Azilsartan ameliorates ox-LDL-induced endothelial dysfunction via promoting the expression of KLF2.

BACKGROUND: Oxidized LDL(Ox-LDL) mediated endothelial dysfunction is involved in the pathogenesis of various cardiovascular diseases, including atherosclerosis. Azilsartan is a potent agent for the treatment of hypertension as the antagonist of the angiotensin II receptor. This study will investigate whether Azilsartan possesses a beneficial effect against endothelial cell dysfunction induced by ox-LDL and explore the underlying preliminary mechanism. METHODS: Ox-LDL was applied to construct an in vitro endothelial dysfunction model in human umbilical vascular endothelial cells (HUVECs). The expression of lectin-type oxidized LDL receptor 1 (LOX-1), endothelial nitric oxide synthase (eNOS), tight junction protein occludin, and transcriptional factor Krüppel-like factor 2 (KLF2) was detected using qRT-PCR and Western blot. ELISA and qRT-PCR were utilized to evaluate the production of chemokine monocyte chemotactic protein 1 (MCP-1) and chemokine (C-X-C motif) Ligand 1 Protein (CXCL1) in treated HUVECs. The generation of nitro oxide (NO) was determined using DAF-FM DA staining assay. KLF2 was silenced by transfecting the cells with specific Small interfering RNA (siRNA). FITC-dextran permeation assay was used to check the endothelial monolayer permeability of treated HUVECs. RESULTS: Firstly, the elevated expressions of LOX-1, MCP-1, and CXCL-1 induced by stimulation with ox-LDL were significantly suppressed by Azilsartan. The downregulated eNOS and reduced production of NO induced by ox-LDL were reversed by the introduction of Azilsartan. Secondly, enlarged endothelial monolayer permeability and decreased expression of occludin stimulated with ox-LDL were greatly reversed by treatment with Azilsartan but were abolished by silencing the expression of KLF2. Lastly, the inhibited expression of KLF2 induced by ox-LDL was significantly elevated by the introduction of Azilsartan. CONCLUSION: Azilsartan might ameliorate ox-LDL-induced endothelial damage via elevating the expression of KLF2.

Synthesis of oxadiazole-2-oxide derivatives as potential drug candidates for schistosomiasis targeting SjTGR.

BACKGROUND: Schistosomiasis is a chronic parasitic disease that affects millions of people's health worldwide. Because of the increasing drug resistance to praziquantel (PZQ), which is the primary drug for schistosomiasis, developing new drugs to treat schistosomiasis is crucial. Oxadiazole-2-oxides have been identified as potential anti-schistosomiasis reagents targeting thioredoxin glutathione reductase (TGR). METHODS: In this work, one of the oxadiazole-2-oxides derivatives furoxan was used as the lead compound to exploit a series of novel furoxan derivatives for studying inhibitory activity against both recombinant Schistosoma japonicum TGR containing selenium (rSjTGR-Sec) and soluble worm antigen protein (SWAP) containing wild-type Schistosoma japonicum TGR (wtSjTGR), in order to develop a new leading compound for schistosomiasis. Thirty-nine novel derivatives were prepared to test their activity toward both enzymes. The docking method was used to detect the binding site between the active molecule and SjTGR. The structure-activity relationship (SAR) of these novel furoxan derivatives was preliminarily analyzed. RESULTS: It was found that several new derivatives, including compounds 6a-6d, 9ab, 9bd and 9be, demonstrated greater activity toward rSjTGR-Sec or SWAP containing wtSjTGR than did furoxan. Interestingly, all intermediates bearing hydroxy (6a-6d) showed excellent inhibitory activity against both enzymes. In particular, compound 6d with trifluoromethyl on a pyridine ring was found to have much higher inhibition toward both rSjTGR-Sec (half-maximal inhibitory concentration, IC50,7.5nM) and SWAP containing wtSjTGR (IC50 55.8nM) than furoxan. Additionally, the docking method identified the possible matching sites between 6d and Schistosoma japonicum TGR (SjTGR), which theoretically lends support to the inhibitory activity of 6d. CONCLUSION: The data obtained herein showed that 6d with trifluoromethyl on a pyridine ring could be a valuable leading compound for further study.

1,3,4-oxadiazole and its derivatives: A review on recent progress in anticancer activities.

The 1,3,4-oxadiazole nucleus is a biologically imperative scaffold possesses numerous biological activities. The broad and potent activity of 1,3,4-oxadiazole and their derivatives has established them as important pharmacological scaffolds especially in the treatment of cancer disease. Several di-, tri-, aromatic, and heterocyclic substituted 1,3,4-oxadiazole derivatives have been reported to possess potent anticancer activity. These substituted 1,3,4-oxadiazoles had shown different mechanism of action and participated in anticancer drug discovery and development. This review is complementary to earlier reviews and aims to review the work reported on anticancer activities of 1,3,4-oxadiazole derivatives from year 2000 to the beginning of 2020.

Evaluation and Docking Study of Pyrazine Containing 1, 3, 4-Oxadiazoles Clubbed with Substituted Azetidin-2-one: A New Class of Potential Antimicrobial and Antitubercular.

BACKGROUND: Tuberculosis (TB) caused by Mycobacterium tuberculosis is one of the main killers of people all over the world. The major hurdles with existing therapy are the lengthy regimen and appearance of multi drug resistant (MDR) and extensively drug resistant (XDR) strains of M.tuberculosis. AIMS: The present work was aimed to synthesize and determine antitubercular and antimicrobial potential of some novel 3-chloro-4-aryl-1-[4-(5-pyrazin-2-yl[1,3,4]oxadiazole-2-ylmethoxy)-phenyl]-azetidin-2-one derivatives 7: (A: -H: ) from pyrazinoic acid as precursor, which is a well-established antitubercular agent. Here we report the synthesis of a new class of heterocyclic molecules in which pyrazine, 1, 3, 4-oxadiazole and azetidinone moieties were present in one frame work. METHODS: Pyrazinoic acid (1: ) was esterified first (2: ) followed by amination to produce hydrazide (3: ) which was refluxed with POCl3 to obtain 2-chloromethyl-5pyrazino-1, 3, 4-oxadiazole (4: ). This was then further reacted with 4-amino phenol to obtain 4-[5-pyrazino-1, 3, 4-oxadiazol-2-yl-methoxy]-phenyl amine (5: ) which on condensation with various aromatic aldehydes afforded a series Schiff's bases 6(A-H): . Dehydrative annulations of 6(A-H): in the presence of chloroacetyl chloride and triethylamine yielded 3-chloro-4-aryl-1-[4-(5-pyrazin-2-yl-[1, 3, 4]oxadiazole-2-ylmethoxy)-phenyl]-azetidin-2-one derivatives 7(A-H): . Antibacterial, antifungal and antitubercular potential of all the synthesized compounds were assessed. Docking study was performed using the software VLife Engine tools of Vlifemds 4.6 on the protein lumazine synthase of M. tuberculosis (PDB entry code 2C92). RESULTS: The present studies demonstrated that synthesized oxadiazole derivatives have good antimicrobial activity against the various microorganisms. Among the synthesized derivative, 7B: and 7G: were found to be prominent compounds which have potential antibacterial, antifungal and antitubercular activity (with MIC 3.12 µg/ml and high dock score ranging from -59.0 to -54.0) against Mycobacterium tuberculosis. CONCLUSIONS: Derivatives 7B: and 7G: would be effective lead candidates for tuberculosis therapy.

Evaluation of Biological Activity of Derivatives of 1,3,4-Thiadiazole, 1,3,4-Oxadiazole and 1,2,4-Triazole.

OBJECTIVE: 1,3,4-thiadiazole (A), 1,3,4-oxadiazole (B) and 1,2,4-triazole (C) derivatives have been known for their immense pharmacotherapeutic potential. The current research article attempts to further explore and understand the probable biochemical mechanism related to antiinflammatory activity of derivatives. METHODS: The screened A, B and C derivatives were investigated for both in-vitro (Erythrocyte Membrane stabilization activity, Proteinase enzyme inhibitory activities) and in-vivo correlation using acute and chronic anti-inflammatory potential by carrageenan induced rats paw edema and cotton pellet granuloma methods, respectively. The activity was studied after interpreting acute toxicity studies results. RESULTS: In vitro studies in the case of Erythrocyte Membrane stability and Proteinase enzyme inhibitory activities exhibited by A, B, and C at 100 ppm were found to be 48.89%, 51.08% and 50.08% and 66.78%, 76.91% and 57.41%, respectively. The maximum toxic dose was found to be 2000 mg/kg. The derivatives were studied for two-dose levels viz; Lower (100 mg/kg) and higher dose (200 mg/kg). In rat paw edema, maximum decrease was obtained for A (50.05%), B (50.05%) and C (51.06%) at lower and higher dose at 68.76%, 55.61%, and 65.26%, respectively for effect up to 24 h. In the chronic model of cotton pellet granuloma viz; higher and lower doses of A, B and C exhibited 38.15%, 33.19% and 30.25 % and 19.45%, 18.55% and 17.55 %, respectively. CONCLUSION: The studied models depicted that derivatives A, B and C have the probable potential as anti-inflammatory agents. Further studies need to be undertaken to explore their potential in the different therapeutic areas.

Furoxan derivatives demonstrated in vivo efficacy by reducing Mycobacterium tuberculosis to undetectable levels in a mouse model of infection.

OBJECTIVES: The most recent survey conducted by the World Health Organization described Tuberculosis (TB) as one of the top 10 causes of death and the leading cause of death from a single infectious agent. The increasing number of TB-resistant cases has contributed to this scenario. In light of this, new strategies to control and treat the disease are necessary. Our research group has previously described furoxan derivatives as promising scaffolds to be explored as new antitubercular drugs. RESULTS: Two of these furoxan derivatives, (14b) and (14c), demonstrated a high selectivity against Mycobacterium tuberculosis. The compounds (14b) and (14c) were also active against a latent M. tuberculosis strain, with MIC90 values of 6.67 µM and 9.84 µM, respectively; they were also active against monoresistant strains (MIC90 values ranging from 0.61 to 20.42 µM) and clinical MDR strains (MIC90 values ranging from 3.09 to 42.95 µM). Time-kill experiments with compound (14c) showed early bactericidal effects that were superior to those of the first- and second-line anti-tuberculosis drugs currently used in therapy. The safety of compounds (14b) and (14c) was demonstrated by the Ames test because these molecules were not mutagenic under the tested conditions. Finally, we confirmed the safety, and high efficacy of compounds (14b) and (14c), which reduced M. tuberculosis to undetectable levels in a mouse aerosol model of infection. CONCLUSION: Altogether, we have identified two advanced lead compounds, (14b) and (14c), as novel promising candidates for the treatment of TB infection.

Uncovering the Pharmacological Mechanism of Stemazole in the Treatment of Neurodegenerative Diseases Based on a Network Pharmacology Approach.

Stemazole exerts potent pharmacological effects against neurodegenerative diseases and protective effects in stem cells. However, on the basis of the current understanding, the molecular mechanisms underlying the effects of stemazole in the treatment of Alzheimer's disease and Parkinson's disease have not been fully elucidated. In this study, a network pharmacology-based strategy integrating target prediction, network construction, gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses, and molecular docking was adopted to predict the targets of stemazole relevant to the treatment of neurodegenerative diseases and to further explore the involved pharmacological mechanisms. The majority of the predicted targets were highly involved in the mitogen-activated protein kinase (MAPK) signaling pathway. RAC-alpha serine/threonine-protein kinase (AKT1), caspase-3 (CASP3), caspase-8 (CASP8), mitogen-activated protein kinase 8 (MAPK8), and mitogen-activated protein kinase 14 (MAPK14) are the core targets regulated by stemazole and play a central role in its anti-apoptosis effects. This work provides a scientific basis for further elucidating the mechanism underlying the effects of stemazole in the treatment of neurodegenerative diseases.

Design, Synthesis, and Efficacy Testing of Nitroethylene- and 7-Nitrobenzoxadiazol-Based Flavodoxin Inhibitors against Helicobacter pylori Drug-Resistant Clinical Strains and in Helicobacter pylori-Infected Mice.

Helicobacter pylori (Hp) infection is the main cause of peptic ulcer and gastric cancer. Hp eradication rates have fallen due to increasing bacterial resistance to currently used broad-spectrum antimicrobials. We have designed, synthesized, and tested redox variants of nitroethylene- and 7-nitrobenzoxadiazole-based inhibitors of the essential Hp protein flavodoxin. Derivatives of the 7-nitrobenzoxadiazole lead, carrying reduced forms of the nitro group and/or oxidized forms of a sulfur atom, display high therapeutic indexes against several reference Hp strains. These inhibitors are effective against metronidazole-, clarithromycin-, and rifampicin-resistant Hp clinical isolates. Their toxicity for mice after oral administration is low, and, when administered individually at single daily doses for 8 days in a mice model of Hp infection, they decrease significantly Hp gastric colonization rates and are able to eradicate the infection in up to 60% of the mice. These flavodoxin inhibitors constitute a novel family of Hp-specific antimicrobials that may help fight the constant increase of Hp antimicrobial-resistant strains.

1,2,4-Oxadiazole/2-Imidazoline Hybrids: Multi-target-directed Compounds for the Treatment of Infectious Diseases and Cancer.

Replacement of amide moiety with the 1,2,4-oxadiazole core in the scaffold of recently reported efflux pump inhibitors afforded a novel series of oxadiazole/2-imidazoline hybrids. The latter compounds exhibited promising antibacterial activity on both Gram-positive (Staphylococcus aureus, Bacillus subtilis) and Gram-negative (Escherichia coli, Pseudomonas fluorescens) strains. Furthermore, selected compounds markedly inhibited the growth of certain drug-resistant bacteria. Additionally, the study revealed the antiproliferative activity of several antibacterial frontrunners against pancreas ductal adenocarcinoma (PANC-1) cell line, as well as their type-selective monoamine oxidase (MAO) inhibitory profile.

Sinusoidal protection by sphingosine-1-phosphate receptor 1 agonist in liver ischemia-reperfusion injury.

BACKGROUND: Functional and structural damages in sinusoidal endothelial cells (SECs) have a crucial role during hepatic ischemia-reperfusion injury (IRI). In regulating endothelial function, sphingosine-1-phosphate receptor 1 (S1PR1), which is a G protein-coupled receptor, has an important role. The present study aimed to clarify whether SEW2871, a selective S1PR1 agonist, can attenuate hepatic damage caused by hepatic IRI, focusing on SEC functions. METHODS: In vivo, using a 60-min partial-warm IRI model, mice were treated with SEW2871 or without it (with vehicle). In vitro, isolated SECs pretreated with SEW2871 or without it (with vehicle) were incubated with hydrogen peroxide. RESULTS: Compared with the IRI + vehicle group, SEW2871 administration significantly improved serum transaminase levels and liver damage, attenuated infiltration of Ly-6G and mouse macrophage antigen-1-positive cells, suppressed the expression of vascular cell adhesion molecule-1 and proinflammatory cytokines in the liver, and enhanced the expressions of endothelial nitric oxide synthase (eNOS) and vascular endothelial (VE) cadherin in the liver (eNOS/ß-actin [median]: 0.24 versus 0.53, P = 0.008; VE-cadherin/ß-actin [median]: 0.21 versus 0.94, P = 0.008). In vitro, compared with the vehicle group, pretreatment of SECs with SEW2871 significantly increased the expressions of eNOS and VE-cadherin (eNOS/ß-actin [median]: 0.22 versus 0.29, P = 0.008; VE-cadherin/ß-actin [median]: 0.38 versus 0.67, P = 0.008). As results of investigation of prosurvival signals, SEW2871 significantly increased Akt phosphorylation in SECs and decreased lactate dehydrogenase levels in supernatants of SECs. CONCLUSIONS: These results indicate that S1PR1 agonist induces attenuation of hepatic IRI, which might be provided by preventing SEC damage. S1PR1 may be a therapeutic target for the prevention of early sinusoidal injury after hepatic IRI.

Discovery of Novel and Potent Stearoyl Coenzyme A Desaturase 1 (SCD1) Inhibitors as Anticancer Agents.

A lead compound A was identified previously as an stearoyl coenzyme A desaturase (SCD) inhibitor during research on potential treatments for obesity. This compound showed high SCD1 binding affinity, but a poor pharmacokinetic (PK) profile and limited chemical accessibility, making it suboptimal for use in anticancer research. To identify potent SCD1 inhibitors with more promising PK profiles, we newly designed a series of 'non-spiro' 4, 4-disubstituted piperidine derivatives based on molecular modeling studies. As a result, we discovered compound 1a, which retained moderate SCD1 binding affinity. Optimization around 1a was accelerated by analyzing Hansch-Fujita and Hammett constants to obtain 4-phenyl-4-(trifluoromethyl)piperidine derivative 1n. Fine-tuning of the azole moiety of 1n led to compound 1o (T-3764518), which retained nanomolar affinity and exhibited an excellent PK profile. Reflecting the good potency and PK profile, orally administrated compound 1o showed significant pharmacodynamic (PD) marker reduction (at 0.3mg/kg, bid) in HCT116 mouse xenograft model and tumor growth suppression (at 1mg/kg, bid) in 786-O mouse xenograft model. In conclusion, we identified a new series of SCD1 inhibitors, represented by compound 1o, which represents a promising new chemical tool suitable for the study of SCD1 biology as well as the potential development of novel anticancer therapies.

Virtual screening and biological evaluation of novel antipyretic compounds.

Due to the absence of safety of the antipyretics to patients with cardiovascular dysfunction, new targets to treat inflammation have been pursued. mPGES-1 is a promising target because its inhibition would not cause the side-effects related to COX inhibition. To identify novel inhibitors of mPGES-1, we developed a ligand-based pharmacophore model that differentiates true inhibitors from decoys and enlightens the structure-activity relationships for known mPGES-1 inhibitors. The model (four hydrophobic centers, two hydrogen bond acceptor and two hydrogen bond donor points) was employed to select lead-like compounds from ZINC database for in vivo evaluation. Among the 18 compounds selected, five inhibited the fever induced by LPS. The most potent compound (5-(4-fluorophenyl)-3-({6-methylimidazo[1,2-a]pyridin-2-yl}methyl)-2,3dihydro-1,3,4-oxadiazol-2-one) is active peripherally (i.v.) or centrally (i.c.v.) (82.18% and 112% reduction, respectively) and reduces (69.13%) hypothalamic PGE2 production, without significant COX-1/2 inhibition. In conclusion, our in silico approach leads to the selection of a compound that presents the chemical features to inhibit mPGES-1 and reduces fever induced by LPS. Furthermore, the in vivo and in vitro results support the hypothesis that its mechanism of action does not depend on COX inhibition. Hence, it can be considered a promising lead compound for antipyretic development, once it would not have the side-effects of COX-1/2 inhibitors.

The oxadiazole antibacterials.

The oxadiazoles are a class of antibacterials discovered by in silico docking and scoring of compounds against the X-ray structure of a penicillin-binding protein. These antibacterials exhibit activity against Gram-positive bacteria, including against methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus (VRE). They show in vivo efficacy in murine models of peritonitis/sepsis and neutropenic thigh MRSA infection. They are bactericidal and orally bioavailable. The oxadiazoles show promise in treatment of MRSA infection.

The CRF1 Antagonist Verucerfont in Anxious Alcohol-Dependent Women: Translation of Neuroendocrine, But not of Anti-Craving Effects.

Blockade of corticotropin-releasing factor receptor 1 (CRF1) suppresses stress-induced alcohol seeking in rodents, but clinical translation remains. Here, we first showed that the CRF1 antagonist verucerfont potently blocks hypothalamic-pituitary adrenal (HPA) axis activation in adrenalectomized rats. We then evaluated verucerfont for its ability to block HPA axis activation and reduce stress-induced alcohol craving in alcohol-dependent patients. Anxious, alcohol-dependent women (age 21-65 years, n=39) were admitted to the NIH Clinical Center and completed withdrawal treatment before enrollment if needed. One-week single-blind placebo was followed by randomized double-blind verucerfont (350 mg per day) or placebo for 3 weeks. Verucerfont effects on the HPA axis were evaluated using the dexamethasone-CRF test. Craving was evaluated using two established protocols, one that combines a social stressor with physical alcohol cue exposure, and one that uses guided imagery to present personalized stress, alcohol, or neutral stimuli. An fMRI session examined brain responses to negative affective stimuli and alcohol cues. In contrast to our recent observations with another CRF1 antagonist, pexacerfont, verucerfont potently blocked the HPA axis response to the dexamethasone-CRF test, but left alcohol craving unaffected. Right amygdala responses to negative affective stimuli were significantly attenuated by verucerfont, but responses to alcohol-associated stimuli were increased in some brain regions, including left insula. Discontinuation rates were significantly higher in the verucerfont group. Our findings provide the first translational evidence that CRF1 antagonists with slow receptor dissociation kinetics may have increased efficacy to dampen HPA axis responses. The findings do not support a clinical efficacy of CRF1 blockade in stress-induced alcohol craving and relapse.

18F-FAZA PET imaging response tracks the reoxygenation of tumors in mice upon treatment with the mitochondrial complex I inhibitor BAY 87-2243.

PURPOSE: We describe a noninvasive PET imaging method that monitors early therapeutic efficacy of BAY 87-2243, a novel small-molecule inhibitor of mitochondrial complex I as a function of hypoxia-inducible factor-1α (HIF1α) activity. EXPERIMENTAL DESIGN: Four PET tracers [(18)F-FDG, (18)F-Fpp(RGD)2, (18)F-FLT, and (18)F-FAZA] were assessed for uptake into tumor xenografts of drug-responsive (H460, PC3) or drug-resistant (786-0) carcinoma cells. Mice were treated with BAY 87-2243 or vehicle. At each point, RNA from treated and vehicle H460 tumor xenografts (n = 3 each) was isolated and analyzed for target genes. RESULTS: Significant changes in uptake of (18)F-FAZA, (18)F-FLT, and (18)F-Fpp(RGD)2 (P < 0.01) occurred with BAY 87-2243 treatment with (18)F-FAZA being the most prominent. (18)F-FDG uptake was unaffected. (18)F-FAZA tumor uptake declined by 55% to 70% (1.21% ± 0.10%ID/g to 0.35 ± 0.1%ID/g; n = 6, vehicle vs. treatment) in both H460 (P < 0.001) and PC3 (P < 0.05) xenografts 1 to 3 days after drug administration. (18)F-FAZA uptake in 786-0 xenografts was unaffected. Decline occurred before significant differences in tumor volume, thus suggesting (18)F-FAZA decrease reflected early changes in tumor metabolism. BAY 87-2243 reduced expression of hypoxia-regulated genes CA IX, ANGPTL4, and EGLN-3 by 99%, 93%, and 83%, respectively (P < 0.001 for all), which corresponds with reduced (18)F-FAZA uptake upon drug treatment. Heterogeneous expression of genes associated with glucose metabolism, vessel density, and proliferation was observed. CONCLUSIONS: Our studies suggest suitability of (18)F-FAZA-PET as an early pharmacodynamic monitor on the efficacy of anticancer agents that target the mitochondrial complex I and intratumor oxygen levels (e.g., BAY 87-2243).

GSK356278, a potent, selective, brain-penetrant phosphodiesterase 4 inhibitor that demonstrates anxiolytic and cognition-enhancing effects without inducing side effects in preclinical species.

Small molecule phosphodiesterase (PDE) 4 inhibitors have long been known to show therapeutic benefit in various preclinical models of psychiatric and neurologic diseases because of their ability to elevate cAMP in various cell types of the central nervous system. Despite the registration of the first PDE4 inhibitor, roflumilast, for the treatment of chronic obstructive pulmonary disease, the therapeutic potential of PDE4 inhibitors in neurologic diseases has never been fulfilled in the clinic due to severe dose-limiting side effects such as nausea and vomiting. In this study, we describe the detailed pharmacological characterization of GSK356278 [5-(5-((2,4-dimethylthiazol-5-yl)methyl)-1,3,4-oxadiazol-2-yl)-1-ethyl-N-(tetrahydro-2H-pyran-4-yl)-1H-pyrazolo[3,4-b]pyridin-4-amine], a potent, selective, and brain-penetrant PDE4 inhibitor that shows a superior therapeutic index to both rolipram and roflumilast in various preclinical species and has potential for further development in the clinic for the treatment of psychiatric and neurologic diseases. GSK356278 inhibited PDE4B enzyme activity with a pIC50 of 8.8 and bound to the high-affinity rolipram binding site with a pIC50 of 8.6. In preclinical models, the therapeutic index as defined in a rodent lung inflammation model versus rat pica feeding was >150 compared with 0.5 and 6.4 for rolipram and roflumilast, respectively. In a model of anxiety in common marmosets, the therapeutic index for GSK356278 was >10 versus <1 for rolipram. We also demonstrate that GSK356278 enhances performance in a model of executive function in cynomolgus macaques with no adverse effects, a therapeutic profile that supports further evaluation of GSK356278 in a clinical setting.

A novel p38 MAPK docking-groove-targeted compound is a potent inhibitor of inflammatory hyperalgesia.

The MAPK (mitogen-activated protein kinase) p38 is an important mediator of inflammation and of inflammatory and neuropathic pain. We have described recently that docking-groove-dependent interactions are important for p38 MAPK-mediated signal transduction. Thus virtual screening was performed to identify putative docking-groove-targeted p38 MAPK inhibitors. Several compounds of the benzo-oxadiazol family were identified with low micromolar inhibitory activity both in a p38 MAPK activity assay, and in THP-1 human monocytes acting as inhibitors of LPS (lipopolysaccharide)-induced TNFα (tumour necrosis factor α) secretion. Positions 2 and 5 in the phenyl ring are essential for the described inhibitory activity with a chloride in position 5 and a methyl group in position 2 yielding the best results, giving an IC50 value of 1.8 µM (FGA-19 compound). Notably, FGA-19 exerted a potent and long-lasting analgesic effect in vivo when tested in a mouse model of inflammatory hyperalgesia. A single intrathecal injection of FGA-19 completely resolved hyperalgesia, being 10-fold as potent and displaying longer lasting effects than the established p38 MAPK inhibitor SB239063. FGA-19 also reversed persistent pain in a model of post-inflammatory hyperalgesia in LysM (lysozyme M)-GRK2 (G-protein-coupled-receptor kinase)(+/-) mice. These potent in vivo effects suggested p38 MAPK docking-site-targeted inhibitors as a potential novel strategy for the treatment of inflammatory pain.

High-throughput screening for GPR119 modulators identifies a novel compound with anti-diabetic efficacy in db/db mice.

G protein-coupled receptor 119 (GPR119) is highly expressed in pancreatic ß cells and enteroendocrine cells. It is involved in glucose-stimulated insulin secretion and glucagon-like peptide-1 (GLP-1) release, thereby representing a promising target for the treatment of type 2 diabetes. Although a number of GPR119 agonists were developed, no positive allosteric modulator (PAM) to this receptor has been reported. Here we describe a high-throughput assay for screening GPR119 PAMs and agonists simultaneously. Following screening of a small molecule compound library containing 312,000 synthetic and natural product-derived samples, one potent GPR119 agonist with novel chemical structure, MW1219, was identified. Exposure of MIN6 and GLUTag cells to MW1219 enhanced glucose-stimulated insulin secretion and GLP-1 release; once-daily oral dosing of MW1219 for 6 weeks in diabetic db/db mice reduced hemoglobin A1c (HbA1c) and improved plasma glucose, insulin and GLP-1 levels; it also increased glucose tolerance. The results demonstrate that MW1219 is capable of effectively controlling blood glucose level and may have the potential to be developed as a new class of anti-diabetic agents.

In vitro read-through of phenylalanine hydroxylase (PAH) nonsense mutations using aminoglycosides: a potential therapy for phenylketonuria.

Phenylketonuria (PKU, OMIM 261600) is an autosomal recessive inborn error of phenylalanine metabolism, predominantly caused by mutations in the phenylalanine hydroxylase (PAH) gene. Approximately 10% of patients carry a nonsense mutation, which results in an inactive or unstable truncated protein. In some genetic disorders, including cystic fibrosis and Duchenne muscular dystrophy, restoration of full-length protein has been achieved by aminoglycoside antibiotics, such as gentamicin and G-418 (Geneticin). More recently, nonsense read-through has been induced at greater rates using a non-aminoglycoside drug, PTC124 (Ataluren), which has the advantage of being non-toxic in contrast to the antibiotics. The efficacy of read-through induced by three compounds, aminoglycosides G418 and gentamicin, and PTC124 were evaluated for four nonsense mutations of PAH in an in vitro expression system in two mammalian cell lines (COS-7 and HEK293). The production of full-length PAH was investigated using western blotting and the functionality confirmed by enzyme activity. Gentamicin and G-418 induced read-through of nonsense PAH mutations in HEK293 cells. The read-through product partially restored enzymatic activity, which was significantly less than that of wild-type, but comparable to a missense mutation of PAH associated with less severe forms of PKU. Treatment with PTC124 up to 100 µM did not result in full-length PAH polypeptide. Nonsense read-through drugs are a potential form of treatment for PKU, although the high dosage of aminoglycosides used is not appropriate in a clinical setting. In vitro studies with new non-toxic read-through agents as well as in vivo studies would also be essential to determine the extent of read-through required to restore normal phenylalanine levels.

Design, synthesis, and pharmacological evaluation of novel hybrid compounds to treat sickle cell disease symptoms. part II: furoxan derivatives.

Phthalimide derivatives containing furoxanyl subunits as nitric oxide (NO)-donors (3a-g) were designed, synthesized, and evaluated in vitro and in vivo for their potential uses in the oral treatment of sickle cell disease symptoms. All compounds (3a-g) demonstrated NO-donor properties at different levels. Moreover, compounds 3b and 3c demonstrated analgesic activity. Compound 3b was determined to be a promising drug candidate for the aforementioned uses, and it was further evaluated in K562 culture cells to determine its ability to increase levels of γ-globin expression. After 96 h at 5 µM, compound 3b was able to induce γ-globin expression by nearly three times. Mutagenic studies using micronucleus tests in peripheral blood cells of mice demonstrated that compound 3b reduces the mutagenic profile as compared with hydroxyurea. Compound 3b has emerged as a new leading drug candidate with multiple beneficial effects for the treatment of sickle cell disease symptoms and provides an alternative to hydroxyurea treatment.