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

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

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

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

• Klíčová slova
• antiretrovirová léčba,
• MeSH
• antivirové látky * MeSH
• HIV infekce * diagnóza   etiologie   farmakoterapie   prevence a kontrola   MeSH
• HIV reverzní transkriptasa biosyntéza   účinky léků   MeSH
• inhibitory proteas aplikace a dávkování   škodlivé účinky   terapeutické užití   MeSH
• inhibitory reverzní transkriptasy aplikace a dávkování   škodlivé účinky   terapeutické užití   MeSH
• klinické zkoušky jako téma MeSH
• kontraceptiva ženská aplikace a dávkování   MeSH
• lidé MeSH
• nežádoucí účinky léčiv metabolismus   MeSH
• socioekonomické faktory MeSH
• těhotenství účinky léků   MeSH
• ženy MeSH
• Check Tag
• lidé MeSH
• těhotenství účinky léků   MeSH
• ženské pohlaví * MeSH
• Publikační typ
• práce podpořená grantem MeSH
• přehledy MeSH

• MeSH
• dějiny lékařství MeSH
• HIV reverzní transkriptasa dějiny   izolace a purifikace   MeSH
• Publikační typ
• novinové články MeSH

In this study, 27 HIV-1-positive patients on long-term highly active antiretroviral therapy (HAART) in the Czech Republic were followed for a period of up to 7 years. Variability of the HIV-1 protease (PR) sequence common in the Czech Republic was observed. Under the pressure of inhibitors of protease (PRIs) and reverse transcriptase (RTIs) mutations in PR were detected. Development of resistance to PRIs was followed by a decrease in CD4 count and increase in viral load. The dynamics of viral load closely corresponded to the accumulation of specific primary mutations in PR and RT. Out of 27 patients 18 developed resistance to PRIs and the prolonged therapy led to the accumulation of a higher number of amino acid changes associated with the resistance and, consequently, cross-resistance to several PRIs was observed. These multi-resistant variants of HIV-1 with mutations in PR could not be inhibited sufficiently with PRIs that are currently available in clinical practice. Efficient yet temporary suppression of viral replication was achieved by a lopinavir (LPV) treatment.

• MeSH
• financování organizované MeSH
• genotyp MeSH
• HIV infekce farmakoterapie   virologie   MeSH
• HIV reverzní transkriptasa genetika   MeSH
• HIV-1 genetika   izolace a purifikace   účinky léků   MeSH
• HIV-proteasa genetika   MeSH
• inhibitory HIV-proteasy aplikace a dávkování   terapeutické užití   MeSH
• látky proti HIV aplikace a dávkování   terapeutické užití   MeSH
• lidé MeSH
• počet CD4 lymfocytů MeSH
• progrese nemoci MeSH
• substituce aminokyselin MeSH
• virová léková rezistence genetika   MeSH
• vysoce aktivní antiretrovirová terapie MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH
• Geografické názvy
• Česká republika MeSH

V podmínkách ČR během léčby HAART u HIV+ pacientů hodnotit restituci imunitních funkcí stanovením subtypů, pomocí exprese cytokinů a stupně aktivace T lymfocytů. Zhodnocení a optimalizace stávajících profylaktických schémat oportunních infekcí asociujících se na HIV infekci se zaměřením na subklinické infekce (CMV, MAC) před nasazením HAART. Sledování toxicity léčby a hledání dalších farmakologických možností vedoucích k zmírnění především lipodystrofie.; Research on the restitution of some functions of the immune system of HIV positive patients treated with HAART and its impact on prophylaxis of opportunistic infections. We will evaluate the current prohylaxis of opportunistic infections and we will search a new scheme of this prophylaxis in the Czech Republic, as well.

• MeSH
• antivirové látky MeSH
• HIV infekce farmakoterapie   MeSH
• HIV reverzní transkriptasa MeSH
• inhibitory reverzní transkriptasy MeSH
• zidovudin MeSH

• Konspekt
• Patologie. Klinická medicína
• NLK Obory
• dermatovenerologie
• NLK Publikační typ
• závěrečné zprávy o řešení grantu IGA MZ ČR

Studium resistentních kmenů HIV v pacientech léčených inhibitory proteasy. Sekvenování, klonování a charakterisace rekombinantních mutantních proteas. Návrh specifických inhibitorů. Evoluce proteasy a dalších virových genů u HIV positivních pacientů.; Resistant HIV strains in patients treated by protease inhibitors. Sequencing, cloning and analysis of recombinant mutant proteases. Design of a broad specificity inhibitor. Co-evolution of protease and pther viral genes in HIV under selection pressure.

• MeSH
• DNA virů MeSH
• HIV reverzní transkriptasa MeSH
• HIV-1 genetika   MeSH
• virová léková rezistence MeSH

• Konspekt
• Patologie. Klinická medicína
• NLK Obory
• dermatovenerologie
• biologie
• NLK Publikační typ
• závěrečné zprávy o řešení grantu IGA MZ ČR