gw-5074 and Disease-Models--Animal

Necroptosis is a form of regulated necrotic cell death that is independent of caspases. Receptor-interacting protein kinase 3 (RIPK3) has been identified as a key regulator for necroptosis, and has been proposed as a potential therapeutic target for the treatment of diseases associated with necroptosis. In this report, we describe the design, synthesis, and evaluation of a series of novel RIPK3 inhibitors. The lead compound 38 exhibited potent activity (EC

Topics: Animals; Antineoplastic Agents; Cell Death; Cell Proliferation; Cell Survival; Cells, Cultured; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Humans; Hypothermia; Injections, Intravenous; Mice; Mice, Inbred C57BL; Molecular Structure; Protein Kinase Inhibitors; Receptor-Interacting Protein Serine-Threonine Kinases; Structure-Activity Relationship; Systemic Inflammatory Response Syndrome; Tumor Necrosis Factor-alpha

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 pineal hormone melatonin influences the secretion of insulin by pancreatic islets via the G‑protein‑coupled melatonin receptors 1 and 2 that are expressed in pancreatic β‑cells. Genome‑wide association studies indicate that melatonin receptor 1B (MTNR1B) single nucleotide polymorphisms are tightly associated with type 2 diabetes mellitus (T2DM). However, the underlying mechanism is unclear. Raf‑1 serves a critical role in the mitogen‑activated protein kinase (MAPK) pathways in β‑cell survival and proliferation and, therefore, may be involved in the mechanism by which melatonin impacts on T2DM through MTNR1B. In the present study, the mRNA expression of the two mouse insulin genes Ins1 and Ins2 was investigated in MIN6 cells treated with different concentrations of melatonin, and insulin secretion was detected under the same conditions. Following the overexpression or silencing of MTNR1B, the activities of components of the MAPK signaling pathway, including Raf‑1 and ERK, were evaluated. The impact of MTNR1B knockdown on the melatonin‑regulated insulin gene expression and insulin secretion were also investigated. The results demonstrated that exogenous melatonin inhibited the expression of insulin mRNA in the MIN6 cells. Insulin secretion by the MIN6 cells, however, was not affected by melatonin. The MAPK signaling pathway was inhibited in MIN6 cells by treatment with melatonin or the overexpression of MTNR1B. The knockdown of MTNR1B totally attenuated the regulating effect of melatonin on insulin gene expression. Additionally, the inductive effect of melatonin on the expression of insulin mRNA was attenuated when the activities of Raf‑1 or ERK were blocked using the chemical inhibitors GW5074 and U0126, respectively. It may be concluded that melatonin exerts an inhibitory effect on insulin transcription via MTNR1B and the downstream MAPK signaling pathway.

Topics: Animals; Butadienes; Diabetes Mellitus, Type 2; Disease Models, Animal; Gene Expression Regulation; Humans; Indoles; Insulin; Islets of Langerhans; MAP Kinase Signaling System; Melatonin; Mice; Nitriles; Phenols; Proto-Oncogene Proteins c-raf; Receptor, Melatonin, MT2

The role of mitogen-activated protein kinase (MAPK) family has been well defined in neuropathic pain. Ras and c-Raf constitute an important part of MAP kinase family as Ras/Raf/MEK/ERK2 signaling cascade. The present study was designed to investigate the analgesic potential of farnesyl thiosalicylic acid, a novel Ras inhibitor, and GW 5074, a selective c-Raf1 inhibitor, in chronic constriction-induced injury (CCI)-induced peripheral neuropathic pain. Neuropathic pain was induced by placing four loose ligatures around the sciatic nerve. The development of pain was assessed on 14th day in terms of cold allodynia; mechanical hyperalgesia and mechanical allodynia by performing acetone test, pinprick and Von Frey tests, respectively. Farnesyl thiosalicylic acid (2.5, 5 and 10 μg) and GW 5074 (1, 2 and 4 μg) were injected intrathecally on 14th day following nerve ligature to assess their analgesic potential in CCI model. Nerve ligature-induced CCI produced significant neuropathic pain manifestations in terms of cold and mechanical allodynia, and mechanical hyperalgesia. Single intrathecal administration of farnesyl thiosalicylic acid (5 and 10 μg) as well as GW 5074 (2 and 4 μg) significantly attenuated CCI-induced hyperalgesia and allodynia. The analgesic effects of farnesyl thiosalicylic acid and GW 5074 in CCI model suggests that pharmacological inhibition of Ras and c-Raf-1 signaling may be potentially useful for managing neuropathic pain.

Topics: Analgesics; Animals; Constriction, Pathologic; Disease Models, Animal; Dose-Response Relationship, Drug; Female; Hyperalgesia; Indoles; Injections, Spinal; Male; Pain Measurement; Pain Threshold; Phenols; Polyisoprenyl Phosphates; Rats; Rats, Wistar; Sciatica

Mutations in leucine-rich repeat kinase 2 (LRRK2) have been identified as a genetic cause of familial Parkinson's disease (PD) and have also been found in the more common sporadic form of PD, thus positioning LRRK2 as important in the pathogenesis of PD. Biochemical studies of the disease-causing mutants of LRRK2 implicates an enhancement of kinase activity as the basis of neuronal toxicity and thus possibly the pathogenesis of PD due to LRRK2 mutations. Previously, a chemical library screen identified inhibitors of LRRK2 kinase activity. Here, two of these inhibitors, GW5074 and sorafenib, are shown to protect against G2019S LRRK2-induced neurodegeneration in vivo in Caenorhabditis elegans and in Drosophila. These findings indicate that increased kinase activity of LRRK2 is neurotoxic and that inhibition of LRRK2 activity can have a disease-modifying effect. This suggests that inhibition of LRRK2 holds promise as a treatment for PD.

Topics: Animals; Animals, Genetically Modified; Benzenesulfonates; Caenorhabditis elegans; Cell Survival; Disease Models, Animal; Dopaminergic Neurons; Drosophila; Enzyme Activation; Humans; Indoles; Motor Activity; Mutation; Niacinamide; Oxidopamine; Parkinson Disease; Phenols; Phenotype; Phenylurea Compounds; Protein Kinase Inhibitors; Protein Serine-Threonine Kinases; Pyridines; Sorafenib; Synucleins

Human tissue transglutaminase (TGM2) is a calcium-dependent crosslinking enzyme involved in the posttranslational modification of intra- and extracellular proteins and implicated in several neurodegenerative diseases. To find specific inhibitors to TGM2, two structurally diverse chemical libraries (LOPAC and Prestwick) were screened. We found that ZM39923, a Janus kinase inhibitor, and its metabolite ZM449829 were the most potent inhibitors with IC(50) of 10 and 5 nM, respectively. In addition, two other inhibitors, including tyrphostin 47 and vitamin K(3), were found to have an IC(50) in the micromolar range. These agents used in part a thiol-dependent mechanism to inhibit TGM2, consistent with the activation of TGM2 by reduction of an intramolecular disulfide bond. These inhibitors were tested in a polyglutamine-expressing Drosophila model of neurodegeneration and found to improve survival. The TGM2 inhibitors we discovered may serve as valuable lead compounds for the development of orally active TGM2 inhibitors to treat human diseases.

Topics: Animals; Calcium; Combinatorial Chemistry Techniques; Disease Models, Animal; Drosophila melanogaster; Drug Evaluation, Preclinical; Enzyme Inhibitors; Factor XIIIa; GTP-Binding Proteins; Guanosine Triphosphate; Humans; Machado-Joseph Disease; Molecular Structure; Naphthoquinones; Octoxynol; Protein Glutamine gamma Glutamyltransferase 2; Transglutaminases; Tyrphostins

Cerebellar granule neurons undergo apoptosis when switched from a medium containing high potassium (HK) to one that has low potassium (LK). LK-induced cell death is blocked by GW5074 [5-Iodo-3-[(3,5-dibromo-4-hydroxyphenyl) methylene]-2-indolinone], a synthetic drug that inhibits c-Raf activity in vitro. GW5074 has no direct effect on the activities of several apoptosis-associated kinases when assayed in vitro. In contrast to its effect in vitro, treatment of neurons with GW5074 causes c-Raf activation (when measured in vitro in the absence of the drug) and stimulates the Raf-MEK-ERK pathway. Treatment of neurons with GW5074 also leads to an increase in the activity of B-Raf, which is not inhibited by GW5074 in vitro at concentrations at which the drug exerts its neuroprotective effect. PD98059 and U0126, two distinct inhibitors of MEK, block the activation of ERK by GW5074 but have no effect on its ability to prevent cell death. Overexpression of a dominant-negative form of Akt does not reduce the efficacy of GW5074, demonstrating an Akt-independent mechanism of action. Neuroprotection is inhibited by SN-50, a specific inhibitor of nuclear factor-kappa B (NF-kappaB) and by the Ras inhibitor S-trans, trans-farnesylthiosalicylic acid (FTS) implicating NF-kappaB and Ras in the neuroprotective signaling pathway activated by GW5074. In addition to preventing LK-induced apoptosis, treatment with GW5074 protects against the neurotoxic effects of MPP+ and methylmercury in cerebellar granule neurons, and glutathione depletion-induced oxidative stress in cortical neurons. Furthermore, GW5074 prevents neurodegeneration and improves behavioral outcome in an animal model of Huntington's disease. Given its neuroprotective effect on distinct types of cultured neurons, in response to different neurotoxic stimuli, and in an animal model of neurodegeneration, GW5074 could have therapeutic value against neurodegenerative pathologies in humans.

Topics: Animals; Cell Death; Cells, Cultured; Disease Models, Animal; Enzyme Inhibitors; Farnesol; Huntington Disease; Indoles; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; Neurodegenerative Diseases; Neurons; Neuroprotective Agents; Neurotoxins; Nitro Compounds; Phenols; Propionates; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-raf; Rats; Rats, Sprague-Dawley; Salicylates; Signal Transduction