l-165041 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

There is a major clinical need for new therapies for the treatment of chronic itch. Many of the molecular components involved in itch neurotransmission are known, including the neuropeptide NPPB, a transmitter required for normal itch responses to multiple pruritogens in mice. Here, we investigated the potential for a novel strategy for the treatment of itch that involves the inhibition of the NPPB receptor NPR1 (natriuretic peptide receptor 1). Because there are no available effective human NPR1 (hNPR1) antagonists, we performed a high-throughput cell-based screen and identified 15 small-molecule hNPR1 inhibitors. Using in vitro assays, we demonstrated that these compounds specifically inhibit hNPR1 and murine NPR1 (mNPR1). In vivo, NPR1 antagonism attenuated behavioral responses to both acute itch- and chronic itch-challenged mice. Together, our results suggest that inhibiting NPR1 might be an effective strategy for treating acute and chronic itch.

Topics: Animals; Behavior, Animal; Cell-Free System; Dermatitis, Contact; Disease Models, Animal; Ganglia, Spinal; Humans; Mice, Inbred C57BL; Mice, Knockout; Neurons; Pruritus; Receptors, Atrial Natriuretic Factor; Reproducibility of Results; Signal Transduction; Small Molecule Libraries

The arachidonic acid preferred long-chain acyl-CoA synthetase 4 (ACSL4) is a key enzyme for fatty acid metabolism in various metabolic tissues. In this study, we utilized hamsters fed a normal chow diet, a high-fat diet or a high cholesterol and high fat diet (HCHFD) as animal models to explore novel transcriptional regulatory mechanisms for ACSL4 expression under hyperlipidemic conditions. Through cloning hamster ACSL4 homolog and tissue profiling ACSL4 mRNA and protein expressions we observed a selective upregulation of ACSL4 in testis and liver of HCHFD fed animals. Examination of transcriptional activators of the ACSL family revealed an increased hepatic expression of PPARδ but not PPARα in HCHFD fed hamsters. To explore a role of PPARδ in dietary cholesterol-mediated upregulation of ACSL4, we administered a PPARδ specific agonist L165041 to normolipidemic and dyslipidemic hamsters. We observed significant increases of hepatic ACSL4 mRNA and protein levels in all L165041-treated hamsters as compared to control animals. The induction of ACSL4 expression by L165041 in liver tissue in vivo was recapitulated in human primary hepatocytes and hepatocytes isolated from hamster and mouse. Moreover, employing the approach of adenovirus-mediated gene knockdown, we showed that depletion of PPARδ in hamster hepatocytes specifically reduced ACSL4 expression. Finally, utilizing HepG2 as a model system, we demonstrate that PPARδ activation leads to increased ACSL4 promoter activity, mRNA and protein expression, and consequently higher arachidonoyl-CoA synthetase activity. Taken together, we have discovered a novel PPARδ-mediated regulatory mechanism for ACSL4 expression in liver tissue and cultured hepatic cells.

Topics: Animals; Cholesterol, Dietary; Cloning, Molecular; Coenzyme A Ligases; Diet, High-Fat; Disease Models, Animal; Enzyme Induction; Gene Expression Profiling; HEK293 Cells; Hep G2 Cells; Hepatocytes; Humans; Hyperlipidemias; Male; Mesocricetus; Mice, Inbred C57BL; Phenoxyacetates; PPAR gamma; Promoter Regions, Genetic; RNA Interference; RNA, Messenger; Testis; Transcription, Genetic; Transcriptional Activation; Transfection

The mechanisms regarding hepatic steatosis related to hepatic insulin resistance have been well documented. However, the agents for treatment of hepatic steatosis and insulin resistance remain poorly developed. Peroxisome proliferator-activated receptors (PPARs) are transcription factors that are responsible for the regulation of glucose and/or lipid metabolism. There are 3 distinct isoforms of PPARs family: PPARα, PPARγ, and PPARδ. Both PPARα and PPARγ agonists are widely used in clinic for the treatment of hyperlipidemia and hyperglycemia. However, the therapeutic efficacy of PPARδ agonists for diabetic disorders remains obscure. In the present study, we used L-165041 as PPARδ agonist to treat the high fat diet (HFD) fed mice. Administration of L-165041 improved the hepatic steatosis and increased the insulin sensitivity in HFD-mice. In addition to the histological identification of hepatic steatosis, the improvement of insulin sensitivity was characterized by the enhanced insulin signals and the increase of hepatic glycogen content. This is the first report showing that pharmacological activation of PPARδ improves insulin resistance in diet-induced diabetic mice. Thus, we suggest that pharmacological activation of PPARδ may be a new strategy for the treatment of diabetic patients with hepatic steatosis.

Topics: Animals; Diabetes Mellitus, Type 2; Diet, High-Fat; Disease Models, Animal; Fatty Liver; Humans; Insulin; Insulin Resistance; Male; Mice; Mice, Inbred C57BL; Phenoxyacetates; PPAR delta

The peroxisome proliferator-activated receptor (PPAR) family of nuclear hormone receptors consists of three subtypes (alpha, beta/delta, and gamma). PPAR delta is ubiquitously expressed and involved in lipid and glucose metabolism. However, the effect of PPAR delta on vascular smooth muscle cell (VSMC) proliferation and migration has not been fully elucidated yet. Here, we investigated the effect of L-165041, a selective ligand for PPAR delta, on PDGF-induced rat VSMC proliferation. Our data show that L-165041 inhibited rat VSMC proliferation in a dose dependent manner by blocking G(1) to S phase progression and repressing the phosphorylation of retinoblastoma protein (Rb). Furthermore, L-165041 inhibited PDGF-induced expression of cyclin D1 and CDK4. These effects less likely involve PPAR gamma pathway because PPAR gamma antagonist GW9662 pretreatment failed to reverse the inhibitory effect of L-165041 on rVSMC proliferation and migration. For in vivo studies, L-165041 was administered to Sprague-Dawley rats using osmotic pumps before and after the carotid balloon injury, and L-165041 decreased neointima formation after the carotid injury. In conclusion, our results suggest that PPAR delta ligand L-165041 can be a therapeutic agent to control pathologic cardiovascular conditions such as restenosis and atherosclerosis.

Topics: Angioplasty, Balloon; Animals; Aorta, Thoracic; Carotid Artery Injuries; Cell Cycle; Cell Division; Cell Movement; Cells, Cultured; Disease Models, Animal; Extracellular Signal-Regulated MAP Kinases; Muscle, Smooth, Vascular; Phenoxyacetates; Phosphorylation; PPAR delta; Rats; Rats, Sprague-Dawley; src-Family Kinases

Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear receptor superfamily and function as ligand-modulated transcription factors that regulate gene expression in many important biological processes. The PPARdelta subtype has the highest expression in the brain and is postulated to play a major role in neuronal cell function; however, the precise physiological roles of this receptor remain to be elucidated. Herein, we show that the high-affinity PPARdelta agonists L-165041 [4-[3-(4-acetyl-3-hydroxy-2-propylphenoxy)-propoxyl]phenoxy]-acetic acid] and GW501516 [2-methyl4-((4-methyl-2-(4-trifluoromethylphenyl)-1,3-triazol-5-yl)-methylsulfanyl)phenoxy acetic acid] protect against cytotoxin-induced SH-SY5Y cell injury in vitro and both ischemic brain injury and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) neurotoxicity in vivo. In the SH-SY5Y studies, treatment with L-165041 or GW501516 significantly and concentration-dependently attenuated cell death following thapsigargin, 1-methyl-4-phenylpyridinium, or staurosporine exposure, with the extent of damage correlated with the level of caspase-3 inhibition. In the transient (90 min) middle cerebral artery occlusion model of ischemic brain injury in rats, i.c.v. infusion of L-165041 or GW501516 significantly attenuated the ischemic brain damage measured 24 h after reperfusion. Moreover, the PPARdelta agonists also significantly attenuated MPTP-induced depletion of striatal dopamine and related metabolite contents in mouse brain. These results demonstrate that subtype-selective PPARdelta agonists possess antiapoptotic properties in vitro, which may underlie their potential neuroprotective potential in in vivo experimental models of cerebral ischemia and Parkinson's disease (PD). These findings suggest that PPARdelta agonists could be useful tools for understanding the role of PPARdelta in other neurodegenerative disorders, as well as attractive therapeutic candidates for stroke and neurodegenerative diseases such as PD.

Topics: Acetates; Animals; Brain; Brain Ischemia; Caspases; Cell Line, Tumor; Cell Survival; Disease Models, Animal; Dopamine; Genes, Reporter; Humans; Ligands; Male; Mice; Mice, Inbred C57BL; Neuroprotective Agents; Parkinson Disease; Phenols; Phenoxyacetates; PPAR delta; Rats; Rats, Wistar; Substrate Specificity; Thiazoles