Cathepsin L is a key host cysteine protease utilized by coronaviruses for cell entry and is a promising drug target for novel antivirals against SARS-CoV-2. The marine natural product gallinamide A and several synthetic analogues were identified as potent inhibitors of cathepsin L with IC50 values in the picomolar range. Lead molecules possessed selectivity over other cathepsins and alternative host proteases involved in viral entry. Gallinamide A directly interacted with cathepsin L in cells and, together with two lead analogues, potently inhibited SARS-CoV-2 infection in vitro, with EC50 values in the nanomolar range. Reduced antiviral activity was observed in cells overexpressing transmembrane protease, serine 2 (TMPRSS2); however, a synergistic improvement in antiviral activity was achieved when combined with a TMPRSS2 inhibitor. These data highlight the potential of cathepsin L as a COVID-19 drug target as well as the likely need to inhibit multiple routes of viral entry to achieve efficacy.

• MeSH
• antivirové látky chemická syntéza   chemie   farmakologie   MeSH
• biologické přípravky chemická syntéza   chemie   farmakologie   MeSH
• buňky A549 MeSH
• Cercopithecus aethiops MeSH
• COVID-19 metabolismus   MeSH
• farmakoterapie COVID-19 MeSH
• inhibitory cysteinových proteinas chemická syntéza   chemie   farmakologie   MeSH
• kathepsin L antagonisté a inhibitory   metabolismus   MeSH
• kationické antimikrobiální peptidy chemická syntéza   chemie   farmakologie   MeSH
• lidé MeSH
• mikrobiální testy citlivosti MeSH
• molekulární konformace MeSH
• proteomika MeSH
• SARS-CoV-2 účinky léků   MeSH
• Vero buňky MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• vztahy mezi strukturou a aktivitou MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

Host-cell cysteine proteases play an essential role in the processing of the viral spike protein of SARS coronaviruses. K777, an irreversible, covalent inactivator of cysteine proteases that has recently completed phase 1 clinical trials, reduced SARS-CoV-2 viral infectivity in several host cells: Vero E6 (EC50< 74 nM), HeLa/ACE2 (4 nM), Caco-2 (EC90 = 4.3 μM), and A549/ACE2 (<80 nM). Infectivity of Calu-3 cells depended on the cell line assayed. If Calu-3/2B4 was used, EC50 was 7 nM, but in the ATCC Calu-3 cell line without ACE2 enrichment, EC50 was >10 μM. There was no toxicity to any of the host cell lines at 10-100 μM K777 concentration. Kinetic analysis confirmed that K777 was a potent inhibitor of human cathepsin L, whereas no inhibition of the SARS-CoV-2 cysteine proteases (papain-like and 3CL-like protease) was observed. Treatment of Vero E6 cells with a propargyl derivative of K777 as an activity-based probe identified human cathepsin B and cathepsin L as the intracellular targets of this molecule in both infected and uninfected Vero E6 cells. However, cleavage of the SARS-CoV-2 spike protein was only carried out by cathepsin L. This cleavage was blocked by K777 and occurred in the S1 domain of the SARS-CoV-2 spike protein, a different site from that previously observed for the SARS-CoV-1 spike protein. These data support the hypothesis that the antiviral activity of K777 is mediated through inhibition of the activity of host cathepsin L and subsequent loss of cathepsin L-mediated viral spike protein processing.

• MeSH
• antivirové látky farmakologie   MeSH
• Cercopithecus aethiops MeSH
• fenylalanin farmakologie   MeSH
• glykoprotein S, koronavirus chemie   metabolismus   MeSH
• inhibitory cysteinových proteinas farmakologie   MeSH
• internalizace viru účinky léků   MeSH
• kathepsin L antagonisté a inhibitory   metabolismus   MeSH
• lidé MeSH
• mikrobiální testy citlivosti MeSH
• nádorové buněčné linie MeSH
• piperaziny farmakologie   MeSH
• proteinové domény MeSH
• proteolýza MeSH
• SARS-CoV-2 účinky léků   MeSH
• tosylové sloučeniny farmakologie   MeSH
• Vero buňky MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Research Support, N.I.H., Extramural MeSH

The gills of the common carp, whose mucosal surface belongs to the key defence mechanisms of piscine immunity, can be infested with both the larval and adult stage of Eudiplozoon nipponicum (Monogenea). Although on their own, monogeneans do not considerably compromise their hosts' health status, fish with epithelial barriers damaged in parasite feeding and attachment sites are at an increased risk of bacterial challenge with possible harmful consequences. Several studies suggest that helminth parasites of teleost fish evade and manipulate host immune system via their excretory-secretory products, but our knowledge of these processes in the monogeneans is limited. Cysteine peptidase inhibitors (CPI), which are found in the secretions of numerous parasites, often induce immunosuppression by subverting Th1 mechanisms and drawing the immune system towards a Th2/Treg response. We employed the qPCR to test the effect of recently characterised CPI of E. nipponicum (rEnStef) on the mRNA expression of pro-inflammatory cytokine TNF-α and anti-inflammatory cytokine IL-10 produced by porcine macrophages in vitro. After an initial preincubation with rEnStef, we stimulated the macrophages using LPS. By inducing a Th1 pro-inflammatory response, we imitated the immune reaction during a bacterial challenge in tissue damaged by the feeding and attachment of E. nipponicum. We observed a significant dose-dependent downregulation of the expression of TNF-α and IL-10 cytokines. The observed suppression of TNF-alpha expression by rEnStef could result in decreased pathogen control, which might in turn lead to increased rates of secondary bacterial infections in fish infected by E. nipponicum.

• MeSH
• cytokiny * účinky léků   metabolismus   MeSH
• imunomodulace MeSH
• inhibitory cysteinových proteinas farmakologie   MeSH
• interleukin-10 metabolismus   MeSH
• kapři parazitologie   MeSH
• makrofágy * účinky léků   metabolismus   MeSH
• prasata MeSH
• rekombinantní proteiny farmakologie   MeSH
• TNF-alfa účinky léků   metabolismus   MeSH
• Trematoda imunologie   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Several diseases induce hypermetabolism, which is characterized by increases in resting energy expenditures (REE) and whole body protein loss. Exaggerated protein degradation is thought to be the driving force underlying this response. The effects of caspase and calpain inhibitors on REE in physiological and hypermetabolic conditions, however, are unknown. Thus, we studied whether MDL28170 (calpain inhibitor) or z-VAD-fmk (caspase inhibitor) affect REE under physiological conditions and during hypermetabolism post-burn. Rats were treated five times weekly and observed for 6 weeks. Treatment was started 2 h (early) or 48 h (late) after burn. In normal rats, MDL28170 transiently increased REE to 130 % of normal during week 2-4. z-VAD-fmk reduced REE by 20-25 % throughout the observation period. Within 14 days after burns, REE increased to 130+/-5 %. Whereas MDL28170/early treatment did not affect REE, MDL28170/late transiently increased REE to 180+/-10 % of normal by week 4 post-burn. In contrast, with z-VAD-fmk/early REE remained between 90-110 % of normal post-burn. z-VAD-fmk/late did not affect burn-induced increases in REE. These data suggest that caspase cascades contribute to the development of hypermetabolism and that burn-induced hypermetabolism can be pharmacologically modulated. Our data point towards caspase cascades as possible therapeutic targets to attenuate hypermetabolism after burns, and possibly in other catabolic disease processes.

• MeSH
• chloromethylketony aminokyselin farmakologie   terapeutické užití   MeSH
• dipeptidy farmakologie   terapeutické užití   MeSH
• energetický metabolismus účinky léků   MeSH
• inhibitory cysteinových proteinas farmakologie   terapeutické užití   MeSH
• inhibitory kaspas farmakologie   terapeutické užití   MeSH
• metabolické nemoci farmakoterapie   etiologie   MeSH
• pilotní projekty MeSH
• popálení komplikace   MeSH
• potkani Sprague-Dawley MeSH
• preklinické hodnocení léčiv MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Ticks, as obligate hematophagous ectoparasites, impact greatly on animal and human health because they transmit various pathogens worldwide. Over the last decade, several cystatins from different hard and soft ticks were identified and biochemically analyzed for their role in the physiology and blood feeding lifestyle of ticks. All these cystatins are potent inhibitors of papain-like cysteine proteases, but not of legumain. Tick cystatins were either detected in the salivary glands and/or the midgut, key tick organs responsible for blood digestion and the expression of pharmacologically potent salivary proteins for blood feeding. For example, the transcription of two cystatins named HlSC-1 and Sialostatin L2 was highly upregulated in these tick tissues during feeding. Vaccinating hosts against Sialostatin L2 and Om-cystatin 2 as well as silencing of a cystatin gene from Amblyomma americanum significantly inhibited the feeding ability of ticks. Additionally, Om-cystatin 2 and Sialostatin L possessed strong host immunosuppressive properties by inhibiting dendritic cell maturation due to their interaction with cathepsin S. These two cystatins, together with Sialostatin L2 are the first tick cystatins with resolved three-dimensional structure. Sialostatin L, furthermore, showed preventive properties against autoimmune diseases. In the case of the cystatin Hlcyst-2, experimental evidence showed its role in tick innate immunity, since increased Hlcyst-2 transcript levels were detected in Babesia gibsoni-infected larval ticks and the protein inhibited Babesia growth. Other cystatins, such as Hlcyst-1 or Om-cystatin 2 are assumed to be involved in regulating blood digestion. Only for Bmcystatin was a role in tick embryogenesis suggested. Finally, all the biochemically analyzed tick cystatins are powerful protease inhibitors, and some may be novel antigens for developing anti-tick vaccines and drugs of medical importance due to their stringent target specificity.

• MeSH
• cystatiny farmakologie   MeSH
• inhibitory cysteinových proteinas farmakologie   MeSH
• klíšťata účinky léků   fyziologie   MeSH
• lidé MeSH
• molekulární modely MeSH
• molekulární sekvence - údaje MeSH
• sekvence aminokyselin MeSH
• sekvenční seřazení MeSH
• stravovací zvyklosti účinky léků   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Research Support, N.I.H., Extramural MeSH

Porcine oocyte-cumulus complexes (OCCs) form an expanded cumulus extracellular matrix (ECM) in response to gonadotropins during meiotic maturation. Essential components of ECM are hyaluronan (HA), tumor necrosis factor α-induced protein 6 (TNFAIP6) and heavy chains (HC) of interalpha-trypsin inhibitor. To form expanded cumulus ECM, intermediate complexes (TNFAIP6-HC) must bind to HA to allow HC transfer onto HA. Protein turnover by the ubiquitin-proteasome pathway is poorly characterized in this process. It is known that the specific proteasomal inhibitor MG132 prevents cumulus expansion and formation of ECM. To determine whether inhibition of proteasomal proteolysis with MG132 affects cumulus cell steroidogenesis and expression of the cumulus expansion-related components (hyaluronan synthase type 2, HAS2, TNFAIP6) we cultured porcine OCCs and granulosa cells (GCs) in a medium supplemented with FSH/LH. Methods performed included real-time reverse transcription PCR, immunofluorescence and RIAs. The expression of TNFAIP6 and HAS2 transcripts increased significantly after the stimulation of OCCs and GCs with FSH/LH. In contrast, treatment with MG132 reduced the expression of TNFAIP6 and HAS2. Hyaluronan was detected with biotinylated HA-binding proteins within FSH/LH-stimulated expanded OCCs but not in those treated with MG132. Progesterone production, although increased almost three times after OCCs stimulation with FSH/LH, was significantly suppressed by MG132. The FSH/LH-stimulated a 40-fold increase in progesterone secretion by GCs was inhibited in the presence of MG132. In conclusion, MG132 affects progesterone secretion and expression of cumulus expansion-related components by cumulus and GCs, suggesting the requirement of ubiquitin-proteasome pathway-regulated protein turnover for formation of ECM during cumulus expansion in the preovulatory period in the pig.

• MeSH
• extracelulární matrix účinky léků   metabolismus   MeSH
• inhibitory cysteinových proteinas farmakologie   MeSH
• inhibitory proteasomu MeSH
• kumulární buňky účinky léků   metabolismus   MeSH
• kvantitativní polymerázová řetězová reakce veterinární   MeSH
• leupeptiny farmakologie   MeSH
• messenger RNA biosyntéza   genetika   MeSH
• molekuly buněčné adheze biosyntéza   MeSH
• oocyty účinky léků   metabolismus   MeSH
• prasata MeSH
• progesteron biosyntéza   MeSH
• proteasomový endopeptidasový komplex metabolismus   MeSH
• zvířata MeSH
• Check Tag
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH

The effects of the pan-caspase inhibitor Q-VD-OPh on caspase activity, DNA fragmentation, PARP cleavage, 7A6 exposition, and cellular adhesivity to fibronectin were analyzed in detail in three different apoptotic systems involving two cell lines (JURL-MK1 and HL60) and two apoptosis inducers (imatinib mesylate and suberoylanilide hydroxamic acid). Q-VD-OPh fully inhibited caspase-3 and -7 activity at 0.05  µM concentration as indicated both by the measurement of the rate of Ac-DEVD-AFC cleavage and anti-caspase immunoblots. Caspase-8 was also inhibited at low Q-VD-OPh concentrations. On the other hand, significantly higher Q-VD-OPh dose (10 µM) was required to fully prevent the cleavage of PARP-1. DNA fragmentation and disruption of the cell membrane functionality (Trypan blue exclusion test) were both prevented at 2 µM Q-VD-OPh while 10 µM inhibitor was needed to inhibit the drug-induced loss of cellular adhesivity to fibronectin which was observed in JURL-MK1 cells. The exposition of the mitochondrial antigen 7A6 occurred independently of Q-VD-OPh addition and may serve to the detection of cumulative incidence of the cells which have initiated the apoptosis. Our results show that Q-VD-OPh efficiency in the inhibition of caspase-3 activity and DNA fragmentation in the whole-cell environment is about two orders of magnitude higher than that of z-VAD-fmk. This difference is not due to a slow permeability of the latter through the cytoplasmic membrane.

• MeSH
• apoptóza účinky léků   MeSH
• buněčná adheze MeSH
• chinoliny farmakologie   MeSH
• chloromethylketony aminokyselin farmakologie   MeSH
• elektroforéza v polyakrylamidovém gelu MeSH
• inhibitory cysteinových proteinas farmakologie   MeSH
• inhibitory kaspas MeSH
• kaspasy MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• western blotting MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Cytotoxicity and apoptosis induced by etoposide were studied during 72 hr in human melanoma cells. Etoposide initiated DNA-damage signaling via ATM kinase and activated p53 pathway and caspase-2. In response to treatment with etoposide, mitochondria of melanoma cells first increased their abundance and activity, and at later treatment intervals their dynamic behavior and functions became suppressed. Observed mitochondrial perturbation was not preceded by membrane potential loss but cytochrome c release was observed together with a rise in caspase-9 and caspase-3 activities. The pharmacological inhibition of relevant induced targets proved the importance of ATM and caspase-2 in etoposide-mediated cytotoxicity and apoptosis.

• MeSH
• adenosintrifosfát metabolismus   MeSH
• antitumorózní látky fytogenní farmakologie   MeSH
• apoptóza účinky léků   MeSH
• benzothiazoly farmakologie   MeSH
• cyklosporin farmakologie   MeSH
• cysteinové endopeptidasy metabolismus   MeSH
• DNA vazebné proteiny antagonisté a inhibitory   metabolismus   MeSH
• etoposid farmakologie   MeSH
• inhibitory cysteinových proteinas farmakologie   MeSH
• inhibitory kaspas MeSH
• inhibitory proteinkinas farmakologie   MeSH
• kaspasa 2 metabolismus   MeSH
• kaspasa 3 metabolismus   MeSH
• kaspasa 9 metabolismus   MeSH
• lidé MeSH
• melanom patologie   MeSH
• mitochondrie účinky léků   MeSH
• nádorové buňky kultivované účinky léků   MeSH
• nádorové proteiny antagonisté a inhibitory   metabolismus   MeSH
• nádorové supresorové proteiny antagonisté a inhibitory   metabolismus   MeSH
• nádorový supresorový protein p53 metabolismus   MeSH
• poškození DNA MeSH
• protein X asociovaný s bcl-2 metabolismus   MeSH
• protein-serin-threoninkinasy antagonisté a inhibitory   metabolismus   MeSH
• proteiny buněčného cyklu metabolismus   MeSH
• superoxidy metabolismus   MeSH
• toluen analogy a deriváty   farmakologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• práce podpořená grantem MeSH

The block of hematopoietic differentiation program in acute myeloid leukemia cells can be overcome by differentiating agent like retinoic acid, but it has several side effects. A study of other differentiation signaling pathways is therefore useful to predict potential targets of anti-leukemic therapy. We demonstrated previously that the co-treatment of HL-60 cells with Tumor necrosis factor-alpha (TNF-alpha) (1 ng/mL) and inhibitor of 5-lipoxygenase MK886 (5 microm) potentiated both monocytic differentiation and apoptosis. In this study, we detected enhanced activation of three main types of mitogen-activated protein kinases (MAPKs) (p38, c-Jun amino-terminal kinase [JNK], extracellular signal-regulated kinase [ERK]), so we assessed their role in differentiation using appropriate pharmacologic inhibitors. The inhibition of pro-apoptotic MAPKs (p38 and JNK) suppressed the effect of MK886 + TNF-alpha co-treatment. On the other hand, down-regulation of pro-survival ERK pathway led to increased differentiation. Those effects were accompanied by increased activation of caspases in cells treated by MK886 + TNF-alpha. Pan-caspase inhibitor ZVAD-fmk significantly decreased both number of apoptotic and differentiated cells. The same effect was observed after inhibition of caspase 9, but not caspase 3 and 8. To conclude, we evidenced that the activation of apoptotic processes and pathways supporting apoptosis (p38 and JNK MAPKs) is required for the monocytic differentiation of HL-60 cells.

• MeSH
• aktivace enzymů účinky léků   MeSH
• akutní promyelocytární leukemie farmakoterapie   metabolismus   patologie   MeSH
• apoptóza fyziologie   účinky léků   MeSH
• buněčná diferenciace účinky léků   MeSH
• chloromethylketony aminokyselin farmakologie   MeSH
• HL-60 buňky MeSH
• indoly farmakologie   MeSH
• inhibitory cysteinových proteinas farmakologie   MeSH
• inhibitory kaspas MeSH
• inhibitory lipoxygenas farmakologie   MeSH
• kaspasy metabolismus   MeSH
• lidé MeSH
• MAP kinasový signální systém účinky léků   MeSH
• monocyty patologie   účinky léků   MeSH
• NF-kappa B antagonisté a inhibitory   MeSH
• TNF-alfa farmakologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• práce podpořená grantem MeSH

The toxic modes of action of non-dioxin-like polychlorinated biphenyls (PCBs) in liver cells are still only partially understood. Several recent studies have indicated that PCBs may interfere with cell membrane protein functions. Therefore, we analyzed in the present study the effects of di-ortho-substituted 2,2',4,4',5,5'-hexachlorobiphenyl (PCB 153) on proteins involved in the formation of adherens junctions in a model of rat liver progenitor cells - WB-F344 cell line. PCB 153, at micromolar concentrations, induced a gradual degradation of E-cadherin, beta-catenin or plakoglobin (gamma-catenin) proteins. This effect was not due to changes in gene expression, as PCB 153 had no effect on mRNA levels of the above mentioned proteins. Moreover, apart from the reduction of total beta-catenin pool, PCB 153 also decreased levels of the active beta-catenin form, dephosphorylated at residues Ser37 and Thr41, which is the key co-activator of Wnt-induced TCF/LEF-dependent gene expression. Therefore, we also evaluated the impact of PCB 153 on expression of Axin2, a known transcriptional target of canonical Wnt signaling. PCB 153 reduced basal Axin2 mRNA levels and it inhibited induction of Axin2 expression by recombinant mouse Wnt3a. Nevertheless, PCB 153 had no effect on phosphorylation of glycogen synthase kinase-3beta (GSK-3beta), which is supposed to target beta-catenin for its proteasomal degradation. This suggested that GSK-3beta activity is not modulated by PCB 153 and, consequently, not involved in the observed PCB 153-induced decrease of both total and active beta-catenin levels. Protein levels of E-cadherin and beta-catenin were partially restored with lysosomal inhibitor leupeptin, thus suggesting a possible role of lysosomes in the observed degradation of adherens junction proteins. Taken together, the present data suggest that PCB 153 may interfere with functions of adherens junction proteins involved in both cell-to-cell communication and intracellular signaling. Such mechanisms might be involved in the effects of non-dioxin-like PCBs contributing to liver tumor promotion.

• MeSH
• adhezní spoje metabolismus   účinky léků   MeSH
• beta-katenin antagonisté a inhibitory   biosyntéza   genetika   MeSH
• buněčné linie MeSH
• epitelové buňky cytologie   metabolismus   účinky léků   MeSH
• fosforylace účinky léků   MeSH
• gama-katenin biosyntéza   genetika   metabolismus   MeSH
• genetická transkripce účinky léků   MeSH
• inhibitory cysteinových proteinas farmakologie   MeSH
• játra cytologie   metabolismus   účinky léků   MeSH
• kadheriny biosyntéza   genetika   metabolismus   MeSH
• kinasa 3 glykogensynthasy metabolismus   MeSH
• kmenové buňky cytologie   metabolismus   účinky léků   MeSH
• krysa rodu rattus MeSH
• leupeptiny farmakologie   MeSH
• lyzozomy metabolismus   MeSH
• messenger RNA biosyntéza   genetika   MeSH
• polychlorované bifenyly toxicita   MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• represorové proteiny biosyntéza   genetika   metabolismus   MeSH
• western blotting MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• zvířata MeSH
• Publikační typ
• práce podpořená grantem MeSH