Design and Synthesis of Novel Spiro Derivatives as Potent and Reversible Monoacylglycerol Lipase (MAGL) Inhibitors: Bioisosteric Transformation from 3-Oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl Moiety.

The therapeutic potential of monoacylglycerol lipase (MAGL) inhibitors in central nervous system-related diseases has attracted attention worldwide. However, the availability of reversible-type inhibitor is still limited to clarify the pharmacological effect. Herein, we report the discovery of novel spiro chemical series as potent and reversible MAGL inhibitors with a different binding mode to MAGL using Arg57 and His121. Starting from hit compound 1 and its co-crystal structure with MAGL, structure-based drug discovery (SBDD) approach enabled us to generate various spiro scaffolds like 2a (azetidine-lactam), 2b (cyclobutane-lactam), and 2d (cyclobutane-carbamate) as novel bioisosteres of 3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl moiety in 1 with higher lipophilic ligand efficiency (LLE). Optimization of the left hand side afforded 4f as a promising reversible MAGL inhibitor, which showed potent in vitro MAGL inhibitory activity (IC50 6.2 nM), good oral absorption, blood-brain barrier penetration, and significant pharmacodynamic changes (2-arachidonoylglycerol increase and arachidonic acid decrease) at 0.3-10 mg/kg, po. in mice.

Design, Synthesis, and Evaluation of (4 R)-1-{3-[2-(18F)Fluoro-4-methylpyridin-3-yl]phenyl}-4-[4-(1,3-thiazol-2-ylcarbonyl)piperazin-1-yl]pyrrolidin-2-one ([18F]T-401) as a Novel Positron-Emission Tomography Imaging Agent for Monoacylglycerol Lipase.

Monoacylglycerol lipase (MAGL) is a cytosolic serine hydrolase involved in endocannabinoid and inflammatory signaling. Positron-emission tomography (PET) imaging of MAGL serves to validate target engagement of therapeutic MAGL inhibitors as well as to investigate MAGL levels under normal and disease conditions. However, PET radioligands with reversible binding kinetics for MAGL, which allow quantitative assessment of MAGL, are hitherto unavailable. In this study, we designed and synthesized fluoro-containing PET probes starting from a recently identified piperazinyl pyrrolidine-2-one derivative with reversible binding to MAGL. By tailoring the lipophilicity of the molecule to optimize nonspecific binding and blood-brain barrier permeability, we successfully identified two compounds that show high uptake to regions enriched with MAGL. PET imaging of wild-type and MAGL-deficient mice as well as a macaque monkey indicated that [18F]5 ((4 R)-1-{3-[2-(18F)fluoro-4-methylpyridin-3-yl]phenyl}-4-[4-(1,3-thiazol-2-ylcarbonyl)piperazin-1-yl]pyrrolidin-2-one, [18F]T-401) specifically binds to MAGL with adequate reversibility, yielding a high contrast for MAGL within an appropriate imaging time.

VaDE: a manually curated database of reproducible associations between various traits and human genomic polymorphisms.

Genome-wide association studies (GWASs) have identified numerous single nucleotide polymorphisms (SNPs) associated with the development of common diseases. However, it is clear that genetic risk factors of common diseases are heterogeneous among human populations. Therefore, we developed a database of genomic polymorphisms that are reproducibly associated with disease susceptibilities, drug responses and other traits for each human population: 'VarySysDB Disease Edition' (VaDE; http://bmi-tokai.jp/VaDE/). SNP-trait association data were obtained from the National Human Genome Research Institute GWAS (NHGRI GWAS) catalog and RAvariome, and we added detailed information of sample populations by curating original papers. In addition, we collected and curated original papers, and registered the detailed information of SNP-trait associations in VaDE. Then, we evaluated reproducibility of associations in each population by counting the number of significantly associated studies. VaDE provides literature-based SNP-trait association data and functional genomic region annotation for SNP functional research. SNP functional annotation data included experimental data of the ENCODE project, H-InvDB transcripts and the 1000 Genome Project. A user-friendly web interface was developed to assist quick search, easy download and fast swapping among viewers. We believe that our database will contribute to the future establishment of personalized medicine and increase our understanding of genetic factors underlying diseases.

First synthesis of saponarin, 6-C- and 7-O-di-ß-D-glucosylapigenin.

Saponarin, apigenin 6-C- and 7-O-bis-ß-D-glucoside, was synthesized in an overall yield of 37% via 11 steps, which included the C-glycosylation of 2,4-O-dibenzylphloroacetophenone, the introduction of a cinnamoyl residue by aldol condensation, the formation of a flavone by regioselective deprotection, and oxidative ring-closure to the final regioselective deprotection and stereoselective O-glycosylation.

Development of spontaneous neuropathy in NF-κBp50-deficient mice by calcineurin-signal involving impaired NF-κB activation.

PURPOSE: The transcriptional regulator, nuclear factor-kappa B (NF-κB)/Rel family are involved in neuronal cell death and survival. Previously, we reported that NF-κBp50-deficient (p50-deficient) mice exhibit many features resembling human normal tension glaucoma (NTG). The developmental mechanism of human NTG is not clearly understood, and a radical curative treatment has yet to be established. Our aim is to elucidate the signal cascade which mediates the spontaneous optic neuropathy in p50-deficient mice as a model of NTG. METHODS: To demonstrate the expression and activation of pro-apoptotic factors, which mediate the death of retinal ganglion cells (RGCs) in p50-deficient mice, western blot (WB) and luciferase reporter assays with retinas from p50-deficient and wild type mice, and cultured RGC-5 cells were performed. Furthermore, we tested the neuroprotective effects of chemical reagents (memantine, lomerizine, and tacrolimus) against N-methyl-D-aspartate (NMDA)-susceptible RGC damage according to in vitro experiments with RGC-5 cells. To elucidate the NF-κB-mediated death signaling, the effects of chemical reagents on spontaneous optic neuropathy were examined by histopathological studies. RESULTS: WB experiments and luciferase reporter assays showed that NF-κB-inducible BCL2-associated X protein (Bax) and a pro-apoptotic factor, activated caspase 3 were expressed in the retina of p50-deficient mice as well as NMDA-treated RGC-5 cells. Further, the constitutively active cleaved forms of calcineurin (CaN), which have been reported to lead to apoptosis, were detected in the retina of p50-deficient mice as well as NMDA-treated RGC-5 cells. Pre-treatment with tacrolimus markedly protected RGC-5 cells from NMDA-induced neurotoxicity, and then both spontaneous RGC death and degenerative changes to the optic nerve in p50-deficient mice were significantly reduced by the chronic administration of tacrolimus. The experiments with cultured RGC-5 cells supported the results of histological examinations with p50-deficient mice, suggesting that CaN activation leads to NF-κB-induced Bax activation and caspase 3 activation, and mediates spontaneous optic neuropathy in p50-deficient mice. CONCLUSIONS: Research findings show that the chronic administration of tacrolimus significantly reduces spontaneous optic neuropathy in p50-deficient mice. We demonstrated a potential CaN signal cascade, which spontaneously induces age-dependent RGC death and degenerative optic nerve changes in p50-deficient mice.

Inflammasome activation of cardiac fibroblasts is essential for myocardial ischemia/reperfusion injury.

Background- Inflammation plays a key role in the pathophysiology of myocardial ischemia/reperfusion (I/R) injury; however, the mechanism by which myocardial I/R induces inflammation remains unclear. Recent evidence indicates that a sterile inflammatory response triggered by tissue damage is mediated through a multiple-protein complex called the inflammasome. Therefore, we hypothesized that the inflammasome is an initial sensor for danger signal(s) in myocardial I/R injury. Methods and Results- We demonstrate that inflammasome activation in cardiac fibroblasts, but not in cardiomyocytes, is crucially involved in the initial inflammatory response after myocardial I/R injury. We found that inflammasomes are formed by I/R and that its subsequent activation of inflammasomes leads to interleukin-1ß production, resulting in inflammatory responses such as inflammatory cell infiltration and cytokine expression in the heart. In mice deficient for apoptosis-associated speck-like adaptor protein and caspase-1, these inflammatory responses and subsequent injuries, including infarct development and myocardial fibrosis and dysfunction, were markedly diminished. Bone marrow transplantation experiments with apoptosis-associated speck-like adaptor protein-deficient mice revealed that inflammasome activation in bone marrow cells and myocardial resident cells such as cardiomyocytes or cardiac fibroblasts plays an important role in myocardial I/R injury. In vitro experiments revealed that hypoxia/reoxygenation stimulated inflammasome activation in cardiac fibroblasts, but not in cardiomyocytes, and that hypoxia/reoxygenation-induced activation was mediated through reactive oxygen species production and potassium efflux. Conclusions- Our results demonstrate the molecular basis for the initial inflammatory response after I/R and suggest that the inflammasome is a potential novel therapeutic target for preventing myocardial I/R injury.

An investigation of the risk factors for infection with methicillin-resistant Staphylococcus aureus among patients in a neonatal intensive care unit.

BACKGROUND: The aims of this study were to investigate the risk factors of methicillin-resistant Staphylococcus aureus (MRSA) infection among infants to establish effective infection control measures for neonatal intensive care unit (NICU). METHODS: Data were prospectively collected from 961 infants hospitalized in a teaching hospital in Japan, from July 2002 through December 2005. RESULTS: Among all infants, 28 (2.9%) developed MRSA infections. Multivariate logistic regression analyses demonstrated the risk factors for developing MRSA infections to include a low birth weight (odds ratio [OR], 0.91; 95% confidence interval [CI]: 0.93-0.99), the presence of eye mucous (OR, 6.78; 95% CI: 2.87-16.01), the practice of kangaroo mother care (OR, 3.82; 95% CI: 1.11-13.13), and the MRSA colonization rate (OR, 11.12; 95% CI: 1.32-93.89). CONCLUSION: The risk factors for developing a MRSA infection among infants in NICU were a low birth weight, the presence of eye mucous, the practice of kangaroo mother care, and a high MRSA colonization rate. Therefore, extra attention should be given to infants in high-risk groups demonstrating a low birth weight and the presence of eye mucous and who have undergone kangaroo mother care. As a result, the cohort isolation of infants with MRSA may therefore be an effective strategy to prevent MRSA infections.

Critical role of bone marrow apoptosis-associated speck-like protein, an inflammasome adaptor molecule, in neointimal formation after vascular injury in mice.

BACKGROUND: Inflammatory cytokines such as interleukin (IL)-1 beta and IL-18 play an important role in the development of atherosclerosis and restenosis. Apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) is an adaptor protein that regulates caspase-1-dependent IL-1 beta and IL-18 generation; however, the role of ASC in vascular injury remains undefined. Here, we investigated the contribution of ASC to neointimal formation after vascular injury in ASC-deficient (ASC(-/-)) mice. METHODS AND RESULTS: Wire-mediated vascular injury was produced in the femoral artery of ASC(-/-) and wild-type mice. Immunohistochemical analysis revealed that ASC was markedly expressed at the site of vascular injury. Neointimal formation was significantly attenuated in ASC(-/-) mice after injury. IL-1 beta and IL-18 were expressed in the neointimal lesion in wild-type mice but showed decreased expression in the lesion of ASC(-/-) mice. To investigate the contribution of bone marrow-derived cells, we developed bone marrow-transplanted mice and found that neointimal formation was significantly decreased in wild-type mice in which bone marrow was replaced with ASC(-/-) bone marrow cells. Furthermore, in vitro experiments showed that the proliferation activity of ASC(-/-) vascular smooth muscle cells was not impaired. CONCLUSIONS: These findings suggest that bone marrow-derived ASC is critical for neointimal formation after vascular injury and identify ASC as a novel therapeutic target for atherosclerosis and restenosis.

Divergent synthesis of multifunctional molecular probes to elucidate the enzyme specificity of dipeptidic gamma-secretase inhibitors.

Divergent synthesis of multifunctional molecular probes based on caprolactam-derived dipeptidic gamma-secretase inhibitors (GSIs), Compound E (CE) and LY411575 analogue (DBZ), was efficiently accomplished by means of Cu(I)-catalyzed azide/alkyne fusion reaction. Photoaffinity labeling experiments using these derivatives coupled to photoactivatable and biotin moieties provided direct evidence that the molecular targets of CE and DBZ are the N-terminal fragment of presenilin 1 within the gamma-secretase complex. Moreover, these photoprobes directly targeted signal peptide peptidase. These data suggest that the divergent synthesis of molecular probes has been successfully applied to characterize the interaction of GSIs with their molecular targets and define the structural requirements for inhibitor binding to intramembrane-cleaving proteases.

Synthesis of biotinylated photoaffinity probes based on arylsulfonamide gamma-secretase inhibitors.

Synthesis and biological evaluation of an arylsulfonamide class of gamma-secretase inhibitors are described. Design, synthesis, and biological evaluation of multifunctional molecular probes harboring a benzophenone photophore as a cross-linking group and a biotin tag are also reported.

Novel gamma-secretase inhibitors discovered by library screening of in-house synthetic natural product intermediates.

Screening of our in-house compound library comprised of intermediates of natural product synthesis projects resulted in discovering two novel gamma-secretase inhibitors, which coincidently had similar moieties, that is, cyclohexenone and two aryl groups arranged on the core six-membered ring. Structure-activity relationship studies of these compounds were also developed.

Efficiency of hand drying for removing bacteria from washed hands: comparison of paper towel drying with warm air drying.

OBJECTIVE: To evaluate warm air and paper towel drying for removing bacteria from washed hands. METHODS: After hands were washed with non-antibacterial soap, they were dried using warm air with and without ultraviolet light, while being rubbed or held stationary, or paper towels. Each method was performed as a randomized trial using 30 hands. RESULTS: Log colony-forming units (CFU) on palms and fingers increased significantly when hands were dried with warm air while being rubbed for 15 seconds (P < .001), and many bacteria remained at 30 seconds without ultraviolet light (P < .001) Holding hands stationary while drying significantly decreased log CFU on palms, fingers, and fingertips (P < .01 or < .001). Few CFU were detected on palms and fingers dried with ultraviolet light. Although log CFU of palms and fingers did not decrease after drying with three sheets of paper towel, those of fingertips decreased significantly (P < .001). For palms and fingers, log reductions were greater with warm air drying while holding hands stationary, paper towels, and warm air drying while rubbing hands. For fingertips, the log reduction was often greater with paper towels than with warm air. CONCLUSIONS: Holding hands stationary and not rubbing them was desirable for removing bacteria. Ultraviolet light reinforced the removal of bacteria during warm air drying. Paper towels were useful for removing bacteria from fingertips but not palms and fingers.

[Short and long-term hypotensive effect of timolol-gel and latanoprost instillation in normal-tension glaucoma].

PURPOSE: To confirm the predictive value of the results of a 4-week trial of latanoprost alone, timolol-gel alone, or a combination of the two in normal-tension glaucoma (NTG) patients, when compared with the hypotensive response after a 6-month trial using the same combination of eye drops for the same patients. METHODS: One eye each of 45 NTG patients was used in a prospective 4-week trial of latanoprost alone, timolol-gel alone, or a combination of the two. Patients continued using the eye drops for 6 months, according to the results of the trial. The correlation of the results of the 6-moth use and the baseline data, and the baseline data and the results of the 4-week trial were evaluated by the paired-t test. RESULTS: The intraocular pressure(IOP)s of patients using timolol-gel alone were 13.9 mmHg at the base line, 9.7 mmHg after the trial, and 12.0 mmHg after 6-month use(baseline, trial: p < 0.05). IOPs when using latanoprost alone were 15.3 mmHg at baseline, 11.7 mmHg after the trial, and 11.5 mmHg after 6-month use(baseline: p < 0.05, trial: p = 0.33). IOPs using timolol-gel and latanoprost in combination were 14.8 mmHg at baseline, 11.4 mmHg after the trial, and 12.0 mmHg after 6-month use (baseline: p < 0:05, trial: p = 0.14). CONCLUSION: The result of the 4-weeks trial of latanoprost alone or in combination with timolol-gel can be indicative of the IOP after 6-month use.

Sulindac sulfide is a noncompetitive gamma-secretase inhibitor that preferentially reduces Abeta 42 generation.

Nonsteroidal anti-inflammatory drugs (NSAIDs) have been known to reduce risk for Alzheimer's disease. In addition to the anti-inflammatory effects of NSAIDs to block cylooxygenase, it has been shown recently that a subset of NSAIDs selectively inhibits the secretion of highly amyloidogenic Abeta42 from cultured cells, although the molecular target(s) of NSAIDs in reducing the activity of gamma-secretase for Abeta42 generation (gamma(42)-secretase) still remain unknown. Here we show that sulindac sulfide (SSide) directly acts on gamma-secretase and preferentially inhibits the gamma(42)-secretase activity derived from the 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate-solubilized membrane fractions of HeLa cells, in an in vitro gamma-secretase assay using recombinant amyloid beta precursor protein C100 as a substrate. SSide also inhibits activities for the generation of Abeta40 as well as for Notch intracellular domain at higher concentrations. Notably, SSide displayed linear noncompetitive inhibition profiles for gamma(42)-secretase in vitro. Our data suggest that SSide is a direct inhibitor of gamma-secretase that preferentially affects the gamma(42)-secretase activity.

The role of presenilin cofactors in the gamma-secretase complex.

Mutations in presenilin genes account for the majority of the cases of the familial form of Alzheimer's disease (FAD). Presenilin is essential for gamma-secretase activity, a proteolytic activity involved in intramembrane cleavage of Notch and beta-amyloid precursor protein (betaAPP). Cleavage of betaAPP by FAD mutant presenilin results in the overproduction of highly amyloidogenic amyloid beta42 peptides. gamma-Secretase activity requires the formation of a stable, high-molecular-mass protein complex that, in addition to the endoproteolysed fragmented form of presenilin, contains essential cofactors including nicastrin, APH-1 (refs 15-18) and PEN-2 (refs 16, 19). However, the role of each protein in complex formation and the generation of enzymatic activity is unclear. Here we show that Drosophila APH-1 (Aph-1) increases the stability of Drosophila presenilin (Psn) holoprotein in the complex. Depletion of PEN-2 by RNA interference prevents endoproteolysis of presenilin and promotes stabilization of the holoprotein in both Drosophila and mammalian cells, including primary neurons. Co-expression of Drosophila Pen-2 with Aph-1 and nicastrin increases the formation of Psn fragments as well as gamma-secretase activity. Thus, APH-1 stabilizes the presenilin holoprotein in the complex, whereas PEN-2 is required for endoproteolytic processing of presenilin and conferring gamma-secretase activity to the complex.

Sequence-dependent synergistic cytotoxicity of ecteinascidin-743 and paclitaxel in human breast cancer cell lines in vitro and in vivo.

Ecteinascidin 743 (ET-743) is a potent antitumor agent from the Caribbean tunicate Ecteinascidia turbinata and is presently in clinical trials for human cancers. The aim of this study was to assess the nature of the interaction between ET-743 and other antineoplastic agents using the combination index method of Chou and Talalay to better understand how ET-743 might be used clinically. We examined the cytotoxic effect of ET-743 combined with six other antineoplastic agents on human breast cancer cell lines, MX-1, MCF7, and P-glycoprotein overexpressing MCF7/DXR to different schedules. Pretreatment with paclitaxel for 24 h before ET-743 was the most effective combination regimen in all three breast cancer cell lines. Furthermore, sequential treatment with paclitaxel followed by ET-743 increased the antitumor effects in nude mice bearing MX-1 mammary carcinoma xenografts without increasing toxicity. These results suggest that the combination of ET-743 and paclitaxel should be assessed in clinical trials for the treatment of breast cancer.

The mechanism of gamma-secretase activities through high molecular weight complex formation of presenilins is conserved in Drosophila melanogaster and mammals.

Mutations in presenilin 1 (PS1) and PS2 genes contribute to the pathogenesis of early onset familial Alzheimer's disease by increasing secretion of the pathologically relevant Abeta42 polypeptides. PS genes are also implicated in Notch signaling through proteolytic processing of the Notch receptor in Caenorhabditis elegans, Drosophila melanogaster, and mammals. Here we show that Drosophila PS (Psn) protein undergoes endoproteolytic cleavage and forms a stable high molecular weight (HMW) complex in Drosophila S2 or mouse neuro2a (N2a) cells in a similar manner to mammalian PS. The loss-of-function recessive point mutations located in the C-terminal region of Psn, that cause an early pupal-lethal phenotype resembling Notch mutant in vivo, disrupted the HMW complex formation, and abolished gamma-secretase activities in cultured cells. The overexpression of Psn in mouse embryonic fibroblasts lacking PS1 and PS2 genes rescued the Notch processing. Moreover, disruption of the expression of Psn by double-stranded RNA-mediated interference completely abolished the gamma-secretase activity in S2 cells. Surprisingly, gamma-secretase activity dependent on wild-type Psn was associated with a drastic overproduction of Abeta1-42 from human betaAPP in N2a cells, but not in S2 cells. Our data suggest that the mechanism of gamma-secretase activities through formation of HMW PS complex, as well as its abolition by loss-of-function mutations located in the C terminus, are highly conserved features in Drosophila and mammals.