Low solubility of reactants or products in aqueous solutions can result in the enzymatic catalytic reactions that can occur in non-aqueous solutions. In current study we investigated aqueous solutions containing different organic solvents / deep eutectic solvents (DESs) that can influence the protease enzyme's activity, structural, and thermal stabilities. Retroviral aspartic protease enzyme is responsible for the cleavage of the polypeptide precursors into mature viral components, a very crucial step for virus life cycle. In molecular dynamic simulations (MDS), the complex of the protease enzyme with Darunavirwas found highly stable in urea aqueous solution compared to when with the ethylene glycol (EG) or glycerol solvents. Particularly, in different organic solvents the presence of Darunavir induced protein-protein interactions within the protease homodimer. For the systems with EG or glycerol solvents, the flap domains of the enzyme formed an "open" conformation which lead to a weak binding affinity with the drug. Conserved D25 and G27 residues among this family of the aspartic protease enzymes made a stable binding with Darunavir in the urea systems. Unfolding of the protease dimer was initiated due to self-aggregation for the EG or glycerol organic solvents, which formed an "open" conformation for the flap domains. On the contrary lack of such clustering in urea solvent, the protease showed conventional structural folding in the presence or absence of the drug molecule. These novel findings may help to better understand the protease enzymes, which could be controlled by deep eutectic solvents.

• MeSH
• darunavir MeSH
• ethylenglykol * MeSH
• glycerol * MeSH
• močovina farmakologie   chemie   MeSH
• proteasy MeSH
• rozpouštědla chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Darunavir is the most recently approved human immunodeficiency virus (HIV) protease (PR) inhibitor (PI) and is active against many HIV type 1 PR variants resistant to earlier-generation PIs. Darunavir shows a high genetic barrier to resistance development, and virus strains with lower sensitivity to darunavir have a higher number of PI resistance-associated mutations than viruses resistant to other PIs. In this work, we have enzymologically and structurally characterized a number of highly mutated clinically derived PRs with high levels of phenotypic resistance to darunavir. With 18 to 21 amino acid residue changes, the PR variants studied in this work are the most highly mutated HIV PR species ever studied by means of enzyme kinetics and X-ray crystallography. The recombinant proteins showed major defects in substrate binding, while the substrate turnover was less affected. Remarkably, the overall catalytic efficiency of the recombinant PRs (5% that of the wild-type enzyme) is still sufficient to support polyprotein processing and particle maturation in the corresponding viruses. The X-ray structures of drug-resistant PRs complexed with darunavir suggest that the impaired inhibitor binding could be explained by change in the PR-inhibitor hydrogen bond pattern in the P2' binding pocket due to a substantial shift of the aminophenyl moiety of the inhibitor. Recombinant virus phenotypic characterization, enzyme kinetics, and X-ray structural analysis thus help to explain darunavir resistance development in HIV-positive patients.

• MeSH
• genové produkty env - virus lidské imunodeficience metabolismus   MeSH
• genové produkty gag - virus lidské imunodeficience metabolismus   MeSH
• HIV infekce virologie   MeSH
• HIV-1 izolace a purifikace   účinky léků   MeSH
• HIV-proteasa genetika   chemie   metabolismus   MeSH
• inhibitory HIV-proteasy farmakologie   MeSH
• krystalografie rentgenová MeSH
• lidé MeSH
• missense mutace MeSH
• molekulární modely MeSH
• molekulární sekvence - údaje MeSH
• mutační analýza DNA MeSH
• polyproteiny metabolismus   MeSH
• sekvence aminokyselin MeSH
• substituce aminokyselin MeSH
• sulfonamidy farmakologie   MeSH
• terciární struktura proteinů MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• virová léková rezistence MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• práce podpořená grantem MeSH

UNLABELLED: We report enzymologic, thermodynamic and structural analyses of a series of six clinically derived mutant HIV proteases (PR) resistant to darunavir. As many as 20 mutations in the resistant PRs decreased the binding affinity of darunavir by up to 13 000-fold, mostly because of a less favorable enthalpy of binding that was only partially compensated by the entropic contribution. X-ray structure analysis suggested that the drop in enthalpy of darunavir binding to resistant PR species was mostly the result of a decrease in the number of hydrogen bonds and a loosening of the fit between the inhibitor and the mutated enzymes. The favorable entropic contribution to darunavir binding to mutated PR variants correlated with a larger burial of the nonpolar solvent-accessible surface area upon inhibitor binding. We show that even very dramatic changes in the PR sequence leading to the loss of hydrogen bonds with the inhibitor could be partially compensated by the entropy contribution as a result of the burial of the larger nonpolar surface area of the mutated HIV PRs. DATABASE: Atomic coordinates and structure factors for the crystal structures PRwt-DRV and PRDRV2-DRV complex have been deposited in the Protein Data Bank under accession codes 4LL3 and 3TTP, respectively. STRUCTURED DIGITAL ABSTRACT: • PR and PR bind by x-ray crystallography (View interaction).

• MeSH
• HIV-proteasa chemie   genetika   metabolismus   MeSH
• inhibitory HIV-proteasy chemie   farmakologie   MeSH
• molekulární sekvence - údaje MeSH
• mutace * MeSH
• sekvence aminokyselin MeSH
• simulace molekulového dockingu * MeSH
• sulfonamidy chemie   farmakologie   MeSH
• termodynamika MeSH
• vazba proteinů MeSH
• virová léková rezistence genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

HIV-1 protease (PR) is a homodimeric enzyme that is autocatalytically cleaved from the Gag-Pol precursor. Known PR inhibitors bind the mature enzyme several orders of magnitude more strongly than the PR precursor. Inhibition of PR at the precursor level, however, may stop the process at its rate-limiting step before the proteolytic cascade is initiated. Due to its structural heterogeneity, limited solubility and autoprocessing, the PR precursor is difficult to access by classical methods, and limited knowledge regarding precursor inhibition is available. Here, we describe a cell-based assay addressing precursor inhibition. We used a reporter molecule containing the transframe (TFP) and p6* peptides, PR, and N-terminal fragment of reverse transcriptase flanked by the fluorescent proteins mCherry and EGFP on its N- and C- termini, respectively. The level of FRET between EGFP and mCherry indicates the amount of unprocessed reporter, allowing specific monitoring of precursor inhibition. The inhibition can be quantified by flow cytometry. Additionally, two microscopy techniques confirmed that the reporter remains unprocessed within individual cells upon inhibition. We tested darunavir, atazanavir and nelfinavir and their combinations against wild-type PR. Shedding light on an inhibitor's ability to act on non-mature forms of PR may aid novel strategies for next-generation drug design.

• MeSH
• atazanavir sulfát farmakologie   MeSH
• buněčné linie MeSH
• darunavir farmakologie   MeSH
• fluorescenční barviva MeSH
• HIV-1 enzymologie   MeSH
• inhibitory HIV-proteasy farmakologie   MeSH
• látky proti HIV farmakologie   MeSH
• lidé MeSH
• nelfinavir farmakologie   MeSH
• proteinové prekurzory antagonisté a inhibitory   MeSH
• proteolýza MeSH
• průtoková cytometrie MeSH
• rezonanční přenos fluorescenční energie MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

• Klíčová slova
• dolutegravir, tenofovir alafenamid fumarát, elvitegravir,
• MeSH
• adenin analogy a deriváty   terapeutické užití   MeSH
• AIDS etiologie   farmakoterapie   patofyziologie   MeSH
• antiretrovirové látky škodlivé účinky   terapeutické užití   MeSH
• chinolony škodlivé účinky   terapeutické užití   MeSH
• darunavir škodlivé účinky   terapeutické užití   MeSH
• fixní kombinace léků MeSH
• heterocyklické sloučeniny tricyklické škodlivé účinky   terapeutické užití   MeSH
• HIV infekce * etiologie   farmakoterapie   patofyziologie   MeSH
• inhibitory HIV-integrasy * škodlivé účinky   terapeutické užití   MeSH
• látky proti HIV * škodlivé účinky   terapeutické užití   MeSH
• lidé MeSH
• rilpivirin škodlivé účinky   terapeutické užití   MeSH
• Check Tag
• lidé MeSH

High-pressure methods have become an interesting tool of investigation of structural stability of proteins. They are used to study protein unfolding, but dissociation of oligomeric proteins can be addressed this way, too. HIV-1 protease, although an interesting object of biophysical experiments, has not been studied at high pressure yet. In this study HIV-1 protease is investigated by high pressure (up to 600 MPa) fluorescence spectroscopy of either the inherent tryptophan residues or external 8-anilino-1-naphtalenesulfonic acid at 25°C. A fast concentration-dependent structural transition is detected that corresponds to the dimer-monomer equilibrium. This transition is followed by a slow concentration independent transition that can be assigned to the monomer unfolding. In the presence of a tight-binding inhibitor none of these transitions are observed, which confirms the stabilizing effect of inhibitor. High-pressure enzyme kinetics (up to 350 MPa) also reveals the stabilizing effect of substrate. Unfolding of the protease can thus proceed only from the monomeric state after dimer dissociation and is unfavourable at atmospheric pressure. Dimer-destabilizing effect of high pressure is caused by negative volume change of dimer dissociation of -32.5 mL/mol. It helps us to determine the atmospheric pressure dimerization constant of 0.92 μM. High-pressure methods thus enable the investigation of structural phenomena that are difficult or impossible to measure at atmospheric pressure.

• MeSH
• anilin-naftalen sulfonáty metabolismus   MeSH
• atmosférický tlak MeSH
• darunavir metabolismus   MeSH
• dimerizace MeSH
• fluorescenční spektrometrie MeSH
• HIV-proteasa chemie   metabolismus   MeSH
• inhibitory HIV-proteasy metabolismus   MeSH
• kinetika MeSH
• konformace proteinů MeSH
• lidé MeSH
• molekulární modely MeSH
• multimerizace proteinu MeSH
• sbalování proteinů * MeSH
• stabilita proteinů účinky léků   MeSH
• termodynamika MeSH
• tryptofan metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

• MeSH
• atazanavir sulfát škodlivé účinky   MeSH
• chronická nemoc MeSH
• darunavir MeSH
• dospělí MeSH
• HIV infekce * farmakoterapie   MeSH
• látky proti HIV * škodlivé účinky   terapeutické užití   MeSH
• ledviny účinky léků   MeSH
• lidé středního věku MeSH
• lidé MeSH
• Lopinavir škodlivé účinky   MeSH
• nemoci ledvin * chemicky indukované   MeSH
• senioři MeSH
• tenofovir škodlivé účinky   MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• senioři MeSH
• Publikační typ
• přehledy MeSH

Infekcie vírusom HPV (Human Papilloma Virus) patria medzi najčastejšie infekcie kože a slizníc. Niektoré z nich možno zaradiť medzi pohlavne prenosné infekcie, s najvyšším výskytom vo vekovo mladšej sexuálne aktívnej populácii. Plne funkčný imunitný systém sa dokáže v niektorých prípadoch aj bez terapeutickej intervencie vysporiadať aj s vysoko rizikovými typmi vírusu. U imunodeficientných jedincov môže mať však infekcia HPV odlišný klinický obraz a torpídny priebeh. Autori prezentujú prípad 48-ročného HIV pozitívneho pacienta s početnými bradavicovitými útvarmi na tvári. Histopatologický obraz lézií potvrdil diagnózu verrucae vulgares. Molekulárno-genetickými metódami (PCR) bola dokázaná prítomnosť vysoko onkogénneho HPV 16, ktorý sa nepovažuje za vyvolávateľa verrucae vulgares, ale býva vyvolávateľom dysplastických zmien lokalizovaných na cervixe, vagíne, vulve, penise, anuse a oropharynxe. Pre HIV infekciu je pacient liečený trojkombináciou virostatík (lamivudin, etravirin, darunavir) s iniciálnou hodnotou CD4 T lymfocytov 30/?l. Antivirotická liečba nebola počas lokálnej liečby HPV infekcie prerušená. Aplikácia lokálneho imunomodulansu 5 % imiquimodu účinne eradikovala kožné lézie počas 1-mesačnej liečby. Počas 1 roka od skončenia liečby bol pacient bez kožných prejavov HPV infekcie.

HPV infections (Human Papilloma Virus) are the most common infections of the skin and mucous membranes. Some of them can be classified as sexually transmitted infections with the highest incidence in the younger sexually active population. Fully functional immune system is able to deal with the high-risk virus infection in some cases. In immunodeficient individuals HPV infection can present with different clinical picture and torpid course. The authors present a case of 48-year-old HIV-positive patient with numerous papular lesions on his face. Histopathologic examination confirmed the diagnosis of vulgar warts. Molecular genetic method (PCR) demonstrated the presence of highly oncogenic HPV 16, which is not considered as an etiological agent in verrucae vulgares. HPV 16 may lead to dysplastic changes localized on the cervix, vagina, vulva, penis, anus and oropharynx. Due to HIV infection, the patient was treated with triple combination of antivirals (lamivudine, etravirine, darunavir), with 30/?l of CD4 T lymphocyte count initially. Antiretroviral treatment was given continuously during topical treatment of HPV infection. Topical application of imiquimod was effective in eradicating of all skin lesions within 1 month of therapy. After one-year follow up, the patient was without any cutaneous manifestations of HPV infection.

• MeSH
• antiretrovirové látky aplikace a dávkování   terapeutické užití   MeSH
• bradavice * diagnóza   etiologie   imunologie   terapie   MeSH
• buněčná imunita MeSH
• HIV infekce * komplikace   MeSH
• imunorestituční zánětlivý syndrom MeSH
• infekce papilomavirem * diagnóza   komplikace   terapie   MeSH
• lidé středního věku MeSH
• lidé MeSH
• lidský papilomavirus 16 izolace a purifikace   účinky léků   MeSH
• polymerázová řetězová reakce MeSH
• průtoková cytometrie MeSH
• RNA virová MeSH
• syfilis MeSH
• Check Tag
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• Publikační typ
• kazuistiky MeSH

The inhibition of P-glycoprotein (ABCB1) could lead to increased drug plasma concentrations and hence increase drug toxicity. The evaluation of a drug's ability to inhibit ABCB1 is complicated by the presence of several transport-competent sites within the ABCB1 binding pocket, making it difficult to select appropriate substrates. Here, we investigate the capacity of antiretrovirals and direct-acting antivirals to inhibit the ABCB1-mediated intestinal efflux of [3H]-digoxin and compare it with our previous rhodamine123 study. At concentrations of up to 100 μM, asunaprevir, atazanavir, daclatasvir, darunavir, elbasvir, etravirine, grazoprevir, ledipasvir, lopinavir, rilpivirine, ritonavir, saquinavir, and velpatasvir inhibited [3H]-digoxin transport in Caco-2 cells and/or in precision-cut intestinal slices prepared from the human jejunum (hPCIS). However, abacavir, dolutegravir, maraviroc, sofosbuvir, tenofovir disoproxil fumarate, and zidovudine had no inhibitory effect. We thus found that most of the tested antivirals have a high potential to cause drug-drug interactions on intestinal ABCB1. Comparing the Caco-2 and hPCIS experimental models, we conclude that the Caco-2 transport assay is more sensitive, but the results obtained using hPCIS agree better with reported in vivo observations. More inhibitors were identified when using digoxin as the ABCB1 probe substrate than when using rhodamine123. However, both approaches had limitations, indicating that inhibitory potency should be tested with at least these two ABCB1 probes.

• Publikační typ
• časopisecké články MeSH

• MeSH
• dítě MeSH
• esenciální léky normy   terapeutické užití   MeSH
• informační služby o lécích MeSH
• spotřeba léčiv MeSH
• Check Tag
• dítě MeSH

• Konspekt
• Farmacie. Farmakologie
• NLK Obory
• farmacie a farmakologie
• NLK Publikační typ
• publikace WHO