BACKGROUND: Resistance to chemotherapy is a major problem in the treatment of patients with triple-negative breast cancer (TNBC). Preclinical data suggest that TNBC is dependent on proteasomes; however, clinical observations indicate that the efficacy of proteasome inhibitors in TNBC may be limited, suggesting the need for combination therapies. METHODS: We compared bortezomib and carfilzomib and their combinations with nelfinavir and lopinavir in TNBC cell lines and primary cells with regard to their cytotoxic activity, functional proteasome inhibition, and induction of the unfolded protein response (UPR). Furthermore, we evaluated the involvement of sXBP1, ABCB1, and ABCG2 in the cytotoxic activity of drug combinations. RESULTS: Carfilzomib, via proteasome β5 + β2 inhibition, is more cytotoxic in TNBC than bortezomib, which inhibits β5 + β1 proteasome subunits. The cytotoxicity of carfilzomib was significantly potentiated by nelfinavir or lopinavir. Carfilzomib with lopinavir induced endoplasmic reticulum stress and pro-apoptotic UPR through the accumulation of excess proteasomal substrate protein in TNBC in vitro. Moreover, lopinavir increased the intracellular availability of carfilzomib by inhibiting carfilzomib export from cells that express high levels and activity of ABCB1, but not ABCG2. CONCLUSION: Proteasome inhibition by carfilzomib combined with nelfinavir/lopinavir represents a potential treatment option for TNBC, warranting further investigation.

• MeSH
• ABC transportér z rodiny G, člen 2 * metabolismus   antagonisté a inhibitory   MeSH
• apoptóza účinky léků   MeSH
• bortezomib * farmakologie   MeSH
• inhibitory HIV-proteasy * farmakologie   MeSH
• inhibitory proteasomu farmakologie   MeSH
• lidé MeSH
• Lopinavir * farmakologie   MeSH
• nádorové buněčné linie MeSH
• nádorové proteiny antagonisté a inhibitory   metabolismus   MeSH
• nelfinavir * farmakologie   MeSH
• oligopeptidy * farmakologie   MeSH
• P-glykoproteiny metabolismus   MeSH
• protokoly protinádorové kombinované chemoterapie farmakologie   MeSH
• signální dráha UPR * účinky léků   MeSH
• stres endoplazmatického retikula účinky léků   MeSH
• synergismus léků * MeSH
• triple-negativní karcinom prsu * farmakoterapie   patologie   MeSH
• XBP1 metabolismus   genetika   MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH

OBJECTIVES: To assess the potential of second-generation proteasome inhibition by carfilzomib and its combination with the human immunodeficiency virus (HIV) protease inhibitors (HIV-PIs) lopinavir and nelfinavir in vitro for improved treatment of clear cell renal cell cancer (ccRCC). MATERIALS AND METHODS: Cytotoxicity, reactive oxygen species (ROS) production, and unfolded protein response (UPR) activation of proteasome inhibitors, HIV-PIs, and their combination were assessed in three cell lines and primary cells derived from three ccRCC tumours by MTS assay, flow cytometry, quantitative reverse transcriptase-polymerase chain reaction and western blot, respectively. Proteasome activity was determined by activity based probes. Flow cytometry was used to assess apoptosis by annexin V/propidium iodide assay and ATP-binding cassette sub-family B member 1 (ABCB1) activity by MitoTrackerTM Green FM efflux assay (Thermo Fisher Scientific, MA, USA). RESULTS: Lopinavir and nelfinavir significantly increased the cytotoxic effect of carfilzomib in all cell lines and primary cells. ABCB1 efflux pump inhibition, induction of ROS production, and UPR pre-activation by lopinavir were identified as underlying mechanisms of this strong synergistic effect. Combined treatment led to unresolved protein stress, increased activation of pro-apoptotic UPR pathway, and a significant increase in apoptosis. CONCLUSION: The combination of the proteasome inhibitor carfilzomib and the HIV-PIs lopinavir and nelfinavir has a strong synergistic cytotoxic activity against ccRCCin vitro at therapeutically relevant drug concentrations. This effect is most likely explained by synergistic UPR triggering and ABCB1-modulation caused by HIV-PIs. Our findings suggest that combined treatment of second-generation proteasome inhibitors and HIV-PIs should be investigated in patients with metastatic RCC within a clinical trial.

• MeSH
• chemorezistence MeSH
• inhibitory HIV-proteasy terapeutické užití   MeSH
• inhibitory proteasomu terapeutické užití   MeSH
• karcinom z renálních buněk farmakoterapie   MeSH
• lidé MeSH
• Lopinavir terapeutické užití   MeSH
• nádorové buněčné linie MeSH
• nádory ledvin farmakoterapie   MeSH
• nelfinavir terapeutické užití   MeSH
• protinádorové látky terapeutické užití   MeSH
• stres endoplazmatického retikula účinky léků   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Nelfinavir mesylate (NFV), a human immunodeficiency virus (HIV) protease inhibitor, is an integral component of highly active anti retro viral therapy (HAART) for management of AIDS. NFV possesses pH-dependent solubility and has low and variable bioavailability hampering its use in therapeutics. Lipid-based particulates have shown to improve solubility of poorly water soluble drugs and oral absorption, thereby aiding in improved bioavailability. The current study compares potential of vesicular and solid lipid nanocarriers of NFV with drug nanocrystallites and microvesicular systems like cochleates in improving bioavailability of NFV. The paper outlines investigation of systems using in vitro models like in vitro lipolysis, in vitro release, and permeation through cell lines to predict the in vivo potential of nanocarriers. Finally, in vivo pharmacokinetic study is reported which provided proof of concept in sync with results from in vitro studies. Graphical Abstract ᅟ.

• MeSH
• biologická dostupnost MeSH
• Caco-2 buňky MeSH
• inhibitory HIV-proteasy chemie   MeSH
• krysa rodu rattus MeSH
• lidé MeSH
• lipidy chemie   MeSH
• nelfinavir chemie   farmakokinetika   MeSH
• potkani Sprague-Dawley MeSH
• rozpustnost MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• lidé MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• srovnávací studie 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

Enzým cytochróm P450 2C19 je jedným z hlavných faktorov farmakokinetickej variability bežne užívaných liečiv. Niekoľko štúdií poukázalo na významný vplyv polymorfizmu enzýmu CYP2C19 s možným výskytom klinicky závažných nežiadúcich účinkov. Cieľom tretej časti tohoto článku je popísať vplyv genetického polymorfizmu cytochrómu P450 2C19 na účinok liečiv.

Cytochrome P450 2C19 is the important cause for great differences in the pharmacokinetics of a number of clinically used drugs. Several studies have demonstrated that the CYP2C19 polymorphism is important for several drugs and may be associated with the occurrence of clinically relevant side effects. The third part of this paper focuses on the influence of genetic polymorphism of cytochrome P450 2C19 on drug effect.

• MeSH
• antidepresiva farmakokinetika   terapeutické užití   MeSH
• antifungální látky farmakokinetika   terapeutické užití   MeSH
• antivirové látky MeSH
• aromatické hydroxylasy genetika   MeSH
• benzodiazepiny farmakokinetika   terapeutické užití   MeSH
• cyklofosfamid farmakokinetika   terapeutické užití   MeSH
• enzymy genetika   MeSH
• farmakogenetika MeSH
• fenytoin farmakokinetika   terapeutické užití   MeSH
• genotyp MeSH
• inhibitory agregace trombocytů farmakokinetika   terapeutické užití   MeSH
• inhibitory protonové pumpy farmakokinetika   terapeutické užití   MeSH
• léčivé přípravky metabolismus   MeSH
• lidé MeSH
• nelfinavir farmakokinetika   terapeutické užití   MeSH
• polymorfismus genetický MeSH
• systém (enzymů) cytochromů P-450 genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• přehledy MeSH

Human immunodeficiency virus (HIV) encodes an aspartic protease (PR) that cleaves viral polyproteins into mature proteins, thus leading to the formation of infectious particles. Protease inhibitors (PIs) are successful virostatics. However, their efficiency is compromised by antiviral resistance. In the PR sequence of viral variants resistant to the PI nelfinavir, the mutations D30N and L90M appear frequently. However, these two mutations are seldom found together in vivo, suggesting that there are two alternative evolutionary pathways leading to nelfinavir resistance. Here we analyze the proteolytic activities, X-ray structures, and thermodynamics of inhibitor binding to HIV-1 PRs harboring the D30N and L90M mutations alone and in combination with other compensatory mutations. Vitality values obtained for recombinant mutant proteases and selected PR inhibitors confirm the crucial role of mutations in positions 30 and 90 for nelfinavir resistance. The combination of the D30N and L90M mutations significantly increases the enzyme vitality in the presence of nelfinavir, without a dramatic decrease in the catalytic efficiency of the recombinant enzyme. Crystal structures, molecular dynamics simulations, and calorimetric data for four mutants (D30N, D30N/A71V, D30N/N88D, and D30N/L90M) were used to augment our kinetic data. Calorimetric analysis revealed that the entropic contribution to the mutant PR/nelfinavir interaction is less favorable than the entropic contribution to the binding of nelfinavir by wild-type PR. This finding is supported by the structural data and simulations; nelfinavir binds most strongly to the wild-type protease, which has the lowest number of protein-ligand hydrogen bonds and whose structure exhibits the greatest degree of fluctuation upon inhibitor binding.

• MeSH
• aktivace enzymů MeSH
• financování organizované MeSH
• HIV-1 enzymologie   genetika   MeSH
• HIV-proteasa genetika   chemie   MeSH
• inhibitory HIV-proteasy chemie   MeSH
• kinetika MeSH
• konformace proteinů MeSH
• krystalografie rentgenová MeSH
• molekulární modely MeSH
• mutace MeSH
• nelfinavir MeSH
• termodynamika MeSH
• vazba proteinů MeSH
• virová léková rezistence MeSH

• Klíčová slova
• VIRACEPT,
• MeSH
• HIV infekce farmakoterapie   MeSH
• lidé MeSH
• nelfinavir MeSH
• Check Tag
• lidé MeSH

• Klíčová slova
• ZALCITABIN, SANQUINAVIR,
• MeSH
• antivirové látky terapeutické užití   MeSH
• dospělí MeSH
• HIV infekce farmakoterapie   terapie   MeSH
• indinavir MeSH
• inhibitory reverzní transkriptasy aplikace a dávkování   terapeutické užití   MeSH
• lamivudin MeSH
• lidé MeSH
• nelfinavir MeSH
• ritonavir MeSH
• stavudin MeSH
• těhotenství MeSH
• zidovudin MeSH
• Check Tag
• dospělí MeSH
• lidé MeSH
• mužské pohlaví MeSH
• těhotenství MeSH
• ženské pohlaví MeSH
• Publikační typ
• přehledy MeSH