Non-nucleoside reverse transcriptase inhibitors (NNRTIs) represent cornerstones of current regimens for treatment of human immunodeficiency virus type 1 (HIV-1) infections. However, NNRTIs usually suffer from low aqueous solubility and the emergence of resistant viral strains. In the present work, novel bicyclic NNRTIs derived from etravirine (ETV) and rilpivirine (RPV), bearing modified purine, tetrahydropteridine, and pyrimidodiazepine cores, were designed and prepared. Compounds 2, 4, and 6 carrying the acrylonitrile moiety displayed single-digit nanomolar activities against the wild-type (WT) virus (EC50 = 2.5, 2.7, and 3.0 nM, respectively), where the low nanomolar activity was retained against HXB2 (EC50 = 2.2-2.8 nM) and the K103N and Y181C mutated strains (fold change, 1.2-6.7×). Most importantly, compound 2 exhibited significantly improved phosphate-buffered saline solubility (10.4 μM) compared to ETV and RPV (≪1 μM). Additionally, the binding modes of compounds 2, 4, and 6 to the reverse transcriptase were studied by X-ray crystallography.

• MeSH
• HIV infekce * farmakoterapie   MeSH
• HIV reverzní transkriptasa metabolismus   MeSH
• HIV-1 * metabolismus   MeSH
• inhibitory reverzní transkriptasy MeSH
• látky proti HIV * chemie   MeSH
• lidé MeSH
• racionální návrh léčiv MeSH
• rilpivirin terapeutické užití   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH

Cryptococcosis is an invasive infection that accounts for 15% of AIDS-related fatalities. Still, treating cryptococcosis remains a significant challenge due to the poor availability of effective antifungal therapies and emergence of drug resistance. Interestingly, protease inhibitor components of antiretroviral therapy regimens have shown some clinical benefits in these opportunistic infections. We investigated Major aspartyl peptidase 1 (May1), a secreted Cryptococcus neoformans protease, as a possible target for the development of drugs that act against both fungal and retroviral aspartyl proteases. Here, we describe the biochemical characterization of May1, present its high-resolution X-ray structure, and provide its substrate specificity analysis. Through combinatorial screening of 11,520 compounds, we identified a potent inhibitor of May1 and HIV protease. This dual-specificity inhibitor exhibits antifungal activity in yeast culture, low cytotoxicity, and low off-target activity against host proteases and could thus serve as a lead compound for further development of May1 and HIV protease inhibitors.

• MeSH
• antifungální látky chemie   metabolismus   farmakologie   MeSH
• aspartátové proteasy antagonisté a inhibitory   genetika   metabolismus   MeSH
• Cryptococcus neoformans enzymologie   MeSH
• fungální proteiny antagonisté a inhibitory   genetika   metabolismus   MeSH
• HIV-proteasa chemie   metabolismus   MeSH
• HIV enzymologie   MeSH
• houby účinky léků   MeSH
• katalytická doména MeSH
• krystalografie rentgenová MeSH
• preklinické hodnocení léčiv MeSH
• rekombinantní proteiny biosyntéza   chemie   izolace a purifikace   MeSH
• simulace molekulární dynamiky MeSH
• substrátová specifita MeSH
• vazebná místa MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

HIV-1 reverse transcriptase (RT) possesses both DNA polymerase activity and RNase H activity that act in concert to convert single-stranded RNA of the viral genome to double-stranded DNA that is then integrated into the DNA of the infected cell. Reverse transcriptase-catalyzed reverse transcription critically relies on the proper generation of a polypurine tract (PPT) primer. However, the mechanism of PPT primer generation and the features of the PPT sequence that are critical for its recognition by HIV-1 RT remain unclear. Here, we used a chemical cross-linking method together with molecular dynamics simulations and single-molecule assays to study the mechanism of PPT primer generation. We found that the PPT was specifically and properly recognized within covalently tethered HIV-1 RT-nucleic acid complexes. These findings indicated that recognition of the PPT occurs within a stable catalytic complex after its formation. We found that this unique recognition is based on two complementary elements that rely on the PPT sequence: RNase H sequence preference and incompatibility of the poly(rA/dT) tract of the PPT with the nucleic acid conformation that is required for RNase H cleavage. The latter results from rigidity of the poly(rA/dT) tract and leads to base-pair slippage of this sequence upon deformation into a catalytically relevant geometry. In summary, our results reveal an unexpected mechanism of PPT primer generation based on specific dynamic properties of the poly(rA/dT) segment and help advance our understanding of the mechanisms in viral RNA reverse transcription.

• MeSH
• DNA primery biosyntéza   chemie   MeSH
• DNA virů MeSH
• HIV reverzní transkriptasa metabolismus   fyziologie   MeSH
• HIV-1 genetika   MeSH
• konformace nukleové kyseliny MeSH
• krystalografie rentgenová metody   MeSH
• nukleové kyseliny MeSH
• poly A MeSH
• poly U MeSH
• polynukleotidy MeSH
• puriny chemie   MeSH
• ribonukleasa H metabolismus   MeSH
• RNA virová chemie   MeSH
• sekvence nukleotidů 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/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

Replication of human immunodeficiency virus 1 (HIV-1) involves conversion of its single-stranded RNA genome to double-stranded DNA, which is integrated into the genome of the host. This conversion is catalyzed by reverse transcriptase (RT), which possesses DNA polymerase and RNase H domains. The available crystal structures suggest that at any given time the RNA/DNA substrate interacts with only one active site of the two domains of HIV-1 RT. Unknown is whether a simultaneous interaction of the substrate with polymerase and RNase H active sites is possible. Therefore, the mechanism of the coordination of the two activities is not fully understood. We performed molecular dynamics simulations to obtain a conformation of the complex in which the unwound RNA/DNA substrate simultaneously interacts with the polymerase and RNase H active sites. When the RNA/DNA hybrid was immobilized at the polymerase active site, RNase H cleavage occurred, experimentally verifying that the substrate can simultaneously interact with both active sites. These findings demonstrate the existence of a transient conformation of the HIV-1 RT substrate complex, which is important for modulating and coordinating the enzymatic activities of HIV-1 RT.

• MeSH
• DNA chemie   metabolismus   MeSH
• HIV reverzní transkriptasa chemie   metabolismus   MeSH
• katalytická doména MeSH
• ribonukleasa H chemie   metabolismus   MeSH
• RNA chemie   metabolismus   MeSH
• simulace molekulární dynamiky MeSH
• Publikační typ
• časopisecké články MeSH

• Klíčová slova
• nezralé částice, inhibitory schopnosti tvořit částice,
• MeSH
• fluorescenční mikroskopie MeSH
• genové produkty gag - virus lidské imunodeficience MeSH
• HIV infekce epidemiologie   etiologie   terapie   MeSH
• HIV-proteasa MeSH
• HIV genetika   patogenita   růst a vývoj   účinky léků   MeSH
• látky proti HIV * farmakologie   MeSH
• léková rezistence MeSH
• lidé MeSH
• mutantní proteiny MeSH
• objevování léků * metody   MeSH
• techniky in vitro MeSH
• testy rezistence k nukleáze MeSH
• vysoce aktivní antiretrovirová terapie ekonomika   škodlivé účinky   MeSH
• Check Tag
• lidé MeSH

To elucidate the structure-geometry-activity relationship in diarylpyrimidine family (DAPYs) containing carbonyl linker between the central pyrimidine core and phenyl type B-arm, a series of (2,6-difluorophenyl)(2-(phenylamino)pyrimidin-4-yl)methanones was designed, prepared and tested for their anti-HIV-1 activity. The carbonyl linker bearing B phenyl arm was successfully attached at both C-2 and C-4 positions of the central pyrimidine ring using a new synthetic approach. Further modifications of target compounds are present at C-5 position of the pyrimidine ring. In vitro anti-HIV-1 activity study performed on a series of 22 compounds confirmed the crucial importance of both conformational rigidity between phenyl B arm and the pyrimidine core linked through the carbonyl bridge, as well as presence of fluoro substituents in ortho-positions of phenyl B moiety. The most potent derivative of the series, compound 17, having almost perpendicular angle within the two planes made from the B aromatic arm and the pyrimidine ring, exhibited low nanomolar anti-HIV-1 activity (EC50 = 4 nM) with no significant toxicity (CC50 > 57.1 μM).

• MeSH
• HIV reverzní transkriptasa antagonisté a inhibitory   chemie   metabolismus   MeSH
• HIV-1 účinky léků   enzymologie   MeSH
• inhibitory reverzní transkriptasy chemie   metabolismus   farmakologie   MeSH
• konformace proteinů MeSH
• pyrimidiny chemie   metabolismus   farmakologie   MeSH
• racionální návrh léčiv * MeSH
• simulace molekulového dockingu MeSH
• Publikační typ
• časopisecké články 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