9-(tetrahydro-2-furyl)-adenine 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

A systems pharmacological approach that capitalizes on the characterization of intracellular signaling networks can transform our understanding of human diseases and lead to therapy development. Here, we applied this strategy to identify pharmacological targets for the treatment of Stargardt disease, a severe juvenile form of macular degeneration. Diverse GPCRs have previously been implicated in neuronal cell survival, and crosstalk between GPCR signaling pathways represents an unexplored avenue for pharmacological intervention. We focused on this receptor family for potential therapeutic interventions in macular disease. Complete transcriptomes of mouse and human samples were analyzed to assess the expression of GPCRs in the retina. Focusing on adrenergic (AR) and serotonin (5-HT) receptors, we found that adrenoceptor α 2C (Adra2c) and serotonin receptor 2a (Htr2a) were the most highly expressed. Using a mouse model of Stargardt disease, we found that pharmacological interventions that targeted both GPCR signaling pathways and adenylate cyclases (ACs) improved photoreceptor cell survival, preserved photoreceptor function, and attenuated the accumulation of pathological fluorescent deposits in the retina. These findings demonstrate a strategy for the identification of new drug candidates and FDA-approved drugs for the treatment of monogenic and complex diseases.

Topics: Adenine; Adenylyl Cyclase Inhibitors; Adrenergic alpha-Agonists; Adrenergic alpha-Antagonists; Alcohol Oxidoreductases; Animals; ATP-Binding Cassette Transporters; Cell Survival; Disease Models, Animal; Doxazosin; Drug Evaluation, Preclinical; Guanabenz; Humans; Light; Macaca fascicularis; Macular Degeneration; Mice; Mice, Inbred BALB C; Mice, Knockout; Molecular Targeted Therapy; Nerve Tissue Proteins; Photoreceptor Cells, Vertebrate; Reactive Oxygen Species; Receptor, Serotonin, 5-HT2A; Receptors, Adrenergic, alpha-2; Receptors, G-Protein-Coupled; Serotonin Antagonists; Signal Transduction; Stargardt Disease

In this study we investigated the activity of the nitric oxide synthase (NOS) in parotid glands from rats with experimental periodontitis and controls.. Periodontitis was produced by a ligature placed around the cervix of the two lower first molar. Experiments were carried out 22 days after the ligature.. Ligation caused an increase in parotid NOS activity. The selective blocker of the inducible isoform of the enzyme partially inhibited its activity in parotid glands from rat with ligature. In controls, the activity was partially inhibited by the antagonists of the selective neural and endothelial isoforms. NOS activity in rats with ligature was cyclic adenosine monophosphate (cAMP)-dependent while in controls it was calcium-dependent. Prostaglandin E₂ concentration was increased in parotid gland from rats with ligature. The inhibitor of prostaglandin production, FR 122047, diminished both, prostaglandin production and NOS activity. In rats with ligature unstimulated amylase released is increased. Both, prostaglandin and NOS were involved in the increment of amylase release.. It can be concluded that in parotid glands from ligated rats, prostaglandin E₂ production is increased and, through cAMP accumulation, activates the inducible NOS isoform. The increment of nitric oxide production participates in the increase in basal amylase release.

Topics: Adenine; Amylases; Animals; Calcium; Cyclic AMP-Dependent Protein Kinase Catalytic Subunits; Cyclooxygenase Inhibitors; Dinoprostone; Disease Models, Animal; Egtazic Acid; Enzyme Inhibitors; Guanidines; Indazoles; Indomethacin; Male; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; omega-N-Methylarginine; Organ Size; Ornithine; Parotid Gland; Periodontitis; Piperazines; Prostaglandin-Endoperoxide Synthases; Rats; Rats, Wistar; Salivary Proteins and Peptides; Thiazoles

The role of hyperpolarization-activated, cyclic nucleotide-modulated (HCN) channel isoforms and hyperpolarization-activated cation current (Ih) for seizure-related burst firing in thalamocortical (TC) neurons was investigated in a rat genetic model of absence epilepsy [Wistar Albino Glaxo rats, bred in Rijswijk (WAG/Rij)]. Burst discharges in TC neurons locked to seizure activity in vivo were prolonged during blockade of Ih by Cs+ and ZD7288 (4-ethylphenylamino-1,2-dimethyl-6-methylaminopyrimidinium chloride). In vitro analyses revealed a hyperpolarizing shift of half-maximal Ih activation (Vh) in WAG/Rij (Vh = -93.2 mV) compared with nonepileptic controls [August x Copenhagen-Irish (ACI) (Vh = -88.0 mV)]. This effect is explained by a shift of the responsiveness of Ih to cAMP toward higher concentrations in TC neurons from WAG/Rij, as revealed by application of 8-bromo-cAMP and the phosphodiesterase inhibitor IBMX. During blockade of adenylyl cyclase activity, Ih activation was similar in the two strains, whereas the difference in cAMP responsiveness persisted, thereby voting against different ambient cAMP levels between strains. Increasing the intracellular cAMP level and shifting Ih activation led to a change from burst to tonic firing mode in WAG/Rij but not in ACI rats. Furthermore, HCN1 expression was significantly increased on mRNA and protein levels, with no changes in HCN2-4 expression. In conclusion, there is an increase in HCN1 expression in the epileptic thalamus, associated with a decrease in cAMP responsiveness of Ih in TC neurons and resulting impairment to control the shift from burst to tonic firing, which, in turn, will prolong burst activity after recruitment of Ih during absence seizures.

Topics: 1-Methyl-3-isobutylxanthine; 8-Bromo Cyclic Adenosine Monophosphate; Action Potentials; Adenine; Animals; Blotting, Northern; Cerebral Cortex; Cesium; Chlorides; Cyclic Nucleotide-Gated Cation Channels; Disease Models, Animal; Dose-Response Relationship, Drug; Dose-Response Relationship, Radiation; Drug Interactions; Electric Stimulation; Electroencephalography; Enzyme Inhibitors; Epilepsy, Absence; Female; Gene Expression Regulation; Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; Immunohistochemistry; In Situ Hybridization; In Vitro Techniques; Ion Channels; Male; Membrane Potentials; Microinjections; Neurons; Patch-Clamp Techniques; Potassium Channels; Protein Isoforms; Pyrimidines; Rats; Rats, Mutant Strains; Rats, Wistar; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Thalamus

Spinal activation of the cAMP pathway produces mechanical hyperalgesia, sensitizes nociceptive spinal neurons, and phosphorylates the transcription factor cAMP-responsive element binding protein (CREB), which initiates gene transcription. This study examined the role of the cAMP pathway in a model of chronic muscle pain by assessing associated behavioral changes and phosphorylation of CREB. Bilateral mechanical hyperalgesia of the paw was induced by administering two injections of acidic saline, 5 d apart, into the gastrocnemius muscle of male Sprague Dawley rats. Interestingly, the increases in immunoreactivity for CREB and phosphorylated CREB (p-CREB) in the spinal dorsal horn occur 24 hr, but not 1 week, after the second injection of acidic saline compared with pH 7.2 intramuscular injections. Spinal blockade of adenylate cyclase prevents the expected increase in p-CREB that occurs after intramuscular acid injection. The reversal of mechanical hyperalgesia by adenylate cyclase or protein kinase A inhibitors spinally follows a similar pattern with reversal at 24 hr, but not 1 week, compared with the vehicle controls. The p-CREB immunoreactivity in the superficial dorsal horn correlates with the mechanical withdrawal threshold such that increases in p-CREB are associated with decreases in threshold. Therefore, activation of the cAMP pathway in the spinal cord phosphorylates CREB and produces mechanical hyperalgesia associated with intramuscular acid injections. The mechanical hyperalgesia and phosphorylation of CREB depend on early activation of the cAMP pathway during the first 24 hr but are independent of the cAMP pathway by 1 week after intramuscular injection of acid.

Topics: Acids; Adenine; Adenylyl Cyclase Inhibitors; Animals; Behavior, Animal; Chronic Disease; Cyclic AMP; Cyclic AMP Response Element-Binding Protein; Cyclic AMP-Dependent Protein Kinases; Disease Models, Animal; Dose-Response Relationship, Drug; Enzyme Inhibitors; Fibromyalgia; Hyperalgesia; Injections, Intramuscular; Injections, Spinal; Male; Motor Activity; Muscle, Skeletal; Pain Measurement; Phosphorylation; Rats; Rats, Sprague-Dawley; Signal Transduction; Spinal Cord