamoyl pyridone scaffold led to a critical substrate controlled diastereoselective synthetic strategy whereby chiral information from small readily available amino alcohols was employed to control relative and absolute stereochemistry of the final drug candidates. Modest to extremely high levels of stereochemical control were observed depending on ring size and position of the stereocenter. This approach resulted in the discovery of 3 and 4, which are currently in clinical development.

Viral fitness cost prevents HIV-1 from evading dolutegravir drug pressure.

Abstract: Clinical studies have shown that integrase strand transfer inhibitors can be used to treat HIV-1 infection. Although the first-generation integrase inhibitors are susceptible to the emergence of resistance mutations that impair their efficacy in therapy, such resistance has not been identified to date in drug-naive patients who have been treated with the second-generation inhibitor dolutegravir. During previous in vitro selection study, we identified a R263K mutation as the most common substitution to arise in the presence of dolutegravir with H51Y arising as a secondary mutation. Additional experiments reported here provide a plausible explanation for the absence of reported dolutegravir resistance among integrase inhibitor-naive patients to date. We now show that H51Y in combination with R263K increases resistance to dolutegravir but is accompanied by dramatic decreases in both enzymatic activity and viral replication. Since H51Y and R263K may define a unique resistance pathway to dolutegravir, our results are consistent with the absence of resistance mutations in antiretroviral drug-naive patients treated with this drug.

Bromo- and Extraterminal Domain Chromatin Regulators Serve as Cofactors for Murine Leukemia Virus Integration

Abstract: Retroviral integrase (IN) proteins catalyze the permanent integration of proviral genomes into host DNA with the help of cellular cofactors. Lens epithelium-derived growth factor (LEDGF) is a cofactor for lentiviruses, including human immunodeficiency virus type 1 (HIV-1), and targets lentiviral integration toward active transcription units in the host genome. In contrast to lentiviruses, murine leukemia virus (MLV), a gammaretrovirus, tends to integrate near transcription start sites. Here, we show that the bromodomain and extraterminal domain (BET) proteins BRD2, BRD3, and BRD4 interact with gammaretroviral INs and stimulate the catalytic activity of MLV IN in vitro. We mapped the interaction site to a characteristic structural feature within the BET protein extraterminal (ET) domain and to three amino acids in MLV IN. The ET domains of different BET proteins stimulate MLV integration in vitro and, in the case of BRD2, also in vivo. Furthermore, two small-molecule BET inhibitors, JQ1 and I-BET, decrease MLV integration and shift it away from transcription start sites. Our data suggest that BET proteins might act as chromatin-bound acceptors for the MLV preintegration complex. These results could pave a way to redirecting MLV DNA integration as a basis for creating safer retroviral vectors.

Non-catalytic site HIV-1 integrase inhibitors disrupt core maturation and induce a reverse transcription block in target cells.

Abstract: HIV-1 integrase (IN) is the target for two classes of antiretrovirals: i) the integrase strand-transfer inhibitors (INSTIs) and ii) the non-catalytic site integrase inhibitors (NCINIs) NCINIs bind at the IN dimer interface and are thought to interfere primarily with viral DNA (vDNA) integration in the target cell by blocking IN-vDNA assembly as well as the IN-LEDGF/p75 interaction Herein we show that treatment of virus-producing cells, but not of mature virions or target cells, drives NCINI antiviral potency NCINIs target an essential late-stage event in HIV replication that is insensitive to LEDGF levels in the producer cells Virus particles produced in the presence of NCINIs displayed normal Gag-Pol processing and endogenous reverse transcriptase activity, but were defective at initiating vDNA synthesis following entry into the target cell NCINI-resistant virus carrying a T174I mutation in the IN dimer interface was less sensitive to the compound-induced late-stage effects, including the reverse transcription block Wild-type, but not T174I virus, produced in the presence of NCINIs exhibited striking defects in core morphology and an increased level of IN oligomers that was not observed upon treatment of mature cell-free particles Collectively, these results reveal that NCINIs act through a novel mechanism that is unrelated to the previously observed inhibition of IN activity or IN-LEDGF interaction, and instead involves the disruption of an IN function during HIV-1 core maturation and assembly

LEDGINs inhibit late stage HIV-1 replication by modulating integrase multimerization in the virions

Abstract: Background: LEDGINs are novel allosteric HIV integrase (IN) inhibitors that target the lens epithelium-derived growth factor (LEDGF)/p75 binding pocket of IN. They block HIV-1 integration by abrogating the interaction between LEDGF/p75 and IN as well as by allosterically inhibiting the catalytic activity of IN. Results: Here we demonstrate that LEDGINs reduce the replication capacity of HIV particles produced in their presence. We systematically studied the molecular basis of this late effect of LEDGINs and demonstrate that HIV virions produced in their presence display a severe replication defect. Both the late effect and the previously described, early effect on integration contribute to LEDGIN antiviral activity as shown by time-of-addition, qPCR and infectivity assays. The late effect phenotype requires binding of LEDGINs to integrase without influencing proteolytic cleavage or production of viral particles. LEDGINs augment IN multimerization during virion assembly or in the released viral particles and severely hamper the infectivity of progeny virions. About 70% of the particles produced in LEDGIN-treated cells do not form a core or display aberrant empty cores with a mislocalized electrondense ribonucleoprotein. The LEDGIN-treated virus displays defective reverse transcription and nuclear import steps in the target cells. The LEDGIN effect is possibly exerted at the level of the Pol precursor polyprotein. Conclusion: Our results suggest that LEDGINs modulate IN multimerization in progeny virions and impair the formation of regular cores during the maturation step, resulting in a decreased infectivity of the viral particles in the target cells. LEDGINs thus profile as unique antivirals with combined early (integration) and late (IN assembly) effects on the HIV replication cycle.

Dual inhibition of HIV-1 replication by integrase-LEDGF allosteric inhibitors is predominant at the post-integration stage.

Abstract: LEDGF/p75 (LEDGF) is the main cellular cofactor of HIV-1 integrase (IN). It acts as a tethering factor for IN, and targets the integration of HIV in actively transcribed gene regions of chromatin. A recently developed class of IN allosteric inhibitors can inhibit the LEDGF-IN interaction. We describe a new series of IN-LEDGF allosteric inhibitors, the most active of which is Mut101. We determined the crystal structure of Mut101 in complex with IN and showed that the compound binds to the LEDGF-binding pocket, promoting conformational changes of IN which explain at the atomic level the allosteric effect of the IN/LEDGF interaction inhibitor on IN functions. In vitro, Mut101 inhibited both IN-LEDGF interaction and IN strand transfer activity while enhancing IN-IN interaction. Time of addition experiments indicated that Mut101 behaved as an integration inhibitor. Mut101 was fully active on HIV-1 mutants resistant to INSTIs and other classes of anti-HIV drugs, indicative that this compound has a new mode of action. However, we found that Mut101 also displayed a more potent antiretroviral activity at a post-integration step. Infectivity of viral particles produced in presence of Mut101 was severely decreased. This latter effect also required the binding of the compound to the LEDGF-binding pocket. Mut101 has dual anti-HIV-1 activity, at integration and post-integration steps of the viral replication cycle, by binding to a unique target on IN (the LEDGF-binding pocket). The post-integration block of HIV-1 replication in virus-producer cells is the mechanism by which Mut101 is most active as an antiretroviral. To explain this difference between Mut101 antiretroviral activity at integration and post-integration stages, we propose the following model: LEDGF is a nuclear, chromatin-bound protein that is absent in the cytoplasm. Therefore, LEDGF can outcompete compound binding to IN in the nucleus of target cells lowering its antiretroviral activity at integration, but not in the cytoplasm where post-integration production of infectious viral particles takes place.

Differential Effects of Human Immunodeficiency Virus Type 1 Capsid and Cellular Factors Nucleoporin 153 and LEDGF/p75 on the Efficiency and Specificity of Viral DNA Integration

Abstract: Retroviruses integrate into cellular DNA nonrandomly. Lentiviruses such as human immunodeficiency virus type 1 (HIV-1) favor the bodies of active genes and gene-enriched transcriptionally active regions of chromosomes. The interaction between lentiviral integrase and the cellular protein lens epithelium-derived growth factor (LEDGF)/p75 underlies the targeting of gene bodies, whereas recent research has highlighted roles for the HIV-1 capsid (CA) protein and cellular factors implicated in viral nuclear import, including transportin 3 (TNPO3) and nucleoporin 358 (NUP358), in the targeting of gene-dense regions of chromosomes. Here, we show that CA mutations, which include the substitution of Asp for Asn74 (N74D), significantly reduce the dependency of HIV-1 on LEDGF/p75 during infection and that this difference correlates with the efficiency of viral DNA integration. The distribution of integration sites mapped by Illumina sequencing confirms that the N74D mutation reduces integration into gene-rich regions of chromosomes and gene bodies and reveals previously unrecognized roles for NUP153 (another HIV-1 cofactor implicated in viral nuclear import) and LEDGF/p75 in the targeting of the viral preintegration complex to gene-dense regions of chromatin. A role for the CA protein in determining the dependency of HIV-1 on LEDGF/p75 during infection highlights a connection between the viral capsid and chromosomal DNA integration.

Impact of Primary Elvitegravir Resistance-Associated Mutations in HIV-1 Integrase on Drug Susceptibility and Viral Replication Fitness

Abstract: Elvitegravir (EVG) is an effective HIV-1 integrase (IN) strand transfer inhibitor (INSTI) in advanced clinical development. Primary INSTI resistance-associated mutations (RAMs) at six IN positions have been identified in HIV-1-infected patients failing EVG-containing regimens in clinical studies: T66I/A/K, E92Q/G, T97A, S147G, Q148R/H/K, and N155H. In this study, the effect of these primary IN mutations, alone and in combination, on susceptibility to the INSTIs EVG, raltegravir (RAL), and dolutegravir (DTG); IN enzyme activities; and viral replication fitness was characterized. Recombinant viruses containing the six most common mutations exhibited a range of reduced EVG susceptibility: 92-fold for Q148R, 30-fold for N155H, 26-fold for E92Q, 10-fold for T66I, 4-fold for S147G, and 2-fold for T97A. Less commonly observed primary IN mutations also showed a range of reduced EVG susceptibilities: 40- to 94-fold for T66K and Q148K and 5- to 10-fold for T66A, E92G, and Q148H. Some primary IN mutations exhibited broad cross-resistance between EVG and RAL (T66K, E92Q, Q148R/H/K, and N155H), while others retained susceptibility to RAL (T66I/A, E92G, T97A, and S147G). Dual combinations of primary IN mutations further reduced INSTI susceptibility, replication capacity, and viral fitness relative to either mutation alone. Susceptibility to DTG was retained by single primary IN mutations but reduced by dual mutation combinations with Q148R. Primary EVG RAMs also diminished IN enzymatic activities, concordant with their structural proximity to the active site. Greater reductions in viral fitness of dual mutation combinations may explain why some primary INSTI RAMs do not readily coexist on the same HIV-1 genome but rather establish independent pathways of resistance to EVG.

LEDGINs inhibit late stage HIV-1 replication by modulating integrase multimerization in the virions and reveal a role for LEDGF/p75 in late stage HIV-1 replication

Abstract: BackgroundLEDGINs are novel allosteric HIV integrase (IN) inhibitors that target the lens epithelium-derived growth factor (LEDGF)/p75 binding pocket of IN. They block HIV-1 integration by abrogating the interaction between LEDGF/p75 and IN as well as by allosterically inhibiting the catalytic activity of IN.ResultsHere we demonstrate that LEDGINs reduce the replication capacity of HIV particles produced in their presence. We systematically studied the molecular basis of this late effect of LEDGINs and demonstrate that HIV virions produced in their presence display a severe replication defect. Both the late effect and the previously described, early effect on integration contribute to LEDGIN antiviral activity as shown by time-of-addition, qPCR and infectivity assays. The late effect phenotype requires binding of LEDGINs to integrase without influencing proteolytic cleavage or production of viral particles. LEDGINs augment IN multimerization during virion assembly or in the released viral particles and severely hamper the infectivity of progeny virions. About 70% of the particles produced in LEDGIN-treated cells do not form a core or display aberrant empty cores with a mislocalized electron-dense ribonucleoprotein. The LEDGIN-treated virus displays defective reverse transcription and nuclear import steps in the target cells. The LEDGIN effect is possibly exerted at the level of the Pol precursor polyprotein.ConclusionOur results suggest that LEDGINs modulate IN multimerization in progeny virions and impair the formation of regular cores during the maturation step, resulting in a decreased infectivity of the viral particles in the target cells. LEDGINs thus profile as unique antivirals with combined early (integration) and late (IN assembly) effects on the HIV replication cycle.

The Hepatitis B Virus Ribonuclease H Is Sensitive to Inhibitors of the Human Immunodeficiency Virus Ribonuclease H and Integrase Enzymes

Abstract: Nucleos(t)ide analog therapy blocks DNA synthesis by the hepatitis B virus (HBV) reverse transcriptase and can control the infection, but treatment is life-long and has high costs and unpredictable long-term side effects. The profound suppression of HBV by the nucleos(t)ide analogs and their ability to cure some patients indicates that they can push HBV to the brink of extinction. Consequently, more patients could be cured by suppressing HBV replication further using a new drug in combination with the nucleos(t)ide analogs. The HBV ribonuclease H (RNAseH) is a logical drug target because it is the second of only two viral enzymes that are essential for viral replication, but it has not been exploited, primarily because it is very difficult to produce active enzyme. To address this difficulty, we expressed HBV genotype D and H RNAseHs in E. coli and enriched the enzymes by nickel-affinity chromatography. HBV RNAseH activity in the enriched lysates was characterized in preparation for drug screening. Twenty-one candidate HBV RNAseH inhibitors were identified using chemical structure-activity analyses based on inhibitors of the HIV RNAseH and integrase. Twelve anti-RNAseH and anti-integrase compounds inhibited the HBV RNAseH at 10 µM, the best compounds had low micromolar IC(50) values against the RNAseH, and one compound inhibited HBV replication in tissue culture at 10 µM. Recombinant HBV genotype D RNAseH was more sensitive to inhibition than genotype H. This study demonstrates that recombinant HBV RNAseH suitable for low-throughput antiviral drug screening has been produced. The high percentage of compounds developed against the HIV RNAseH and integrase that were active against the HBV RNAseH indicates that the extensive drug design efforts against these HIV enzymes can guide anti-HBV RNAseH drug discovery. Finally, differential inhibition of HBV genotype D and H RNAseHs indicates that viral genetic variability will be a factor during drug development.

Fates of retroviral core components during unrestricted and TRIM5-restricted infection.

Abstract: TRIM5 proteins can restrict retroviral infection soon after delivery of the viral core into the cytoplasm. However, the molecular mechanisms by which TRIM5α inhibits infection have been elusive, in part due to the difficulty of developing and executing biochemical assays that examine this stage of the retroviral life cycle. Prevailing models suggest that TRIM5α causes premature disassembly of retroviral capsids and/or degradation of capsids by proteasomes, but whether one of these events leads to the other is unclear. Furthermore, how TRIM5α affects the essential components of the viral core, other than capsid, is unknown. To address these questions, we devised a biochemical assay in which the fate of multiple components of retroviral cores during infection can be determined. We utilized cells that can be efficiently infected by VSV-G-pseudotyped retroviruses, and fractionated the cytosolic proteins on linear gradients following synchronized infection. The fates of capsid and integrase proteins, as well as viral genomic RNA and reverse transcription products were then monitored. We found that components of MLV and HIV-1 cores formed a large complex under non-restrictive conditions. In contrast, when MLV infection was restricted by human TRIM5α, the integrase protein and reverse transcription products were lost from infected cells, while capsid and viral RNA were both solubilized. Similarly, when HIV-1 infection was restricted by rhesus TRIM5α or owl monkey TRIMCyp, the integrase protein and reverse transcription products were lost. However, viral RNA was also lost, and high levels of preexisting soluble CA prevented the determination of whether CA was solubilized. Notably, proteasome inhibition blocked all of the aforementioned biochemical consequences of TRIM5α-mediated restriction but had no effect on its antiviral potency. Together, our results show how TRIM5α affects various retroviral core components and indicate that proteasomes are required for TRIM5α-induced core disruption but not for TRIM5α-induced restriction.

Accuracy and efficiency define Bxb1 integrase as the best of fifteen candidate serine recombinases for the integration of DNA into the human genome

Abstract: Background: Phage-encoded serine integrases, such as φC31 integrase, are widely used for genome engineering. Fifteen such integrases have been described but their utility for genome engineering has not been compared in uniform assays. Results: We have compared fifteen serine integrases for their utility for DNA manipulations in mammalian cells after first demonstrating that all were functional in E. coli. Chromosomal recombination reporters were used to show that seven integrases were active on chromosomally integrated DNA in human fibroblasts and mouse embryonic stem cells. Five of the remaining eight enzymes were active on extra-chromosomal substrates thereby demonstrating that the ability to mediate extra-chromosomal recombination is no guide to ability to mediate site-specific recombination on integrated DNA. All the integrases that were active on integrated DNA also promoted DNA integration reactions that were not mediated through conservative site-specific recombination or damaged the recombination sites but the extent of these aberrant reactions varied over at least an order of magnitude. Bxb1 integrase yielded approximately two-fold more recombinants and displayed about two fold less damage to the recombination sites than the next best recombinase; φC31 integrase. Conclusions: We conclude that the Bxb1 andφC31 integrases are the reagents of choice for genome engineering in vertebrate cells and that DNA damage repair is a major limitation upon the utility of this class of site-specific recombinase.

Exploring the molecular mechanism of cross-resistance to HIV-1 integrase strand transfer inhibitors by molecular dynamics simulation and residue interaction network analysis.

Abstract: The rapid emergence of cross-resistance to the integrase strand transfer inhibitors (INSTIs) has become a serious problem in the therapy of human immunodeficiency virus type 1 (HIV-1) infection. Understanding the detailed molecular mechanism of INSTIs cross-resistance is therefore critical for the development of new effective therapy against cross-resistance. On the basis of the homology modeling constructed structure of tetrameric HIV-1 intasome, the detailed molecular mechanism of the cross-resistance mutation E138K/Q148K to three important INSTIs (Raltegravir (RAL, FDA approved in 2007), Elvitegravir (EVG, FDA approved in 2012), and Dolutegravir (DTG, phase III clinical trials)) was investigated by using molecular dynamics (MD) simulation and residue interaction network (RIN) analysis. The results from conformation analysis and binding free energy calculation can provide some useful information about the detailed binding mode and cross-resistance mechanism for the three INSTIs to HIV-1 intasome. Bindin...

Attachment site recognition and regulation of directionality by the serine integrases

Abstract: Serine integrases catalyze the integration of bacteriophage DNA into a host genome by site-specific recombination between 'attachment sites' in the phage (attP) and the host (attB). The reaction is highly directional; the reverse excision reaction between the product attL and attR sites does not occur in the absence of a phage-encoded factor, nor does recombination occur between other pairings of attachment sites. A mechanistic understanding of how these enzymes achieve site-selectivity and directionality has been limited by a lack of structural models. Here, we report the structure of the C-terminal domains of a serine integrase bound to an attP DNA half-site. The structure leads directly to models for understanding how the integrase-bound attP and attB sites differ, why these enzymes preferentially form attP × attB synaptic complexes to initiate recombination, and how attL × attR recombination is prevented. In these models, different domain organizations on attP vs. attB half-sites allow attachment-site specific interactions to form between integrase subunits via an unusual protruding coiled-coil motif. These interactions are used to preferentially synapse integrase-bound attP and attB and inhibit synapsis of integrase-bound attL and attR. The results provide a structural framework for understanding, testing and engineering serine integrase function.

Quantitative analysis of the time-course of viral DNA forms during the HIV-1 life cycle

Abstract: HIV-1 DNA is found both integrated in the host chromosome and unintegrated in various forms: linear (DNAL) or circular (1-LTRc, 2-LTRc or products of auto-integration). Here, based on pre-established strategies, we extended and characterized in terms of sensitivity two methodologies for quantifying 1-LTRc and DNAL, respectively, the latter being able to discriminate between unprocessed or 3′-processed DNA. Quantifying different types of viral DNA genome individually provides new information about the dynamics of all viral DNA forms and their interplay. For DNAL, we found that the 3′-processing reaction was efficient during the early stage of the replication cycle. Moreover, strand-transfer inhibitors (Dolutegravir, Elvitegravir, Raltegravir) affected 3′-processing differently. The comparisons of 2-LTRc accumulation mediated by either strand-transfer inhibitors or catalytic mutation of integrase indicate that 3′-processing efficiency did not influence the total 2-LTRc accumulation although the nature of the LTR-LTR junction was qualitatively affected. Finally, a significant proportion of 1-LTRc was generated concomitantly with reverse transcription, although most of the 1-LTRc were produced in the nucleus. We describe the fate of viral DNA forms during HIV-1 infection. Our study reveals the interplay between various forms of the viral DNA genome, the distribution of which can be affected by mutations and by inhibitors of HIV-1 viral proteins. In the latter case, the quantification of 3′-processed DNA in infected cells can be informative about the mechanisms of future integrase inhibitors directly in the cell context.

Past and future. Current drugs targeting HIV-1 integrase and reverse transcriptase-associated ribonuclease H activity: single and dual active site inhibitors.

Abstract: Catalytic HIV type-1 (HIV-1) integrase (IN) and ribonuclease H (RNase H) domains belong to the polynucleotidyl transferase superfamily and are characterized by highly conserved motifs that coordinate two divalent Mg(2+) cations and are attractive targets for new antiviral agents. Several structural features of both domains are now available. Drugs targeting the HIV-1 IN are currently approved for anti-HIV therapy, while no drug targeting the HIV-1 RNase H function is yet available. This review describes HIV-1 IN and the RNase H function and structures, compounds targeting their active sites and dual inhibition as a new approach for drug development.

Fragment-Based Discovery of 8-Hydroxyquinoline Inhibitors of the HIV-1 Integrase–Lens Epithelium-Derived Growth Factor/p75 (IN–LEDGF/p75) Interaction

Abstract: On the basis of an initial molecular modeling study suggesting the favorable binding of the "privileged" fragment 8-hydroxyquinoline with HIV-1 integrase (IN) at the IN-lens epithelium-derived growth factor/p75 (LEDGF/p75) interface , we developed a set of modified 8-hydroxyquinoline fragments demonstrating micromolar IC50 values for inhibition of the IN-LEDGF/p75 interaction, but significant cytotoxicity was associated with these initial compounds. Diverse modifications at the C5 and C7 carbons of the 8-hydroxyquinoline core improved potency, but reduction of diversity to only modifications at the C5 position ultimately yielded potent inhibitors with low cytotoxicity. Two of these particular compounds, 5-((p-tolylamino)methyl)quinolin-8-ol and 5-(((3,4-dimethylphenyl)amino)methyl)quinolin-8-ol, inhibited viral replication in MT-4 cells with low micromolar EC50. This is the first study providing evidence for 8-hydroxyquinolines as novel inhibitors of the IN-LEDGF/p75 interaction. Our lead compounds are druglike, have low molecular weights, and are amenable to various substitutions suitable for enhancing their potency and selectivity.

Dolutegravir interactions with HIV-1 integrase-DNA: structural rationale for drug resistance and dissociation kinetics.

Abstract: Signature HIV-1 integrase mutations associated with clinical raltegravir resistance involve 1 of 3 primary genetic pathways, Y143C/R, Q148H/K/R and N155H, the latter 2 of which confer cross-resistance to elvitegravir. In accord with clinical findings, in vitro drug resistance profiling studies with wild-type and site-directed integrase mutant viruses have shown significant fold increases in raltegravir and elvitegravir resistance for the specified viral mutants relative to wild-type HIV-1. Dolutegravir, in contrast, has demonstrated clinical efficacy in subjects failing raltegravir therapy due to integrase mutations at Y143, Q148 or N155, which is consistent with its distinct in vitro resistance profile as dolutegravir’s antiviral activity against these viral mutants is equivalent to its activity against wild-type HIV-1. Kinetic studies of inhibitor dissociation from wild-type and mutant integrase-viral DNA complexes have shown that dolutegravir also has a distinct off-rate profile with dissociative half-lives substantially longer than those of raltegravir and elvitegravir, suggesting that dolutegravir’s prolonged binding may be an important contributing factor to its distinct resistance profile. To provide a structural rationale for these observations, we constructed several molecular models of wild-type and clinically relevant mutant HIV-1 integrase enzymes in complex with viral DNA and dolutegravir, raltegravir or elvitegravir. Here, we discuss our structural models and the posited effects that the integrase mutations and the structural and electronic properties of the integrase inhibitors may have on the catalytic pocket and inhibitor binding and, consequently, on antiviral potency in vitro and in the clinic.

An HIV-1 Replication Pathway Utilizing Reverse Transcription Products That Fail To Integrate

Abstract: Integration is a central event in the replication of retroviruses, yet ≥90% of HIV-1 reverse transcripts fail to integrate, resulting in accumulation of unintegrated viral DNA in cells. However, understanding what role, if any, unintegrated viral DNA plays in the natural history of HIV-1 has remained elusive. Unintegrated HIV-1 DNA is reported to possess a limited capacity for gene expression restricted to early gene products and is considered a replicative dead end. Although the majority of peripheral blood CD4+ T cells are refractory to infection, nonactivated CD4 T cells present in lymphoid and mucosal tissues are major targets for infection. Treatment with cytokine interleukin-2 (IL-2), IL-4, IL-7, or IL-15 renders CD4+ T cells permissive to HIV-1 infection in the absence of cell activation and proliferation and provides a useful model for infection of resting CD4+ T cells. We found that infection of cytokine-treated resting CD4+ T cells in the presence of raltegravir or with integrase active-site mutant HIV-1 yielded de novo virus production following subsequent T cell activation. Infection with integration-competent HIV-1 naturally generated a population of cells generating virus from unintegrated DNA. Latent infection persisted for several weeks and could be activated to virus production by a combination of a histone deacetylase inhibitor and a protein kinase C activator or by T cell activation. HIV-1 Vpr was essential for unintegrated HIV-1 gene expression and de novo virus production in this system. Bypassing integration by this mechanism may allow the preservation of genetic information that otherwise would be lost.

Next-generation integrase inhibitors : where to after raltegravir?

Abstract: The integrase enzyme facilitates the incorporation of HIV-1 proviral DNA into the host cell genome and catalyses a function vital to viral replication. Inhibitors of this enzyme represent the newest class of antiretroviral drugs in our armamentarium to treat HIV-1 infection. Raltegravir, an integrase strand transfer inhibitor, was the first drug of this class approved by the US FDA; it is a potent and well tolerated antiviral agent. However, it has the limitations of twice-daily dosing and a relatively modest genetic barrier to the development of resistance. These qualities have prompted the search for agents with once-daily dosing, a more robust barrier to resistance, and a resistance profile of limited overlap with that of raltegravir. We review a series of integrase inhibitors that are in clinical or advanced pre-clinical studies. Elvitegravir, recently approved by the FDA as part of the elvitegravir/cobicistat/tenofovir disoproxil fumarate/emtricitabine fixed-dose combination pill has the benefit of being part of a one-pill, once-daily regimen, but suffers from extensive cross-resistance with raltegravir. Dolutegravir is the most advanced second-generation integrase inhibitor, and it boasts good tolerability, once-daily dosing with no need for a pharmacological enhancer, and relatively little cross-resistance with raltegravir. S/GSK1265744 has been developed into a long-acting parenteral agent that shows a high barrier to resistance in vitro and the potential for an infrequent dosing schedule. BI 224436 is in early clinical trials, but is unlikely to demonstrate cross-resistance with other integrase inhibitors. The inhibitors of the lens epithelium-derived growth factor (LEDGF)/p75 binding site of integrase (LEDGINs) are extremely early in development. Each of these contributes a new benefit to the class and will extend the treatment options for patients with HIV-1 infection.

Cell-permeable stapled peptides based on HIV-1 integrase inhibitors derived from HIV-1 gene products.

Abstract: HIV-1 integrase (IN) is an enzyme which is indispensable for the stable infection of host cells because it catalyzes the insertion of viral DNA into the genome and thus is an attractive target for the development of anti-HIV agents. Earlier, we found Vpr-derived peptides with inhibitory activity against HIV-1 IN. These Vpr-derived peptides are originally located in an α-helical region of the parent Vpr protein. Addition of an octa-arginyl group to the inhibitory peptides caused significant inhibition against HIV replication associated with an increase in cell permeability but also relatively high cytotoxicity. In the current study, stapled peptides, a new class of stabilized α-helical peptidomimetics were adopted to enhance the cell permeability of the above lead peptides. A series of stapled peptides, which have a hydrocarbon link formed by a ruthenium-catalyzed ring-closing metathesis reaction between successive turns of α-helix, were designed, synthesized, and evaluated for biological activity. In cell-based assays some of the stapled peptides showed potent anti-HIV activity comparable with that of the original octa-arginine-containing peptide (2) but with lower cytotoxicity. Fluorescent imaging experiments revealed that these stapled peptides are significantly cell permeable, and CD analysis showed they form α-helical structures, whereas the unstapled congeners form β-sheet structures. The application of this stapling strategy to Vpr-derived IN inhibitory peptides led to a remarkable increase in their potency in cells and a significant reduction of their cytotoxicity.