ucn-1028-c and Disease-Models--Animal

When Zika virus emerged as a public health emergency there were no drugs or vaccines approved for its prevention or treatment. We used a high-throughput screen for Zika virus protease inhibitors to identify several inhibitors of Zika virus infection. We expressed the NS2B-NS3 Zika virus protease and conducted a biochemical screen for small-molecule inhibitors. A quantitative structure-activity relationship model was employed to virtually screen ∼138,000 compounds, which increased the identification of active compounds, while decreasing screening time and resources. Candidate inhibitors were validated in several viral infection assays. Small molecules with favorable clinical profiles, especially the five-lipoxygenase-activating protein inhibitor, MK-591, inhibited the Zika virus protease and infection in neural stem cells. Members of the tetracycline family of antibiotics were more potent inhibitors of Zika virus infection than the protease, suggesting they may have multiple mechanisms of action. The most potent tetracycline, methacycline, reduced the amount of Zika virus present in the brain and the severity of Zika virus-induced motor deficits in an immunocompetent mouse model. As Food and Drug Administration-approved drugs, the tetracyclines could be quickly translated to the clinic. The compounds identified through our screening paradigm have the potential to be used as prophylactics for patients traveling to endemic regions or for the treatment of the neurological complications of Zika virus infection.

Topics: Animals; Antiviral Agents; Artificial Intelligence; Chlorocebus aethiops; Disease Models, Animal; Drug Evaluation, Preclinical; High-Throughput Screening Assays; Immunocompetence; Inhibitory Concentration 50; Methacycline; Mice, Inbred C57BL; Protease Inhibitors; Quantitative Structure-Activity Relationship; Small Molecule Libraries; Vero Cells; Zika Virus; Zika Virus Infection

Repeated morphine treatment results in a decreased analgesic effect or the development of analgesic tolerance. However, we reported that some inflammatory chronic pain may inhibit morphine tolerance via kappa opioid receptor (KOR) activation. In this study, we further investigated the role of KOR in the inhibition of morphine tolerance in a chronic pain condition with a focus on the regulation of protein kinase C (PKC) activity.. Chronic pain was induced by formalin treatment into the dorsal part of the left hind paws of mice. The analgesic effect of morphine was measured by the tail flick method. We analysed the protein expression of PKC and its activity, and G-protein activity of mu opioid receptor (MOR) under repeated morphine treatment with or without formalin treatment.. We found that conventional subtypes of PKC (cPKC) were up-regulated by repeated morphine treatment. Also, antisense oligonucleotide (AS-ODN) targeting cPKC completely suppressed the development of morphine tolerance. The disappearance of the repeated morphine-induced up-regulation of cPKC was completely reversed by treatment with AS-ODN targeting KOR. In addition, AS-ODN targeting KOR significantly reversed the chronic pain-induced down-regulation of PKC activity or up-regulation of MOR [(35)S]GTPgammaS binding activity after repeated morphine treatment.. These results indicate that KOR plays an important role in the inhibition of repeated morphine-induced cPKC up-regulation under chronic pain condition. Furthermore, this may result in the increase of MOR activity and in the inhibition of morphine tolerance under chronic pain condition.

Topics: Analgesics, Opioid; Animals; Brain; Chronic Disease; Disease Models, Animal; Down-Regulation; Drug Tolerance; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enzyme Activation; Formaldehyde; Guanosine 5'-O-(3-Thiotriphosphate); Male; Mice; Morphine; Naphthalenes; Oligonucleotides, Antisense; Pain; Pain Measurement; Pain Threshold; Protein Kinase C; Protein Kinase Inhibitors; Receptors, Opioid, kappa; Receptors, Opioid, mu; Time Factors

Our previous studies have shown that morphine withdrawal induced hyperactivity of cardiac noradrenergic pathways. The purpose of the present study was to evaluate the effects of morphine withdrawal on site-specific phosphorylation of TH in the heart.. Dependence on morphine was induced by a 7-day s.c. implantation of morphine pellets in rats. Morphine withdrawal was precipitated on day 8 by an injection of naloxone (2 mg kg(-1)). TH phosphorylation was determined by quantitative blot immunolabelling using phosphorylation state-specific antibodies.. Naloxone-induced morphine withdrawal induced phosphorylation of TH at serine (Ser)40 and Ser31 in the right ventricle, associated with both an increase in total TH levels and an enhancement of TH activity. When HA-1004 (PK A inhibitor) was infused, concomitantly with morphine, it diminished the increase in noradrenaline turnover, total TH levels and TH phosphorylation at Ser40 in morphine-withdrawn rats. In contrast, the infusion of calphostin C (PKC inhibitor), did not modify the morphine withdrawal-induced increase in noradrenaline turnover and total TH levels. In addition, we show that the ability of morphine withdrawal to stimulate phosphorylation at Ser31 was reduced by SL327, an inhibitor of ERK 1/2 activation.. The present findings demonstrate that the enhancement of total TH levels and the increased phosphorylation state of TH during morphine withdrawal were dependent on PKA and ERK activities and suggest that these transduction pathways might contribute to the activation of the cardiac catecholaminergic neurons in response to morphine withdrawal.

Topics: Aminoacetonitrile; Animals; Cyclic AMP-Dependent Protein Kinases; Disease Models, Animal; Drug Implants; Isoquinolines; Male; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Morphine; Morphine Dependence; Myocardium; Naloxone; Naphthalenes; Narcotic Antagonists; Norepinephrine; Phosphorylation; Protein Kinase C-delta; Protein Kinase Inhibitors; Rats; Rats, Sprague-Dawley; Serine; Substance Withdrawal Syndrome; Sulfonamides; Tyrosine 3-Monooxygenase

Inducible nitric-oxide synthase (iNOS) plays a central role in the regulation of vascular function and response to injury. A central mediator controlling iNOS expression is transforming growth factor-beta (TGF-beta), which represses its expression through a mechanism that is poorly understood. We have identified a binding site in the iNOS promoter that interacts with the nuclear heterodimer TCF11/MafG using chromatin immunoprecipitation and mutation analyses. We demonstrate that binding at this site acts to repress the induction of iNOS gene expression by cytokines. We show that this repressor is induced by TGF-beta1 and by Smad6-short, which enhances TGF-beta signaling. In contrast, the up-regulation of TCF11/MafG binding could be suppressed by overexpression of the TGF-beta inhibitor Smad7, and a small interfering RNA to TCF11 blocked the suppression of iNOS by TGF-beta. The binding of TCF11/MafG to the iNOS promoter could be enhanced by phorbol 12-myristate 13-acetate and suppressed by the protein kinase C inhibitor staurosporine. Moreover, the induction of TCF11/MafG binding by TGF-beta and Smad6-short could be blocked by staurosporine, and the effect of TGF-beta was blocked by the selective protein kinase C inhibitor calphostin C. Consistent with the in vitro data, we found suppression of TCF11 coincident with iNOS up-regulation in a rat model of endotoxemia, and we observed a highly significant negative correlation between TCF11 and nitric oxide production. Furthermore, treatment with activated protein C, a serine protease effective in septic shock, blocked the down-regulation of TCF11 and suppressed endotoxin-induced iNOS. Overall, our results demonstrate a novel mechanism by which iNOS expression is regulated in the context of inflammatory activation.

Topics: Animals; Carcinogens; Cells, Cultured; Dimerization; Disease Models, Animal; Endotoxemia; Enzyme Inhibitors; Gene Expression Regulation, Enzymologic; Hepatocyte Nuclear Factor 1-beta; Humans; Inflammation; MafG Transcription Factor; Male; Mutation; Naphthalenes; Nitric Oxide Synthase Type II; Protein Kinase C; Rats; Rats, Sprague-Dawley; Repressor Proteins; Response Elements; RNA, Small Interfering; Signal Transduction; Smad6 Protein; Smad7 Protein; Staurosporine; Tetradecanoylphorbol Acetate; Transforming Growth Factor beta1

Certain forms of coronary artery disease do not respond to treatment with Ca2+ channel blockers, and a role for endothelin-1 (ET-1) in Ca2+ antagonist-insensitive forms of coronary vasospasm has been suggested; however, the signaling mechanisms involved are unclear. We tested the hypothesis that a component of ET-1-induced coronary smooth muscle contraction is Ca2+ antagonist-insensitive and involves activation of protein kinase C (PKC). Cell contraction was measured in smooth muscle cells isolated from porcine coronary artery, [Ca2+]i was measured in fura-2 loaded cells, and the cytosolic and particulate fractions were examined for PKC activity and reactivity with isoform-specific PKC antibodies using Western blot analysis. In Hank's solution (1 mmol/L Ca2+), ET-1 (10(-7) mol/L) caused a transient increase in [Ca2+]i (236+/-14 nmol/L) followed by a maintained increase in [Ca2+]i (184+/-8 nmol/L) and 35% cell contraction. The Ca2+ channel blockers verapamil and diltiazem (10(-6) mol/L) abolished the maintained ET-1-induced [Ca2+]i, but only partially inhibited ET-1-induced cell contraction to 18%. The verapamil-insensitive component of ET-1 contraction was inhibited by the PKC inhibitors calphostin C and epsilon-PKCV1-2. ET-1 caused translocation of Ca2+-dependent alpha-PKC and Ca2+-independent epsilon-PKC from the cytosolic to the particulate fraction that was inhibited by calphostin C. Verapamil abolished ET-1-induced translocation of alpha-PKC, but not that of epsilon-PKC. Phorbol 12-myristate 13-acetate (10(-6) mol/L), a direct activator of PKC, caused 22% cell contraction, with no increase in [Ca2+]i, and translocation of epsilon-PKC that was inhibited by calphostin C, but not by verapamil. KCl (51 mmol/L), which stimulates Ca2+ influx, caused 35% cell contraction and increase in [Ca2+]i (291+/-11 nmol/L) that were inhibited by verapamil, but not by calphostin C, and did not cause translocation of alpha- or epsilon-PKC. In Ca2+-free (2 mmol/L EGTA) Hank's solution, ET-1 caused 15% cell contraction, with no increase in [Ca2+]i, and translocation of epsilon-PKC that were inhibited by epsilon-PKC V1-2 inhibitory peptide. Thus, a significant component of ET-1-induced contraction of coronary smooth muscle is Ca2+ antagonist-insensitive and involves activation and translocation of Ca2+-independent epsilon-PKC, and may represent a signaling mechanism of Ca2+ antagonist-resistant forms of coronary vasospasm.

Topics: Animals; Calcium; Calcium Channel Blockers; Coronary Vasospasm; Coronary Vessels; Diltiazem; Disease Models, Animal; Drug Resistance; Endothelin-1; Enzyme Activation; Enzyme Inhibitors; Male; Muscle, Smooth, Vascular; Naphthalenes; Peptide Fragments; Protein Kinase C; Protein Kinase C-epsilon; Protein Transport; Signal Transduction; Swine; Tetradecanoylphorbol Acetate; Vasoconstriction; Verapamil

We examined the effect of diabetes on the fenvalerate-induced nociceptive response in mice. The intrathecal (i.t.) or intraplantar (i.pl.) injection of fenvalerate, a sodium channel activator, induced a characteristic behavioral syndrome mainly consisting of reciprocal hind limb scratching directed towards caudal parts of the body and biting or licking of the hind legs in both non-diabetic and diabetic mice. However, the intensity of such fenvalerate-induced nociceptive responses was significantly greater in diabetic mice than in non-diabetic mice. Calphostin C (3 pmol, i.t.), a selective protein kinase C inhibitor, significantly inhibited intrathecal fenvalerate-induced nociceptive behavior with a rightward shift of the dose-response curve for fenvalerate-induced nociceptive behavior to the level those observed in non-diabetic mice. On the other hand, when non-diabetic mice were pretreated with phorbol-12, 13-dibutyrate (50 pmol, i.t.), the dose-response curve for intrathecal fenvalerate-induced nociceptive behavior was shifted leftward to the level those observed in diabetic mice. These results suggest that the sensitization of sodium channels, probably tetrodotoxin-resistant (TTX-R) sodium channels, by the long-term activation of protein kinase C may play an important role in the enhancement of the duration of fenvalerate-induced nociceptive behavior in diabetic mice.

Topics: Animals; Behavior, Animal; Diabetes Mellitus, Experimental; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Administration Routes; Enzyme Activators; Enzyme Inhibitors; Hindlimb; Injections, Spinal; Male; Mexiletine; Mice; Mice, Inbred ICR; Naphthalenes; Nitriles; Pain Measurement; Phorbol 12,13-Dibutyrate; Protein Kinase C; Pyrethrins; Sodium Channels; Streptozocin

Several recent studies have suggested an ATP-sensitive potassium channel opener (2-nicotinamidoethyl nitrate: 2-NN) may exert a protective effect against the myocardial ischemic/reperfusion injury. This study examines the effects of 2-NN on intracellular signaling by measuring intracellular cyclic AMP, cyclic GMP accumulation and protein kinase C (PKC) activity after 2-NN perfusion.. Ischemia/reperfused hearts were made by LAD occlusion for 30 min followed by 30 min of reperfusion in isolated rat hearts. Hearts were pre-perfused with 0.1 mM 2-NN, 100 nM Calphostin C, or 2-NN plus Calphostin C for 10 min prior to ischemia. The left ventricular function, cyclic AMP, cyclic GMP and LDH were examined to determine the effects of 2-NN on ischemic/reperfusion injury. Four separate groups of hearts were stained with a bisindolylmaleimide PKC inhibitor conjugated to fluorescein (fim, Teflabs) and PKC activity was measured.. 2-NN reduced ischemia/reperfusion injury as evidenced by the enhanced myocardial functional recovery, decreased LDH release after reperfusion, and decreased reperfusion arrhythmias. The PKC inhibitor attenuated myocardial functional recovery but not reperfusion arrhythmias. Cyclic AMP levels decreased after 10 min of 2-NN perfusion, compared to controls. We observed an increase in PKC activity after 2-NN treatment.. These results suggest that PKC plays a significant role in the cardioprotective effect of 2-NN on ischemic and reperfused myocardium. The anti-arrhythmic effect of 2-NN in the reperfusion phase may be linked its action on the ATP-sensitive potassium channel itself rather than its effect on PKC activity.

Topics: Animals; Coronary Circulation; Cyclic AMP; Cyclic GMP; Disease Models, Animal; Heart Function Tests; Hemodynamics; Male; Microscopy, Fluorescence; Myocardial Contraction; Myocardial Ischemia; Myocardial Reperfusion Injury; Naphthalenes; Nicorandil; Probability; Random Allocation; Rats; Rats, Sprague-Dawley; Reference Values; Sensitivity and Specificity