Lens epithelium-derived growth factor/p75 (LEDGF/p75, or PSIP1) is a transcriptional coactivator that tethers other proteins to gene bodies. The chromatin tethering function of LEDGF/p75 is hijacked by HIV integrase to ensure viral integration at sites of active transcription. LEDGF/p75 is also important for the development of mixed-lineage leukemia (MLL), where it tethers the MLL1 fusion complex at aberrant MLL targets, inducing malignant transformation. However, little is known about how the LEDGF/p75 protein interaction network is regulated. Here, we obtained solution structures of the complete interfaces between the LEDGF/p75 integrase binding domain (IBD) and its cellular binding partners and validated another binding partner, Mediator subunit 1 (MED1). We reveal that structurally conserved IBD-binding motifs (IBMs) on known LEDGF/p75 binding partners can be regulated by phosphorylation, permitting switching between low- and high-affinity states. Finally, we show that elimination of IBM phosphorylation sites on MLL1 disrupts the oncogenic potential of primary MLL1-rearranged leukemic cells. Our results demonstrate that kinase-dependent phosphorylation of MLL1 represents a previously unknown oncogenic dependency that may be harnessed in the treatment of MLL-rearranged leukemia.
- MeSH
- adaptorové proteiny signální transdukční genetika metabolismus MeSH
- aminokyselinové motivy MeSH
- fosforylace genetika MeSH
- histonlysin-N-methyltransferasa genetika metabolismus MeSH
- HIV-integrasa genetika metabolismus MeSH
- HIV enzymologie genetika MeSH
- lidé MeSH
- mediátorový komplex - podjednotka 1 genetika metabolismus MeSH
- nádorové buněčné linie MeSH
- protoonkogenní protein MLL genetika metabolismus MeSH
- transkripční faktory genetika metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH
Lens epithelium-derived growth factor (LEDGF/p75) is an epigenetic reader and attractive therapeutic target involved in HIV integration and the development of mixed lineage leukaemia (MLL1) fusion-driven leukaemia. Besides HIV integrase and the MLL1-menin complex, LEDGF/p75 interacts with various cellular proteins via its integrase binding domain (IBD). Here we present structural characterization of IBD interactions with transcriptional repressor JPO2 and domesticated transposase PogZ, and show that the PogZ interaction is nearly identical to the interaction of LEDGF/p75 with MLL1. The interaction with the IBD is maintained by an intrinsically disordered IBD-binding motif (IBM) common to all known cellular partners of LEDGF/p75. In addition, based on IBM conservation, we identify and validate IWS1 as a novel LEDGF/p75 interaction partner. Our results also reveal how HIV integrase efficiently displaces cellular binding partners from LEDGF/p75. Finally, the similar binding modes of LEDGF/p75 interaction partners represent a new challenge for the development of selective interaction inhibitors.
- MeSH
- dimerizace MeSH
- Escherichia coli MeSH
- histonlysin-N-methyltransferasa metabolismus MeSH
- HIV-integrasa metabolismus MeSH
- konsenzuální sekvence MeSH
- Lentivirus enzymologie MeSH
- lidé MeSH
- mezibuněčné signální peptidy a proteiny metabolismus MeSH
- molekulární sekvence - údaje MeSH
- proteiny metabolismus MeSH
- protoonkogenní protein MLL metabolismus MeSH
- represorové proteiny metabolismus MeSH
- sekvence aminokyselin MeSH
- terciární struktura proteinů MeSH
- transposasy metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
OBJECTIVES: The objective of this study was to define the natural genotypic variation of the HIV-1 integrase gene across Europe for epidemiological surveillance of integrase strand-transfer inhibitor (InSTI) resistance. METHODS: This was a multicentre, cross-sectional study within the European SPREAD HIV resistance surveillance programme. A representative set of 300 samples was selected from 1950 naive HIV-positive subjects newly diagnosed in 2006-07. The prevalence of InSTI resistance was evaluated using quality-controlled baseline population sequencing of integrase. Signature raltegravir, elvitegravir and dolutegravir resistance mutations were defined according to the IAS-USA 2014 list. In addition, all integrase substitutions relative to HXB2 were identified, including those with a Stanford HIVdb score ≥ 10 to at least one InSTI. To rule out circulation of minority InSTI-resistant HIV, 65 samples were selected for 454 integrase sequencing. RESULTS: For the population sequencing analysis, 278 samples were retrieved and successfully analysed. No signature resistance mutations to any of the InSTIs were detected. Eleven (4%) subjects had mutations at resistance-associated positions with an HIVdb score ≥ 10. Of the 56 samples successfully analysed with 454 sequencing, no InSTI signature mutations were detected, whereas integrase substitutions with an HIVdb score ≥ 10 were found in 8 (14.3%) individuals. CONCLUSIONS: No signature InSTI-resistant variants were circulating in Europe before the introduction of InSTIs. However, polymorphisms contributing to InSTI resistance were not rare. As InSTI use becomes more widespread, continuous surveillance of primary InSTI resistance is warranted. These data will be key to modelling the kinetics of InSTI resistance transmission in Europe in the coming years.
- MeSH
- genetická variace MeSH
- genotyp MeSH
- HIV infekce farmakoterapie epidemiologie virologie MeSH
- HIV-1 účinky léků genetika MeSH
- HIV-integrasa genetika MeSH
- inhibitory HIV-integrasy farmakologie terapeutické užití MeSH
- lidé MeSH
- počet CD4 lymfocytů MeSH
- průřezové studie MeSH
- rizikové faktory MeSH
- sekvenční analýza DNA MeSH
- surveillance populace MeSH
- virová léková rezistence * MeSH
- virová nálož MeSH
- vysoce aktivní antiretrovirová terapie MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- multicentrická studie MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Evropa MeSH
Heterocyclic substances perform a very unique role in drug design and discovery. This article provides the primary objectives of the analysis within pyrimidine centered new heterocyclic elements chronologically from their finding focusing on one of the essential enzyme of HIV virus particle that is integrase upon suppressing its strand transfer function. The class of compounds reviewed here includes bicyclic pyrimidines, dihydroxypyrimidines, pyrimidine-2,4-dinones, N-methylpyrimidones, pyranopyrimidine, pyridine-quinoline conjugates, pyrimidine-2-carboxamides, N-3 hydroxylated pyrimidine-2,4-diones as well as their various substituted analogues. Such initiatives released an effective drug Raltegravir as a first FDA approved anti-HIV integrase inhibitor as well as several of its derivatives along with other pyrimidones is under clinical or preclinical growth. Some of the provided scaffolds indicated dual anti-HIV efficacies against HIV reverse transcriptase and integrase enzymes at both cites as 3'-processing and strand transfer, while several scaffolds exhibited potency against Raltegravir resistant HIV mutant strains determining themselves a potent class of compounds having appealing upcoming implementations. Connections of the new compounds' molecular structure and HIV viral target has been overviewed to be able to accomplish further growth of promising anti-HIV agents in future drug discovery process.
- MeSH
- HIV-integrasa metabolismus MeSH
- inhibitory HIV-integrasy farmakologie MeSH
- lidé MeSH
- objevování léků metody MeSH
- pyrimidinony farmakologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
Heterocyclic compounds execute a very important role in drug design and discovery. This article provides the basic milestones of the research for pyrroloaryl and pyrroloheteroaryl based components targeting HIV viral replication cycle. Anti-HIV activity is elaborated for several classes of pyrrolo-compounds as pyrrolopyridines, pyrrolopyrimidines, pyrrolopyridazines, pyrrolobenzodiazepinones, pyrrolobenzothiazepines, pyrrolobenzoxazepinones, pyrrolophenanthridines, pyrroloquinoxalines, pyrrolotriazines, pyrroloquinolines, pyrrolopyrazinones, pyrrolothiatriazines, arylthiopyrroles and pyrrolopyrazolones targeting two essential HIV enzymes, reverse transcriptase and integrase as well as attachment/fusion of HIV virons to the host CD-4 cell. Such attempts were resulted in a discovery of highly potent anti-HIV agents suitable for clinical trials, for example, BMS-378806, BMS-585248, BMS-626529, BMS-663068, BMS-488043 and BMS-663749, etc. as anti-HIV attachment agents, triciribine, QX432, BI-1 and BI-2 as HIV RT inhibitors which are in preclinical or clinical development. Mechanism of action of compounds presented in this article towards the suppression of HIV attachment/fusion as well as against the activities of HIV enzymes reverse transcriptase and integrase has been discussed. Relationships of new compounds' molecular framework and HIV viral target has been overviewed in order to facilitate further construction of promising anti-HIV agents in future drug discovery process.
- MeSH
- HIV infekce farmakoterapie virologie MeSH
- HIV-integrasa metabolismus MeSH
- HIV účinky léků enzymologie fyziologie MeSH
- inhibitory HIV fúze chemie farmakologie MeSH
- inhibitory HIV-integrasy chemie farmakologie MeSH
- inhibitory reverzní transkriptasy chemie farmakologie MeSH
- látky proti HIV chemie farmakologie MeSH
- lidé MeSH
- objevování léků MeSH
- pyrroly chemie farmakologie MeSH
- replikace viru účinky léků MeSH
- reverzní transkriptasa metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
x
x
- Klíčová slova
- studie START, plazmatická virémie,
- MeSH
- adherence k farmakoterapii MeSH
- antiretrovirové látky * farmakologie terapeutické užití MeSH
- CD4-pozitivní T-lymfocyty MeSH
- fixní kombinace léků MeSH
- HIV infekce * farmakoterapie virologie MeSH
- HIV séropozitivita farmakoterapie MeSH
- HIV-integrasa farmakologie terapeutické užití MeSH
- HIV genetika účinky léků MeSH
- inhibitory HIV fúze farmakologie terapeutické užití MeSH
- inhibitory HIV-integrasy farmakologie terapeutické užití MeSH
- inhibitory HIV-proteasy farmakologie terapeutické užití MeSH
- inhibitory reverzní transkriptasy farmakologie terapeutické užití MeSH
- kombinovaná farmakoterapie MeSH
- látky proti HIV farmakologie terapeutické užití MeSH
- lidé MeSH
- multicentrické studie jako téma MeSH
- počet CD4 lymfocytů MeSH
- randomizované kontrolované studie jako téma MeSH
- RNA virová krev MeSH
- viremie MeSH
- virová nálož MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- přehledy MeSH
The role of HIV-1 minority variants on transmission, pathogenesis, and virologic failure to antiretroviral regimens has been explored; however, most studies of low-level HIV-1 drug-resistant variants have focused in single target regions. Here we used a novel HIV-1 genotypic assay based on deep sequencing, DEEPGEN (Gibson et al 2014 Antimicrob Agents Chemother 58∶2167) to simultaneously analyze the presence of minority variants carrying mutations associated with reduced susceptibility to protease (PR), reverse transcriptase (RT), and integrase strand transfer integrase inhibitors (INSTIs), as well as HIV-1 coreceptor tropism. gag-p2/NCp7/p1/p6/pol-PR/RT/INT and env/C2V3 PCR products were obtained from twelve heavily treatment-experienced patients experiencing virologic failure while participating in a 48-week dose-ranging study of elvitegravir (GS-US-183-0105). Deep sequencing results were compared with (i) virological response to treatment, (ii) genotyping based on population sequencing, (iii) phenotyping data using PhenoSense and VIRALARTS, and (iv) HIV-1 coreceptor tropism based on the phenotypic test VERITROP. Most patients failed the antiretroviral regimen with numerous pre-existing mutations in the PR and RT, and additionally newly acquired INSTI-resistance mutations as determined by population sequencing (mean 9.4, 5.3, and 1.4 PI- RTI-, and INSTI-resistance mutations, respectively). Interestingly, since DEEPGEN allows the accurate detection of amino acid substitutions at frequencies as low as 1% of the population, a series of additional drug resistance mutations were detected by deep sequencing (mean 2.5, 1.5, and 0.9, respectively). The presence of these low-abundance HIV-1 variants was associated with drug susceptibility, replicative fitness, and coreceptor tropism determined using sensitive phenotypic assays, enhancing the overall burden of resistance to all four antiretroviral drug classes. Further longitudinal studies based on deep sequencing tests will help to clarify (i) the potential impact of minority HIV-1 drug resistant variants in response to antiretroviral therapy and (ii) the importance of the detection of HIV minority variants in the clinical practice.
- MeSH
- buněčné linie MeSH
- fenotyp MeSH
- genotypizační techniky MeSH
- HIV-1 účinky léků enzymologie genetika MeSH
- HIV-integrasa metabolismus MeSH
- inhibitory HIV-integrasy farmakologie MeSH
- látky proti HIV farmakologie MeSH
- lidé MeSH
- mnohočetná léková rezistence účinky léků genetika MeSH
- mutace * MeSH
- myši MeSH
- replikace viru účinky léků genetika MeSH
- tropismus virů účinky léků genetika MeSH
- virová léková rezistence účinky léků genetika MeSH
- vysoce účinné nukleotidové sekvenování MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Mixed lineage leukemia (MLL) fusion-driven acute leukemias represent a genetically distinct subset of leukemias with poor prognosis. MLL forms a ternary complex with the lens epithelium-derived growth factor (LEDGF/p75) and MENIN. LEDGF/p75, a chromatin reader recognizing H3K36me3 marks, contributes to the association of the MLL multiprotein complex to chromatin. Formation of this complex is critical for the development of MLL leukemia. Available X-ray data represent only a partial structure of the LEDGF/p75-MLL-MENIN complex. Using nuclear magnetic resonance spectroscopy, we identified an additional LEDGF/p75-MLL interface, which overlaps with the binding site of known LEDGF/p75 interactors-HIV-1 integrase, PogZ, and JPO2. Binding of these proteins or MLL to LEDGF/p75 is mutually exclusive. The resolved structure, as well as mutational analysis, shows that the interaction is primarily sustained via two aromatic residues of MLL (F148 and F151). Colony-forming assays in MLL-AF9(+) leukemic cells expressing MLL interaction-defective LEDGF/p75 mutants revealed that this interaction is essential for transformation. Finally, we show that the clonogenic growth of primary murine MLL-AF9-expressing leukemic blasts is selectively impaired upon overexpression of a LEDGF/p75-binding cyclic peptide CP65, originally developed to inhibit the LEDGF/p75-HIV-1 integrase interaction. The newly defined protein-protein interface therefore represents a new target for the development of therapeutics against LEDGF/p75-dependent MLL fusion-driven leukemic disorders. Cancer Res; 74(18); 5139-51. ©2014 AACR.
- MeSH
- akutní myeloidní leukemie genetika metabolismus MeSH
- cílená molekulární terapie MeSH
- fúzní onkogenní proteiny genetika metabolismus MeSH
- histonlysin-N-methyltransferasa chemie genetika metabolismus MeSH
- HIV-integrasa chemie metabolismus MeSH
- lidé MeSH
- magnetická rezonanční spektroskopie MeSH
- mezibuněčné signální peptidy a proteiny chemie genetika metabolismus MeSH
- MFC-7 buňky MeSH
- molekulární modely MeSH
- myši MeSH
- protoonkogenní protein MLL chemie genetika metabolismus MeSH
- vazba proteinů MeSH
- vazebná místa MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
HIV integrase (IN) catalyzes the insertion of proviral DNA into the host cell chromosome. While IN has strict sequence requirements for the viral cDNA ends, the integration site preference has been shown to be very diverse. Here, we mapped the HIV IN strand transfer reaction requirements using various short oligonucleotides (ON) that mimic the target DNA. Most double stranded DNA dodecamers served as excellent IN targets with variable integration efficiency depending mostly on the ON sequences. The preferred integration was lost with any changes in the geometry of the DNA double helical structures. Various hairpin-loop-forming ONs also served as efficient integration targets. Similar integration preferences were also observed for ONs, in which the nucleotide hairpin loop was replaced with a flexible aliphatic linker. The integration biases with all target DNA structures tested were significantly influenced by changes in the resulting secondary ON structures.
- MeSH
- dimerizace MeSH
- DNA virů genetika MeSH
- HIV-1 genetika MeSH
- HIV-integrasa genetika izolace a purifikace MeSH
- integrace viru genetika MeSH
- katalýza MeSH
- konformace nukleové kyseliny MeSH
- molekulární sekvence - údaje MeSH
- oligonukleotidy genetika MeSH
- sekvence nukleotidů MeSH
- substrátová specifita genetika MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Publikační typ
- práce podpořená grantem MeSH
Understanding the molecular mechanism of HIV-1 integrase (IN) activity is critical to find functional inhibitors for an effective AIDS therapy. A robust, fast, and sensitive method for studying IN activity is required. In this work, an assay for real-time label-free monitoring of the IN activity based on surface plasmon resonance was developed. This assay enabled direct monitoring of the integration of a viral doubled-stranded (ds) DNA into the host genome. The strand transfer reaction was detected by using two different DNA targets: supercoiled plasmid (pUC 19) and short palindrome oligonucleotide. The effect of the length of the DNA target on the possibility to monitor the actual process of the strand transfer reaction is discussed. The surface density of integrated ds-DNA was determined. IN binding to the oligonucleotide complexes and model DNA triplexes in the presence of various divalent ions as metal cofactors was investigated as well. The assay developed can serve as an important analytical tool to search for potential strand transfer reaction inhibitors as well as for the study of compounds interfering with the binding of ds long terminal repeats-IN complexes with the host DNA.
