Bisphenol S Impaired In Vitro Ovine Early Developmental Oocyte Competence.

INTRODUCTION: Bisphenol A (BPA) is a widespread compound in the plastic industry that is especially used to produce baby bottles, food packaging and metal cans. BPA, an endocrine disruptor, leads to alterations in reproductive function and therefore has been banned from the food industry. Unregulated BPA analogues, particularly Bisphenol S (BPS), have emerged and are now used in the plastic industry. Thus, this study aimed to examine the acute effects of low and environmental doses of BPS on ewe oocyte quality and developmental competence, and its mechanism of action, during in vitro maturation. METHODS: Ewe cumulus-oocyte complexes underwent in vitro maturation in the presence or absence of BPS (1 nM, 10 nM, 100 nM, 1 µM or 10 µM). Oocytes were then subjected to in vitro fertilisation and development. RESULTS: 1 µM BPS induced a 12.7% decrease in the cleavage rate (p = 0.004) and a 42.6% decrease in the blastocyst rate (p = 0.017) compared to control. The blastocyst rate reduction was also observed with 10 nM BPS. Furthermore, 10 µM BPS reduced the oocyte maturation rate, and 1 µM BPS decreased cumulus cell progesterone secretion. PR and AMH gene expression were reduced in cumulus cells. BPS induced a 5-fold increase in MAPK 3/1 activation (p = 0.04). CONCLUSIONS: BPS impaired ewe oocyte developmental competence. The data suggest that BPS might not be a safe BPA analogue. Further studies are required to elucidate its detailed mechanism of action.

Molecular dynamics-guided discovery of an ago-allosteric modulator for GPR40/FFAR1.

The long-chain fatty acid receptor FFAR1/GPR40 binds agonists in both an interhelical site between the extracellular segments of transmembrane helix (TM)-III and TM-IV and a lipid-exposed groove between the intracellular segments of these helices. Molecular dynamics simulations of FFAR1 with agonist removed demonstrated a major rearrangement of the polar and charged anchor point residues for the carboxylic acid moiety of the agonist in the interhelical site, which was associated with closure of a neighboring, solvent-exposed pocket between the extracellular poles of TM-I, TM-II, and TM-VII. A synthetic compound designed to bind in this pocket, and thereby prevent its closure, was identified through structure-based virtual screening and shown to function both as an agonist and as an allosteric modulator of receptor activation. This discovery of an allosteric agonist for a previously unexploited, dynamic pocket in FFAR1 demonstrates both the power of including molecular dynamics in the drug discovery process and that this specific, clinically proven, but difficult, antidiabetes target can be addressed by chemotypes different from existing ligands.

Two key cathepsins, TgCPB and TgCPL, are targeted by the vinyl sulfone inhibitor K11777 in in vitro and in vivo models of toxoplasmosis.

Although toxoplasmosis is one of the most common parasitic infections worldwide, therapeutic options remain limited. Cathepsins, proteases that play key roles in the pathogenesis of toxoplasmosis and many other protozoan infections, are important potential therapeutic targets. Because both TgCPB and TgCPL play a role in T. gondii invasion, we evaluated the efficacy of the potent, irreversible vinyl sulfone inhibitor, K11777 (N-methyl-piperazine-Phe-homoPhe-vinylsulfone-phenyl). The inhibitor's toxicity and pharmacokinetic profile have been well-studied because of its in vitro and in vivo activity against a number of parasites. We found that it inhibited both TgCPB (EC50 = 114 nM) and TgCPL (EC50 = 71 nM) in vitro. K11777 also inhibited invasion of human fibroblasts by RH tachyzoites by 71% (p = 0.003) and intracellular replication by >99% (p<0.0001). In vivo, a single dose of K11777 led to 100% survival of chicken embryos in an model of acute toxoplasmosis (p = 0.015 Cox regression analysis). Therefore, K11777 shows promise as a novel therapeutic agent in the treatment of toxoplasmosis, and may prove to be a broadly effective anti-parasitic agent.

Respiratory syncytial virus (RSV) entry is inhibited by serine protease inhibitor AEBSF when present during an early stage of infection.

BACKGROUND: Host proteases have been shown to play important roles in many viral activities such as entry, uncoating, viral protein production and disease induction. Therefore, these cellular proteases are putative targets for the development of antivirals that inhibit their activity. Host proteases have been described to play essential roles in Ebola, HCV, HIV and influenza, such that specific protease inhibitors are able to reduce infection. RSV utilizes a host protease in its replication cycle but its potential as antiviral target is unknown. Therefore, we evaluated the effect of protease inhibitors on RSV infection. METHODS: To measure the sensitivity of RSV infection to protease inhibitors, cells were infected with RSV and incubated for 18 h in the presence or absence of the inhibitors. Cells were fixed, stained and studied using fluorescence microscopy. RESULTS: Several protease inhibitors, representing different classes of proteases (AEBSF, Pepstatin A, E-64, TPCK, PMSF and aprotinin), were tested for inhibitory effects on an RSV A2 infection of HEp-2 cells. Different treatment durations, ranging from 1 h prior to inoculation and continuing for 18 h during the assay, were evaluated. Of all the inhibitors tested, AEBSF and TPCK significantly decreased RSV infection. To ascertain that the observed effect of AEBSF was not a specific feature related to HEp-2 cells, A549 and BEAS-2B cells were also used. Similar to HEp-2, an almost complete block in the number of RSV infected cells after 18 h of incubation was observed and the effect was dose-dependent. To gain insight into the mechanism of this inhibition, AEBSF treatment was applied during different phases of an infection cycle (pre-, peri- and post-inoculation treatment). The results from these experiments indicate that AEBSF is mainly active during the early entry phase of RSV. The inhibitory effect was also observed with other RSV isolates A1998/3-2 and A2000/3-4, suggesting that this is a general feature of RSV. CONCLUSION: RSV infection can be inhibited by broad serine protease inhibitors, AEBSF and TPCK. We confirmed that AEBSF inhibition is independent of the cell line used or RSV strain. The time point at which treatment with the inhibitor was most potent, was found to coincide with the expected moment of entry of the virion with the host cell.

In vitro effects of cysteine protease inhibitors on Trichomonas foetus-induced cytopathic changes in porcine intestinal epithelial cells.

OBJECTIVE To investigate the effects of specific cysteine protease (CP) inhibitors on cytopathic changes to porcine intestinal epithelial cells induced by Tritrichomonas foetus isolated from naturally infected cats. SAMPLE T foetus isolates from 4 naturally infected cats and nontransformed porcine intestinal epithelial cells. PROCEDURES T foetus isolates were treated with or without 0.1 to 1.0mM of the CP inhibitors antipain, cystatin, leupeptin, and chymostatin and the vinyl sulfone inhibitors WRR-483 and K11777. In-gel gelatin zymography was performed to evaluate the effects of these inhibitors on CP activity of T foetus isolates. Each treated or untreated isolate was also cocultured with monolayers of porcine intestinal epithelial cells for 24 hours, and cytopathic effects of T foetus were evaluated by light microscopy and crystal violet spectrophotometry. RESULTS Results of in-gel gelatin zymography suggested an ability of WRR-483, K11777, and cystatin to target specific zones of CP activity of the T foetus isolates. These inhibitors had no effect on T foetus growth, and the cytopathic changes to the intestinal epithelium induced by all 4 T foetus isolates were significantly inhibited. CONCLUSIONS AND CLINICAL RELEVANCE This study revealed that certain protease inhibitors were capable of inhibiting regions of CP activity (which has been suggested to cause intestinal cell damage in cats) in T foetus organisms and of ameliorating T foetus-induced cytopathic changes to porcine intestinal epithelium in vitro. Although additional research is needed, these inhibitors might be useful in the treatment of cats with trichomonosis.

A selective antagonist reveals a potential role of G protein-coupled receptor 55 in platelet and endothelial cell function.

The G protein-coupled receptor 55 (GPR55) is a lysophosphatidylinositol (LPI) receptor that is also responsive to certain cannabinoids. Although GPR55 has been implicated in several (patho)physiologic functions, its role remains enigmatic owing mainly to the lack of selective GPR55 antagonists. Here we show that the compound CID16020046 ((4-[4-(3-hydroxyphenyl)-3-(4-methylphenyl)-6-oxo-1H,4H,5H,6H-pyrrolo[3,4-c]pyrazol-5-yl] benzoic acid) is a selective GPR55 antagonist. In yeast cells expressing human GPR55, CID16020046 antagonized agonist-induced receptor activation. In human embryonic kidney (HEK293) cells stably expressing human GPR55, the compound behaved as an antagonist on LPI-mediated Ca²âº release and extracellular signal-regulated kinases activation, but not in HEK293 cells expressing cannabinoid receptor 1 or 2 (CB1 or CB2). CID16020046 concentration dependently inhibited LPI-induced activation of nuclear factor of activated T-cells (NFAT), nuclear factor κ of activated B cells (NF-κB) and serum response element, translocation of NFAT and NF-κB, and GPR55 internalization. It reduced LPI-induced wound healing in primary human lung microvascular endothelial cells and reversed LPI-inhibited platelet aggregation, suggesting a novel role for GPR55 in platelet and endothelial cell function. CID16020046 is therefore a valuable tool to study GPR55-mediated mechanisms in primary cells and tissues.

Exogenous estrogen does not attenuate the association between rofecoxib and myocardial infarction in perimenopausal women.

Rofecoxib has been proposed to increase the risk of myocardial infarction (MI) through suppression of cyclooxygenase 2­mediated prostacyclin. Estrogen may have protective effects through augmenting cyclooxygenase 2 expression and subsequently increasing prostacyclin. Estrogen may attenuate the association between rofecoxib and MI. We used 1999­2002 Medicaid claims data to measure the MI hazard ratio (HR) attributed to rofecoxib exposure in estrogen-exposed and unexposed 45- to 65-year-old women.We identified 184,169 female rofecoxib users who contributed 309,504 person-years and experienced 1217 first MIs. Estrogen exposure seemed protective [MI-HR 0.72; 95% confidence interval (CI), 0.62­0.84] in this cohort. Rofecoxib was associated with an elevated MI-HR in both estrogen-exposed (2.01; 95% CI, 1.60­2.54) and estrogen-unexposed women (1.69; 95% CI, 1.43­1.99). The rofecoxib­estrogen interaction ratio was not significantly different from 1 (1.19; 95% CI, 0.91­1.57). Although estrogen use was associated with a lower risk of MI, it did not seem to attenuate the association between rofecoxib and MI.

Selective inhibition of ion transport mechanisms regulating intracellular pH reduces proliferation and induces apoptosis in cholangiocarcinoma cells.

BACKGROUND: Cells within the acidic extracellular environment of solid tumours maintain their intracellular pH through the activity of the Na(+)/H(+) exchanger and the Na(+) dependent Cl(-)/HCO(3)(-) exchanger. The inhibition of these mechanisms could therefore inhibit cancer cell growth. AIM: We evaluated the effect of two selective inhibitors of these transporters (cariporide and S3705) on proliferation and apoptosis of human cholangiocarcinoma cells (HUH-28 and Mz-ChA-1 cells) as a function of external pH (7.4 and 6.8). METHODS/RESULTS: HUH-28 cells incubated for 24h at external pH 7.4 or 6.8 without inhibitors maintained intracellular pH at physiological level, whereas incubation with cariporide and/or S3705 caused the intracellular pH of cells to drop. Incubation of HUH-28 cells with cariporide and/or S3705 was able to reduce proliferation, evaluated by a colorimetric ELISA method, and to induce apoptosis, evaluated by measuring caspase-3 activity and Annexin-V staining, and these effects were more evident at external pH 6.8. S3705 but not cariporide was able to inhibit serum-induced phosphorylation of ERK1/2, AKT and BAD, intracellular molecules involved in cancer cell proliferation and survival. Similar results were obtained in Mz-ChA-1 cells. CONCLUSIONS: (1) Inhibition of intracellular pH regulatory mechanisms by cariporide and S3705 reduces proliferation and induces apoptosis in cholangiocarcinoma cells; and (2) these drugs might have potential therapeutic value against cholangiocarcinoma.

CYP2A5-mediated activation and early ultrastructural changes in the olfactory mucosa: studies on 2,6-dichlorophenyl methylsulfone.

2,6-Dichlorophenyl methylsulfone (2,6-diClPh-MeSO2) is a potent olfactory toxicant reported to induce endoplasmic reticulum (ER) stress, caspase activation, and extensive cell death in mice. The aim of the present study was to examine cytochrome P450 (P450)-dependent bioactivation, nonprotein sulfhydryl (NP-SH) levels, and early ultrastructural changes in mouse olfactory mucosa following an i.p. injection of 2,6-diClPh-MeSO2 (32 mg/kg). A high covalent binding of 2,6-diClPh-14C-MeSO2 in olfactory mucosa S9 fraction was observed, and the CYP2A5/CYP2G1 substrates coumarin and dichlobenil significantly decreased the binding, whereas the CYP2E1 substrate chlorzoxazone had no effects. An increased bioactivation was detected in liver microsomes of mice pretreated with pyrazole, known to induce CYP2A4, 2A5, 2E1, and 2J, and addition of chlorzoxazone reduced this binding. 2,6-DiClPh-14C-MeSO2 showed a marked covalent binding to microsomes of recombinant yeast cells expressing mouse CYP2A5 or human CYP2A6 compared with wild type. One and 4 h after a single injection of 2,6-diClPh-MeSO2, the NP-SH levels in the olfactory mucosa were significantly reduced compared with control, whereas there was no change in the liver. Ultrastructural studies revealed that ER, mitochondria, and secretory granules in nonneuronal cells were early targets 1 h after injection. We propose that lesions induced by 2,6-diClPh-MeSO2 in the mouse olfactory mucosa were initiated by a P450-mediated bioactivation in the Bowman's glands and depletion of NP-SH levels, leading to disruption of ion homeostasis, organelle swelling, and cell death. The high expression of CYP2A5 in the olfactory mucosa is suggested to play a key role for the tissue-specific toxicity induced by 2,6-diClPh-MeSO2.

Effect of rofecoxib on antihypertensive effects of candesartan in experimental models of hypertension.

Given the high prevalence of hypertension, concomitant use of nonsteroidal anti-inflammatory drugs and antihypertensive medications is commonly encountered in clinical practice. The present study was designed to study the effect of indomethacin, nimesulide, and rofecoxib on blood pressure (BP) in normotensive and hypertensive rats and also to investigate the effect of rofecoxib on BP control in candesartan-treated hypertensive rats. Male Wistar rats weighing 150-200 g were divided into three groups: control, DOCA-hypertensive, and L-NAME-hypertensive rats. All the rats were given indomethacin (15 mg/kg body weight), nimesulide (20 mg/kg body weight), rofecoxib (10 mg/kg body weight), or vehicle orally and daily for 6 weeks. Hypertensive rats in separate groups were treated with either candesartan (1 mg/kg body weight) alone or a combination of candesartan (1 mg/kg body weight) and rofecoxib (10 mg/kg body weight) orally and daily for 6 weeks. BP measurements were performed using tail cuff method at baseline and 1-week intervals throughout the treatment period. All the three COX inhibitors resulted in increase in BP, but mean change in BP was the highest with rofecoxib. Rofecoxib-treated L-NAME-hypertensive rats exhibited a significant increase in mean arterial pressure at 6 weeks (168.3+/-5.7 mmHg) as compared with DOCA-hypertensive rats (128.818+/-7.2 mmHg). Administration of Rofecoxib L-NAME-hypertensive rats treated with candesartan resulted in a significant increase in BP. Systolic BP at 0 week (107.0+/-4.2 mmHg) rose to 141.6+/-2.0 mmHg at 6 weeks. Systolic BP at 2, 4, and 6 weeks was significantly higher as compared with (L-NAME+candesartan)- and (rofecoxib+candesartan)-treated group. In conclusion, concomitant use of rofecoxib resulted in poor BP control by candesartan in L-NAME-hypertensive rats.

Sortase from Staphylococcus aureus does not contain a thiolate-imidazolium ion pair in its active site.

Many surface proteins are anchored to the cell wall by the action of sortase enzymes, a recently discovered family of cysteine transpeptidases. As the surface proteins of human pathogens are frequently required for virulence, the sortase-mediated anchoring reaction represents a potential target for new anti-infective agents. It has been suggested that the sortase from Staphylococcus aureus (SrtA), may use a similar catalytic strategy as the papain cysteine proteases, holding its Cys184 side chain in an active configuration through a thiolate-imidazolium ion interaction with residue His120. To investigate the mechanism of transpeptidation, we have synthesized a peptidyl-vinyl sulfone substrate mimic that irreversibly inhibits SrtA. Through the study of the pH dependence of SrtA inhibition and NMR, we have estimated the pKas of the active site thiol (Cys184) and imidazole (His120) to be approximately 9.4 and 7.0, respectively. These measurements are inconsistent with the existence of a thiolate-imidazolium ion pair and suggest a general base catalysis mechanism during transpeptidation.

Expression, purification, crystallization and preliminary X-ray diffraction studies of human cathepsin F complexed with an irreversible vinyl sulfone inhibitor.

Cathepsin F is a cysteine protease believed to be involved in the antigen-presenting process of the class II major histocompatibility complex (MHC-II) in macrophages. It has been expressed, purified and crystallized. A complete data set to a resolution of 2.5 A has been collected at room temperature. The Laue group was determined to be orthorhombic, space group P2(1)2(1)2, with unit-cell parameters a = 68.9, b = 104.8, c = 68.5 A.

Design, synthesis, and in vitro inhibitory activity toward human leukocyte elastase, cathepsin G, and proteinase 3 of saccharin-derived sulfones and congeners.

The inhibitory activity toward human leukocyte elastase (HLE), cathepsin G (Cat G), and proteinase 3 (PR 3) of a series of saccharin derivatives having a sulfinate leaving group was investigated. The results of this study revealed that (a) inhibitory activity is dependent on the nature and pKa of the leaving group, and (b) the synthesized saccharin derivatives exhibit selective inhibition toward HLE and PR 3, with low or no activity toward cathepsin G. The results of exploratory biochemical, HPLC and high-field 13C NMR studies are also described.

Characterization of the binding of [3H]SR 33805 to the slow Ca2+ channel in rat heart sarcolemmal membrane.

SR 33805 is a representative of a new class of compounds (indole sulfone) that inhibits L-type Ca2+ channels. [3H]SR 33805 has been shown to bind with a high affinity (Kd approximately 20 pM calculated from saturation isotherms and association/dissociation kinetics) to a single site in a purified preparation of rat cardiac sarcolemmal membranes. The binding was found to be saturable and reversible. The maximal binding capacity was in approximately 1:1 stoichiometry with that of other Ca2+ channel antagonists. Various cations (Na+, Ca2+, Cd2+, and La3+) were shown to inhibit specific [3H]SR 33805 binding, with La3+ being the most potent. Using a range of receptor or channel ligands (including omega-conotoxin and Na+ and K+ channel modulators), only the L-type Ca2+ channel antagonists were found to displace [3H]SR 33805. However, dihydropyridines, phenylalkylamines, benzothiazepines, and diphenyl-butylpiperidines were found to inhibit [3H]SR 33805 in a non-competitive manner as demonstrated by displacement experiments in addition to dissociation kinetics. In contrast, the interaction of SR 33805 with fantofarone has been found to be competitive. Binding of [3H]SR 33805 (and [3H]fantofarone) is entropy driven as opposed to that of the [3H]nitrendipine which is enthalpy driven. From these results we suggest that SR 33805 binds with a high affinity to a unique site on the L-type Ca2+ channel found in rat cardiac sarcolemmal membranes. This site is equivalent to that of fantofarone and in allosteric interaction with that of the dihydropyridines, phenylalkylamines and benzothiazepines.

Ulcerogenic mechanism of ethanol and the action of sulphanilyl fluoride on the rat stomach in-vivo.

The effects of ethanol alone and in combination with sulphanilyl fluoride on some of the antioxidant defences in the stomach of rats have been examined. These effects were correlated with lesion formation in the gastric mucosa. Oral administration of ethanol induced gastric lesions which were prevented by sulphanilyl fluoride pre-treatment. N-Ethylmaleimide antagonized the anti-lesion action of sulphanilyl fluoride. Ethanol administration lowered the glucose-6-phosphate dehydrogenase activity in the gastric mucosa, an effect potentiated by N-ethylmaleimide pre-treatment. The total superoxide dismutase activity was unaffected by the drugs used in the present study. Ethanol, however, markedly increased mucosal catalase activity which was reduced by sulphanilyl fluoride pretreatment and reversed by N-ethylmaleimide. It is concluded that the ulcerogenic mechanism of ethanol is mediated at least in part by the depression of the hexose monophosphate shunt and the production of active oxygen species, whereas the anti-lesion action of sulphanilyl fluoride is probably not mediated through these mechanisms.