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 (IC(50) 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.
OBJECTIVE: Enhancement of LCAT (lecithin:cholesterol acyltransferase) activity has possibility to be beneficial for atherosclerosis. To evaluate this concept, we characterized our novel, orally administered, small-molecule LCAT activator DS-8190a, which was created from high-throughput screening and subsequent derivatization. We also focused on its mechanism of LCAT activation and the therapeutic activity with improvement of HDL (high-density lipoprotein) functionality. Approach and Results: DS-8190a activated human and cynomolgus monkey but not mouse LCAT enzymes in vitro. DS-8190a was orally administered to cynomolgus monkeys and dose dependently increased LCAT activity (2.0-fold in 3 mg/kg group on day 7), resulting in HDL cholesterol elevation without drastic changes of non-HDL cholesterol. Atheroprotective effects were then evaluated using Ldl-r KOxhLcat Tg mice fed a Western diet for 8 weeks. DS-8190a treatment achieved significant reduction of atherosclerotic lesion area (48.3% reduction in 10 mg/kg treatment group). Furthermore, we conducted reverse cholesterol transport study using Ldl-r KOxhLcat Tg mice intraperitoneally injected with J774A.1 cells loaded with [(3)H]-cholesterol and confirmed significant increases of [(3)H] count in plasma (1.4-fold) and feces (1.4-fold on day 2 and 1.5-fold on day3) in the DS-8190a-treated group. With regard to the molecular mechanism involved, direct binding of DS-8190a to human LCAT protein was confirmed by 2 different approaches: affinity purification by DS-8190a-immobilized beads and thermal shift assay. In addition, the candidate binding site of DS-8190a in human LCAT protein was identified by photoaffinity labeling. CONCLUSIONS: This study demonstrates the potential of DS-8190a as a novel therapeutic for atherosclerosis. In addition, this compound proves that a small-molecule direct LCAT activator can achieve HDL-C elevation in monkey and reduction of atherosclerotic lesion area with enhanced HDL function in rodent.
BACKGROUND: In recent years, systemic chemotherapy has significantly improved the prognosis of metastatic colorectal cancer (CRC); however, different patients have different responses to chemotherapeutics. METHODS: Dipeptidyl peptidase 9 (DPP9) is an enzyme in the dipeptidyl peptidase IV family that has been reported to increase drug sensitivity in acute myeloid leukemia. In this study, we examined the relationship between DPP9 expression and the prognosis of patients with CRC, as well as the role of DPP9 in anticancer drug resistance. Moreover, the effects of the DPP9 inhibitors talabostat and vildagliptin in CRC cell lines and primary cultured cells were assessed. RESULTS: High expression of DPP9 was associated with worse prognosis in 196 patients with CRC. Cell viability was markedly inhibited in CRC cell lines transfected with DPP9 small interfering RNA or small hairpin RNA. Talabostat suppressed proliferation in CRC cell lines and primary cultured cells, and increased their sensitivity to chemotherapy. Vildagliptin, a DPP family inhibitor currently administered for diabetes, also increased the sensitivity of CRC cells to anticancer drugs. CONCLUSION: DPP9 was a poor prognostic factor for CRC and could be a new therapeutic target, while vildagliptin could be used as a repositioned drug for CRC treatment.
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 [(18)F]5 ((4 R)-1-{3-[2-((18)F)fluoro-4-methylpyridin-3-yl]phenyl}-4-[4-(1,3-thiazol-2-ylcarbo nyl)piperazin-1-yl]pyrrolidin-2-one, [(18)F]T-401) specifically binds to MAGL with adequate reversibility, yielding a high contrast for MAGL within an appropriate imaging time.
Monoacylglycerol lipase (MAGL) is a major serine hydrolase that hydrolyzes 2-arachidonoylglycerol (2-AG) to arachidonic acid (AA) and glycerol in the brain. Because 2-AG and AA are endogenous biologically active ligands in the brain, inhibition of MAGL is an attractive therapeutic target for CNS disorders, particularly neurodegenerative diseases. In this study, we report the structure-based drug design of novel piperazinyl pyrrolidin-2-ones starting from our hit compounds 2a and 2b. By enhancing the interaction of the piperazinyl pyrrolidin-2-one core and its substituents with the MAGL enzyme via design modifications, we identified a potent and reversible MAGL inhibitor, compound ( R)-3t. Oral administration of compound ( R)-3t to mice decreased AA levels and elevated 2-AG levels in the brain.
BACKGROUND: Skeletal muscle metabolism is a major determinant of resting energy expenditure (REE). Although the severe muscle loss that characterizes Duchenne muscular dystrophy (DMD) may alter REE, this has not been extensively investigated. METHODS: We studied REE in 77 patients with DMD ranging in age from 10 to 37 years using a portable indirect calorimeter, together with several clinical parameters (age, height, body weight (BW), body mass index (BMI), vital capacity (VC), creatine kinase, creatinine, albumin, cholinesterase, prealbumin), and assessed their influence on REE. In addition, in 12 patients maintaining a stable body weight, the ratio of energy intake to REE was calculated and defined as an alternative index for the physical activity level (aPAL). RESULTS: REE (kcal/day, mean+/-SD) in DMD patients was 1123 (10-11 years), 1186+/-188 (12-14 years), 1146+/-214 (15-17 years), 1006+/-136 (18-29 years) and 1023+/-97 (>/=30 years), each of these values being significantly lower than the corresponding control (p<0.0001). VC (p<0.001) was the parameter most strongly associated with REE, followed by BMI (p<0.01) and BW (p<0.05). The calculated aPAL values were 1.61 (10-11 years), 1.19 (12-14 years), 1.16 (15-17 years), and 1.57 (18-29 years). CONCLUSION: The REE in DMD patients was significantly lower than the normal value in every age group, and strongly associated with VC. Both the low REE and PAL values during the early teens, resulting in a low energy requirement, might be related to the obesity that frequently occurs in this age group. In contrast, the high PAL value in the late stage of the disease, possibly due to the presence of respiratory failure, may lead to a high energy requirement, and thus become one of the risk factors for development of malnutrition.
The design, synthesis, and structure-activity relationships of a new class of potent and orally active non-peptide dipeptidyl peptidase IV (DPP-4) inhibitors, 3-aminomethyl-1,2-dihydro-4-phenyl-1-isoquinolones, are described. We hypothesized that the 4-phenyl group of the isoquinolone occupies the S1 pocket of the enzyme, the 3-aminomethyl group forms an electrostatic interaction with the S2 pocket, and the introduction of a hydrogen bond donor onto the 6- or 7-substituent provides interaction with the hydrophilic region of the enzyme. Based on this hypothesis, intensive research focused on developing new non-peptide DPP-4 inhibitors has been carried out. Among the compounds designed in this study, we identified 2-[(3-aminomethyl-2-(2-methylpropyl)-1-oxo-4-phenyl-1,2-dihydro-6-isoquinolinyl)o xy]acetamide (35a) as a potent, selective, and orally bioavailable DPP-4 inhibitor, which exhibited in vivo efficacy in diabetic model rats. Finally, X-ray crystallography of 35a in a complex with the enzyme validated our hypothesized binding mode and identified Lys554 as a new target-binding site available for DPP-4 inhibitors.
Dipeptidyl peptidase IV (DPP-4) inhibition is a validated therapeutic option for type 2 diabetes, exhibiting multiple antidiabetic effects with little or no risk of hypoglycemia. In our studies involving non-covalent DPP-4 inhibitors, a novel series of quinoline-based inhibitors were designed based on the co-crystal structure of isoquinolone 2 in complex with DPP-4 to target the side chain of Lys554. Synthesis and evaluation of designed compounds revealed 1-[3-(aminomethyl)-4-(4-methylphenyl)-2-(2-methylpropyl)quinolin-6-yl]piperazine- 2,5-dione (1) as a potent, selective, and orally active DPP-4 inhibitor (IC(5)(0)=1.3 nM) with long-lasting ex vivo activity in dogs and excellent antihyperglycemic effects in rats. A docking study of compound 1 revealed a hydrogen-bonding interaction with the side chain of Lys554, suggesting this residue as a potential target site useful for enhancing DPP-4 inhibition.
Inhibition of dipeptidyl peptidase IV (DPP-4) is an exciting new approach for the treatment of diabetes. To date there has been no DPP-4 chemotype possessing a carboxy group that has progressed into clinical trials. Originating from the discovery of the structurally novel quinoline derivative 1, we designed novel pyridine derivatives containing a carboxy group. In our design, the carboxy group interacted with the targeted amino acid residues around the catalytic region and thereby increased the inhibitory activity. After further optimization, we identified a hydrate of [5-(aminomethyl)-6-(2,2-dimethylpropyl)-2-ethyl-4-(4-methylphenyl)pyridin-3-yl]ac etic acid (30c) as a potent and selective DPP-4 inhibitor. The desired interactions with the critical active-site residues, such as a salt-bridge interaction with Arg125, were confirmed by X-ray cocrystal structure analysis. In addition, compound 30c showed a desired preclinical safety profile, and it was encoded as TAK-100.
From postnatal-day-0 to postnatal-day-2, a few acetylcholinesterase (AChE)-active and choline acetytransferase (ChAT)-immunoreactive nerve fibers and relatively numerous vesicular acetylcholine transporter (VAChT)-immunoreactive puncta were observed in the rat adrenal medulla. Despite relatively numerous clear vesicles in the nerve fibers, the synthesis and hydrolysis of acetylcholine may not be fully activated until postnatal-day-2. The number of AChE-active and ChAT-immunoreactive nerve fibers dramatically increased and that of VAChT-immunoreactive puncta gradually increased from postnatal-day-3 to postnatal-week-1. The synthesis and hydrolysis of acetylcholine may be dramatically activated in the nerve fibers of the medulla until postnatal-week-1. From postnatal-week-2 to postnatal-week-3, the number of AChE-active and the ChAT-immunoreactive nerve fibers gradually increased and reached the adult levels. The VAChT-immunoreactive puncta per unit area was maximum number at postnatal-week-2. The synthesis and hydrolysis of acetylcholine in the nerve fibers of the medulla may be completed between postnatal-week-2 to postnatal-week-3. The diameter of the VAChT-immunoreactive puncta gradually increased from postnatal-day-0 with aging. However, the number of the VAChT-immunoreactive puncta gradually decreased from postnatal-week-2 onwards. In electron-microscopy, the VAChT-immunoreactive deposits were seen in clusters of clear vesicles, and the diameter of the nerve fibers and the number of clear vesicles at postnatal-week-8 increased compared with those at postnatal-week-2. The AChE-active, ChAT-immunoreactive, and VAChT-immunoreactive nerve fibers observed around noradrenaline (NA) cells were denser than those around adrenaline (A) cells in the medulla at postnatal-week-8. These suggest that the preferential innervation of NA and A cells may cause the differential secretion NA and A.
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.
As part of an on-going investigation to develop an increasing agent on rhythmic bladder contractions, 1-aryl-3-(1-benzylpiperidin-4-yl)propanones were synthesized and examined as noncarbamate acetylcholinesterase (AChE) inhibitors. Among compounds with various aryl groups, 1,2,5,6-tetrahydro-4H-pyrrolo[3,2,1-ij]quinolin-4-one derivative 9c was found to possess a potent AChE inhibition activity with an IC(50) value of 1.3nM. The compound 9c increased rhythmic bladder contractions in Guinea pigs and rats without affecting the basal intravesical pressure, which suggests that 9c may be useful for the treatment of voiding dysfunction caused by detrusor underactivity.
Sphingomonas paucimobilis SYK-6 degrades syringate to 3-O-methylgallate (3MGA), which is finally converted to pyruvate and oxaloacetate via multiple pathways in which protocatechuate 4,5-dioxygenase, 3MGA dioxygenase, and gallate dioxygenase are involved. Here we isolated the syringate O-demethylase gene (desA), which complemented the growth deficiency on syringate of a Tn5 mutant of the SYK-6 derivative strain. The desA gene is located 929 bp downstream of ferA, encoding feruloyl-coenzyme A synthetase, and consists of a 1,386-bp open reading frame encoding a polypeptide with a molecular mass of 50,721 Da. The deduced amino acid sequence of desA showed 26% identity in a 325-amino-acid overlap with that of gcvT of Escherichia coli, which encodes the tetrahydrofolate (H(4)folate)-dependent aminomethyltransferase involved in glycine cleavage. The cell extract of E. coli carrying desA converted syringate to 3MGA only when H(4)folate was added to the reaction mixture. DesA catalyzes the transfer of the methyl moiety of syringate to H(4)folate, forming 5-methyl-H(4)folate. Vanillate and 3MGA were also used as substrates for DesA; however, the relative activities toward them were 3 and 0.4% of that toward syringate, respectively. Disruption of desA in SYK-6 resulted in a growth defect on syringate but did not affect growth on vanillate, indicating that desA is essential to syringate degradation. In a previous study the ligH gene, which complements the growth deficiency on vanillate and syringate of a chemical-induced mutant of SYK-6, DC-49, was isolated (S. Nishikawa, T. Sonoki, T. Kasahara, T. Obi, S. Kubota, S. Kawai, N. Morohoshi, and Y. Katayama, Appl. Environ. Microbiol. 64:836-842, 1998). Disruption of ligH resulted in the same phenotype as DC-49; its cell extract, however, was found to be able to convert vanillate and syringate in the presence of H(4)folate. The possible role of ligH is discussed.
As a base for human transcriptome and functional genomics, we created the "full-length long Japan" (FLJ) collection of sequenced human cDNAs. We determined the entire sequence of 21,243 selected clones and found that 14,490 cDNAs (10,897 clusters) were unique to the FLJ collection. About half of them (5,416) seemed to be protein-coding. Of those, 1,999 clusters had not been predicted by computational methods. The distribution of GC content of nonpredicted cDNAs had a peak at approximately 58% compared with a peak at approximately 42%for predicted cDNAs. Thus, there seems to be a slight bias against GC-rich transcripts in current gene prediction procedures. The rest of the cDNAs unique to the FLJ collection (5,481) contained no obvious open reading frames (ORFs) and thus are candidate noncoding RNAs. About one-fourth of them (1,378) showed a clear pattern of splicing. The distribution of GC content of noncoding cDNAs was narrow and had a peak at approximately 42%, relatively low compared with that of protein-coding cDNAs.
Tissue distribution of dichlorvos (DDVP) was determined in a case of fatal ingestion using a rapid and simple gas chromatographic (GC) assay. Remarkable autopsy findings were congestion of the lung and kidneys and bleeding ulcer extending from the dorsum of the tongue to the upper pharynx. The serum cholinesterase activity was 2 IU/1, however, miosis was not observed. In the stomach, 250 ml of volatile fluid was found. Tissue distribution of DDVP was determined using a newly developed simple and rapid GC method. DDVP was found in the spleen and heart at higher concentrations (3340 and 815 micrograms/g, respectively), and also detected in the urine at the lowest level (4.5 micrograms/ml). The DDVP concentrations in blood, brain, lung, kidney and liver were 29, 9.7, 81, 80 and 20 micrograms/ml or g, respectively.
A longitudinal characteristic curve of chronic liver disease (LCC-LD) is derived for the first time by a new method of time series data analysis, where a hospital information system is utilized as a clinical research application of the database. It describes a typical pattern of development of disease from the beginning of chronic hepatitis to the final stage of cirrhosis. The LCC-LD is obtained by effectively using patient data with various stages of developments of liver disease and the present method is applicable to derive the LCC of other diseases. The obtained LCC-LD may be useful for a clinical decision making support such as the prospective assessment (for example, the onset time of cirrhosis) of liver disease in individual patients, an evaluation of drug effect, etc.
