Specific forms of the lipid ceramide, synthesized by the ceramide synthase enzyme family, are believed to regulate metabolic physiology. Genetic mouse models have established C16 ceramide as a driver of insulin resistance in liver and adipose tissue. C18 ceramide, synthesized by ceramide synthase 1 (CerS1), is abundant in skeletal muscle and suggested to promote insulin resistance in humans. We herein describe the first isoform-specific ceramide synthase inhibitor, P053, which inhibits CerS1 with nanomolar potency. Lipidomic profiling shows that P053 is highly selective for CerS1. Daily P053 administration to mice fed a high-fat diet (HFD) increases fatty acid oxidation in skeletal muscle and impedes increases in muscle triglycerides and adiposity, but does not protect against HFD-induced insulin resistance. Our inhibitor therefore allowed us to define a role for CerS1 as an endogenous inhibitor of mitochondrial fatty acid oxidation in muscle and regulator of whole-body adiposity. Ceramides are signalling molecules that regulate several physiological functions including insulin sensitivity. Here the authors report a selective ceramide synthase 1 inhibitor that counteracts lipid accumulation within the muscle and adiposity by increasing fatty acid oxidation but without affecting insulin sensitivity in mice fed with an obesogenic diet. Ceramides form the lipid backbone to which a diverse array of headgroup structures are conjugated, forming sphingomyelin (SM), glucosyl- and galactosylceramide (HexCer), gangliosides, and globosides3. Ceramides are also signalling molecules that regulate ER stress4, apoptosis5, insulin sensitivity6, and other physiological functions. At the molecular level, ceramides influence membrane fluidity, modulating the compartmentalisation of cellular signalling processes8, and directly activate specific protein kinases and phosphatases such as the ubiquitous phosphatase PP2A8. Increased ceramide levels are heavily implicated in the pathogenesis of insulin resistance12 and neurodegenerative conditions13, whilst decreased levels fuel cancer cell resistance to therapy3.Ceramide is the central metabolite of the sphingolipid family, structurally comprised of a sphingoid base—generally 18 carbon dihydrosphingosine or sphingosine—with a variable length fatty acyl side-chain1. Studies employing genetic manipulations have demonstrated that different CerS isoforms exhibit strong preference for fatty acyl-CoAs with differing carbon chain lengths. CerS1 exclusively uses 18 carbon (C18) fatty acids, forming C18 (d18:1/18:0) ceramide16, whilst CerS2 preferentially forms d18:1/24:0 (C24:0) and d18:1/24:1 (C24:1) ceramides18. Thus, ceramide is not a single lipid entity; rather it is a family of signalling lipids with important physiological functions, and variation in the ceramide acyl-chain dramatically influences the biological properties of these lipids. Insulin resistance caused by a HFD is alleviated by CerS5 or CerS6 gene deletion, which prevents C16 ceramide synthesis in liver and adipose tissue12. C16 and shorter chain ceramides antagonise the insulin receptor—PI3 kinase—Akt signalling pathway and inhibit fat utilisation as an energy source via β-oxidation19. In direct contrast, C24 ceramides synthesized by CerS2 protect against insulin resistance20. The synthesis of ceramides and other sphingolipids is regulated by availability of fatty acyl-CoA substrates, particularly palmitoyl-CoA derived from the common saturated fatty acid palmitate21. As such, ceramide synthesis may act as a direct metabolic sensor of fatty acid availability, feeding back to regulate metabolic processes.Ceramide synthesis in mammals is catalysed by a family of six ceramide synthases (CerS1-6). These enzymes transfer a variable length fatty acyl-coenzyme A (CoA) to the amine group of a sphingoid base23. CerS1 and its product C18 ceramide are highly abundant in skeletal muscle (SkM)18. Studies comparing obese insulin resistant and insulin sensitive subjects, exercise interventions in type 2 diabetes, and induction of insulin resistance in mice, all show an association between muscle C18:0 ceramide and impairments in insulin action24. Although a relatively minor species in plasma, circulating C18 ceramide is also very significantly correlated with body mass index25 and visceral fat mass22. Similarly, C18 ceramide in SkM is positively correlated with visceral fat, as well as blood pressure22.Another ceramide species implicated in insulin resistance is C18:027. This report describes the discovery and characterisation of the first potent, isoform-selective CerS inhibitor, specifically targeting CerS1. CerS1 inhibition is shown to promote fatty acid oxidation in SkM and reduce overall adiposity in mice fed a HFD.Selective inhibition of CerS5, CerS6, and/or CerS1 would therefore be predicted to produce significant benefits for metabolic health, whilst CerS2 inhibition would have detrimental effects. However, isoform-specific CerS inhibitors with sufficient potency, selectivity, and bioavailability for in vivo use have not yet been developed28. Following its phosphorylation in vivo, Fingolimod is a potent agonist of sphingosine 1-phosphate receptors, however the non-phosphorylated pro-drug also exhibits non-selective inhibition of ceramide synthases as an off-target effect30. We recently established that the non-phosphorylatable, chiral FTY720 analogue AAL(S) and its benzyl tail derivative G024 , show limited selectivity for CerS1 over other CerS isoforms27. However, their degree of selectivity for CerS1 is poor and, importantly, these compounds do not selectively reduce C18 ceramide levels in cultured cells. Using the Topliss tree as a guide32 we examined variations of the benzyl tail of G024, resulting in the identification of (S)-2-amino-4-phenyl)-2-methylbutan-1-ol  as a potent and selective CerS1 inhibitor 33, which inhibited all CerS isoforms with sub-micromolar IC50  without notably affecting substrate affinity  , which is an analogue of the endogenous lipid sphingosinetor Fig. . The IC5lf) Fig. , indicat34 using cortical neuron cultures, a two hour pre-treatment with P053 resulted in dose-dependent inhibition of de novo C18:0 ceramide synthesis from deuterated dihydrosphingosine . Very long chain ceramides  were significantly increased by P053 treatment in the HFD group , and 11% in mice fed a chow diet (not significant). Dihydrosphingosine levels were not notably affected by P053 treatment , C18 dihydroceramide , and 18:2/18:0 ceramide with P053 treatment , and was 60% lower in the HFD + P053 group compared to the HFD vehicle group (P = 0.021). P053 treatment did not affect SkM TAG levels in mice fed normal chow . We noted that the levels of most triacylglycerol (TAG) species were reduced by more than 50% in SkM of the HFD + P053 compared to HFD + vehicle group Fig. . Total Show Fig. . TAGs arP < 0.01 after adjusting for multiple comparisons), including C22:0 and C24:1 ceramide , from which P053 is derived, is a non-phosphorylatable FTY720 analogue37. In cultured HEK293 cells incubated with FTY720, FTY720-phosphate was clearly detected, whereas the phosphate of P053 was not detected in either cultured cells or plasma of mice administered the drug, indicating that like the parent compound AAL(S), P053 is non-phosphorylatable  , in soleus muscles from mice treated for 2 weeks with P053. Incorporation of 14C-palmitate into TAG during the 1 h incubation was unchanged   Fig. . This ef06) Fig. , where C40 . The expression of transcriptional regulators of mitochondrial biogenesis Gabp1α (Nuclear respiratory factor 2)41 and Nfe2l2 (nuclear factor (erythroid-derived 2)-like 2)42 was also significantly increased by P053 treatment   suggest enhanced mitochondrial biogenesis in response to depletion of C18 ceramide in muscle. Other studies have shown down-regulation of genes regulating fatty acid metabolism in the absence of CerS257, associated with reduced β-oxidation of fatty acids9. These effects were attributed to elevation of C16 ceramide in the absence of very long chain ceramide synthesis. Similarly, loss of CerS2 was shown to reduce respiratory enzyme activities and increase mitochondrial oxidative stress, effects that were also attributed to elevated C16 ceramide58. Our data indicates that C18 ceramide behaves similarly to C16 ceramide in suppressing respiratory enzyme levels and fatty acid β-oxidation. Another possible contributor to changes in mitochondrial content/function in response to P053 treatment is reduced mitophagy, since exogenous CerS1 expression and a mitochondrial-targeted analogue of C18 ceramide were both shown to promote mitophagy39. Ceramides have also been shown to induce mitochondrial fission in SkM cells, reducing their oxidative capacity19. Thus, another possibility is that CerS1 inhibition, by lowering the overall ceramide content of SkM, boosts respiratory capacity via inhibition of fission.The precise mechanism through which chronic CerS1 inhibition changes mitochondrial content and/or function requires further investigation. Our findings of elevated expression of PGC-1α and other important transcriptional regulators of mitochondrial content and function  was added dropwise to a solution of freshly distilled (S)-Schöllkopf’s reagent  in freshly distilled THF (22 mL) at −78 °C (dry ice/acetone). The solution was stirred at −78 °C for 15 min, where it had turned dark yellow. A solution of 2-(4′-benzyloxyphenyl)-1-iodoethane60  in freshly distilled THF (21 mL) at −78 °C was added dropwise. The solution was stirred for a further 30 min at −78 °C then allowed to slowly warm to −15 °C over 4 h. The reaction mixture was quenched with saturated aqueous sodium bicarbonate solution and allowed to warm to room temperature. The THF was removed under reduced pressure and the residue extracted with dichloromethane (×4). The organic extracts were combined and dried (Na2SO4). The solvent was removed under reduced pressure and the crude material was purified by flash chromatography on silica gel, eluting with 3% ethyl acetate/n-hexane, to afford the product as a clear colourless oil . 3); 1H NMR  δ 0.70 , 1.06 , 1.92–2.02 , 2.07–2.19 , 2.22–2.32 , 2.48–2.64 , 3.69 , 3.70 , 3.97 , 4.02–4.07 , 5.04 , 6.89 , 7.10–7.12 , 7.29–7.45 ; 13C NMR  δ 16.8, 19.2, 30.2, 32.0, 36.2, 52.5, 55.1, 60.9, 70.2, 114.8, 127.6, 128.0, 128.7, 129.5, 134.7, 137.4, 157.1, 163.7, 163.9; IR  1694 cm−1; HRMS (ESI-MS): m/z calcd for C24H31N2O3 [M + H]+ 395.2334, found 395.2320.A solution of n-butyllithium in hexanes  was added dropwise to a solution of -5-isopropyl-3,6-dimethoxy-2-(4′-benzyloxyphenethyl)-2,5-dihydropyrazine  in freshly distilled THF (47 mL) at −78 °C (dry ice/acetone). The solution was stirred at −78 °C for 15 min, where it had turned dark yellow. Methyl iodide  was added dropwise. The solution was stirred for a further 30 min at −78 °C then allowed to slowly warm to −15 °C over 4 h. The reaction mixture was quenched with saturated aqueous sodium bicarbonate solution and allowed to warm to room temperature. The THF was removed under reduced pressure and the residue extracted with dichloromethane (×4). The organic extracts were combined and dried (Na2SO4). The solvent was removed under reduced pressure and the crude material was purified by flash chromatography on silica gel, eluting with 2% ethyl acetate/n-hexane, to afford the product as a clear colourless oil , with all analytical data matching that reported in the literature27. 3); 1H NMR  δ 0.70 , 1.12 , 1.30 , 1.85 , 2.08 , 2.25 , 2.34–2.48 , 3.70 , 3.71 , 3.93 , 5.04 , 6.87–6.90 , 7.06–7.09 , 7.29–7.44 ; 13C NMR  δ 17.1, 19.7, 28.7, 30.7, 30.8, 42.9, 52.4, 58.4, 60.5, 70.2, 114.8, 127.6, 128.0, 128.7, 129.4, 135.2, 137.4, 157.0, 162.1, 165.7; IR  1694 cm−1; HRMS (ESI-MS): m/z calcd for C25H33N2O3 [M + H]+ 409.2491, found 409.2490.A solution of S,5S)-5-isopropyl-3,6-dimethoxy-2-(4′-benzyloxyphenethyl)-2-methyl-2,5-dihydropyrazine  in acetonitrile (100 mL). The solution was stirred at room temperature for 4 h after which the acetonitrile was removed under reduced pressure. The residue was diluted with water and neutralised with portions of solid sodium bicarbonate, then extracted with dichloromethane (×4). The organic extracts were combined and dried (Na2SO4). The solvent was removed under reduced pressure and the crude material was purified by flash chromatography on silica gel, eluting with ethyl acetate, to afford the product as a clear colourless oil , with all analytical data matching that reported in the literature27. 3); 1H NMR  δ 1.37 , 1.80–1.90 , 1.96–2.06 , 2.41–2.51 , 2.55–2.65 , 3.70 , 5.04 , 6.88–6.91 , 7.07–7.10 , 7.31–7.44 ; 13C NMR  δ 26.6, 29.9, 43.1, 52.3, 57.8, 70.1, 114.9, 127.5, 127.9, 128.6, 129.3, 134.0, 137.2, 157.1, 178.0; IR  1733 cm−1; HRMS (ESI-MS): m/z calcd for C19H24NO3 [M + H]+ 314.1756, found 314.1754.A solution of trifluoroacetic acid  in water (30 mL) was added dropwise to a solution of (2S)-2-amino-4-(4′-benzyloxyphenyl)-2-methylbutanoate  in freshly distilled THF (40 mL) at 0 °C. The solution was stirred at 0 °C for 20 min then the cold bath was removed and the solution stirred at room temperature for 1 h. The reaction was quenched with saturated aqueous sodium sulphate solution and the mixture was extracted with ethyl acetate (×4). The organic extracts were combined and washed with saturated aqueous sodium bicarbonate solution, water and brine, then dried (Na2SO4). The solvent was removed under reduced pressure and the crude material was purified by flash chromatography on silica gel, eluting with 2% methanol/4% triethylamine/dichloromethane, to afford the product as a white solid , with all data matching that reported in the literature27. 3); M.p.: 196.7–197.6 °C; 1H NMR  δ 1.13 , 1.61–1.71 , 2.59 , 3.34 , 5.04 , 6.90 , 7.09–7.12 , 7.29–7.45 ; 13C NMR  δ 24.6, 29.5, 42.2, 53.1, 70.2, 70.3, 115.0, 127.6, 128.0, 128.7, 129.3, 134.8, 137.3, 157.1; IR  3425 cm−1; HRMS (ESI-MS): m/z calcd for C16H28NO4 [M + H]+ 286.1807, found 286.1807.Lithium aluminium hydride  was added as a solid in one portion to a solution of methyl  was added as a solid in one portion to a mixture of (2S)-2-amino-4-(4′-benzyloxyphenyl)-2-methyl-1-butanol  in saturated aqueous sodium bicarbonate solution (12.8 mL) and ethyl acetate (12.8 mL). The mixture was heated at 70 °C for 16 h then allowed to cool to room temperature. The solution was diluted with water and extracted with ethyl acetate (×3). The organic extracts were combined and washed with brine, then dried (Na2SO4). The solvent was removed under reduce pressure and the crude material was purified by flash chromatography on silica gel, eluting with 30% ethyl acetate/n-hexane, to afford the product as a white solid , with all analytical data matching that reported in the literature61. 3); 1H NMR  δ 1.22 , 1.44 , 1.80–1.88 , 1.99–2.06 , 2.53 , 2.63 , 3.62–3.72 , 4.06 , 4.62 , 5.04 , 6.89–6.91 , 7.10–7.12 , 7.30–7.44 .Di-S)-t-butyl(1-hydroxy-4-(4′-benzyloxyphenyl)-2-methylbutan-2-yl)carbamate  in dry methanol (30 mL) at room temperature. A hydrogen balloon was attached to the flask and the flask was evacuated and purged with hydrogen (×3). The solution was stirred at room temperature for 18 h. The reaction mixture was diluted with dichloromethane and filtered through a short pad of Celite, eluting with dichloromethane. The solvent was removed under reduced pressure to afford the product as a white solid , with all the analytical data matching that reported in the literature61. 3); 1H NMR  δ 1.21 , 1.44 , 1.79–1.87 , 2.04 , 2.50 , 2.59 , 3.63–3.76 , 4.30 , 4.65 , 5.33 , 6.75 , 7.02–7.04 .Palladium on carbon  was added to a solution of (2S)-t-butyl(1-hydroxy-4-(4′-hydroxyphenyl)-2-methylbutan-2-yl)carbamate  and potassium carbonate  in dry DMF (0.8 mL). The suspension was stirred at room temperature for 15 h. The solution was diluted with water and extracted with ethyl acetate (×3). The organic extracts were combined and washed with water and brine, then dried (Na2SO4). The solvent was removed under reduced pressure and the crude material was purified by flash chromatography on silica gel, eluting with 30% ethyl acetate/n-hexane. After dissolving the purified material in methanol (0.5 mL), a solution of 2 M methanolic hydrochloric acid  was added dropwise to it and the resulting solution was heated at reflux for 4 h. After cooling to room temperature, the solvent was removed under reduced pressure. The residue was dissolved in chloroform and washed with saturated aqueous sodium bicarbonate solution (×2) and brine, then dried (Na2SO4). The solvent was removed under reduced pressure to afford the product as a white solid . 3); 1H NMR  δ 1.15 , 1.63–1.77 , 1.83 , 2.60 , 3.35 , 3.41 , 4.98 , 6.85–6.87 , 7.10–7.13 , 7.24–7.35 , 7.44 , 7.53 ; 13C NMR  δ 24.4, 29.5, 53.1, 68.8, 70.2, 77.4, 115.0, 126.7, 129.3, 129.5, 130.7, 135.2, 135.51, 135.54, 137.7, 156.6; IR  3425 cm−1; HRMS (ESI-MS): m/z calcd for C18H22NO2Cl2 [M + H]+ 354.1027, found 354.1017.3,4-Dichloro-benzyl bromide  was added dropwise to a suspension of ; and COS cells expressing murine CerS1, 2, and 5. Human CerS expression plasmids (pCMV6 plasmids from Origene) were transfected using FuGene-6 transfection reagent, and cells were harvested 24 h after transfection in 20 mM Hepes pH 7.4, 10 mM KCl, 1 mM dithiothreitol, 3 mM β-glycerophosphate, and EDTA-free complete protease inhibitor cocktail (Roche). Cells were sonicated in a Diagenode Bioruptor sonicating bath for 5 min, after which cell extracts were cleared by centrifugation at 800 × g for 10 min. Protein concentration in the supernatant was assayed using the bicinchoninic acid (BCA) assay, and the supernatant stored at −80 °C. COS cells were infected with lentiviral constructs expressing murine CerS1, 2, or 5, then homogenised as described above.Ceramide synthase assays were carried out using an HPLC-based assay with a fluorescent NBD-dihydrosphingosine substrate2, 0.5 mM dithiothreitol, 0.1% fatty acid free BSA, 10 μM NBD-dihydrosphingosine and 50 μM fatty acid free BSA. C18:0-CoA was used as the fatty acyl substrate for CerS1 and CerS4; C24:1-CoA for CerS2; and C16:0-CoA for CerS5 and CerS6. All reactions were run in triplicate. Test inhibitors were added, after which reactions were started with the addition of 2.5–25 μg lysate protein (optimised depending on the particular CerS isoform being assayed). Assays were run for 30 min at 37 °C, and stopped with the addition of 200 μL methanol. The reaction mixture was transferred to a glass HPLC vial with 300 μL fused glass insert (Thermo Fisher Scientific) and NBD-dihydroceramide reaction products were analysed on a Thermo Scientific Surveyor HPLC connected to a Shimadzu RF-10AXL fluorescent detector (set to gain of 3). A 3 × 150 mm Agilent XDB-C8 column (5 μM pore size) was used. Data was acquired over a 12 min chromatography run using: solvent A, 0.2% formic acid, 2 mM ammonium formate in water; solvent B, 0.2% formic acid, 1 mM ammonium formate in methanol. The gradient started at 20:80 A/B, increasing to 5:95 A/B over 2 min, then to 100% B over 6 min. The gradient was then held at 100% B for 2 min, before re-equilibration to 20:80 A/B for a further 2 min. Peak areas were integrated using Xcalibur software (Thermo Fisher Scientific).The reaction buffer consisted of 20 mM Hepes pH 7.4, 25 mM KCl, 2 mM MgCl64. Reactions containing 50 mM Hepes pH 7.4, 0.01% BSA, 15 mM MgCl2, 2 mM ATP, 10 μM NBD-dihydrosphingosine, and 10 μM FTY720 or P053, were started with addition of 10 ng recombinant human sphingosine kinase 1 or 2 (R&D Systems), and run for 30 min at 37 °C. Reactions were stopped with 100 μL methanol, and 5 μL amounts were resolved on Silica Gel 60 thin layer chromatography (TLC) plates using butanol:acetic acid:water (3:1:1). The fluorescent NBD-dihydrosphingosine 1-phosphate band was quantified using a BioRad ChemiDoc imaging system with excitation by UV light.Sphingosine kinase assays were performed using fluorescent NBD-dihydrosphingosine65 and seeded at 5 × 106 cells/well in poly-D-lysine coated six-well plates. Two weeks after seeding, neurons were pre-treated for 2 h with P053, then incubated for a further 1 h with 1 μM deuterated dihydrosphingosine (D7-dhSph). Lipid extraction and quantification of D7-ceramide using liquid chromatography-tandem mass spectrometry (LC-MS/MS)34 is described below.Mouse cortical neurons were prepared from E16 pups66 for 4–6 weeks. P053 (5 mg/kg) was administered daily by oral gavage, with control animals receiving vehicle (2% DMSO in drinking water). Treatment was commenced at the same time as mice were randomised to dietary groups. The experiments were approved by the UNSW animal care and ethics committee (ACEC 15/48B), and followed guidelines issued by the National Health and Medical Research Council of Australia.Male C57BL6/J mice were obtained from the Animal Resources Centre in Perth . Mice were housed at 22 ± 1 °C with a controlled 12:12 h light-dark cycle and had ad libitum access to water and either chow  or HFD tert-butyl ether (MTBE)/methanol/water 67. Frozen tissue samples (~20 mg) were homogenized in 0.2 mL methanol containing 0.01% butylated hydroxytoluene (BHT), using a Precellys 24 homogenizer and Cryolys cooling unit (Bertin Technologies) with CK14 (1.4-mm ceramic) beads. The homogenates were spiked with an internal standard mixture comprising 5 nmole 19:0/19:0 phosphatidylcholine and 2 nmole each of 17:0/17:0 phosphatidylethanolamine, 17:0/17:0 phosphatidylserine, 17:0/17:0 d5-diacylglycerol, 17:0/17:0 phosphatidylglycerol, 14:0/14:0/14:0/14:0 cardiolipin, 18:1/12:0 SM, 18:1/17:0 ceramide, 18:1/12:0 glucosylceramide, 18:1/12:0 lactosylceramide, 18:1/12:0 sulfatide , and 17:0/17:0/17:0 TAG , then transferred to 10 mL screw cap glass tubes. The homogenization tubes containing beads were then washed with another 0.3 mL methanol, and this wash was combined with the first methanol extract, after which 1.7 mL MTBE was added and the samples were sonicated in an ice-cold sonicating water bath  for 30 min. Phase separation was induced by addition of 417 µL of mass spectrometry-grade water followed by vortexing and centrifugation at 1000 × g for 10 min. The upper organic phase was collected into 5 mL glass tubes. The lower phase was re-extracted by adding 1 mL MTBE, 300 μL methanol, and 250 μL water. The organic phases were combined in the 5 mL glass tubes and dried under vacuum in a Savant SC210 SpeedVac (Thermo Scientific), then reconstituted in 500 μL methanol and stored at −20 °C until analysis.Lipids were extracted from frozen tissue samples or cells using a two-phase procedure with methyl-6 cells/treatment) were treated for 24 h with P053, then washed with PBS and scraped into 0.6 mL methanol on ice. Lipids were then extracted using MTBE/methanol/water as described above67.HEK293 cells  for 2 min at 30 s intervals. Extracts were centrifuged at 1400 rpm for 20 min, 4 °C, to clear debris. Supernatants were transferred to 5 mL glass tubes. The remaining insoluble material was re-extracted with 1 mL 80% methanol as above, and the supernatant combined with the first extract. The extracts were evaporated under vacuum . Dried extracts were reconstituted in 200 μL of 80% methanol/0.2% formic acid and stored at −20 °C. On the day of analysis, the extracts were vortexed thoroughly and centrifuged at 300 × g for 20 min before transferring 100 μL of the supernatant to glass HPLC vials. To extract P053 from tissues, MTBE/methanol/water extraction was performed as described above, with AAL(S) as the internal standard.Plasma samples (20 μL) were extracted with 200 μL methanol containing 20 pmoles AAL(S)−1, increasing to 0.5 mL min−1 from 5–7 min, and column oven 30 °C. P053 was quantified using an external standard curve covering the range 0.1–1000 nM, with all concentrations normalised to the 20 pmole AAL(S) internal standard. Visualisation and quantification of peaks was performed using Thermo Fisher’s XCalibur software v2.2. Precursor/product ion m/z values were as follows: 354.3 and 159.0 for P053; 294.0 and 161.1 for AAL(S); 380.3 and 255.2 for FTY720; 388.2 and 255.2 for FTY720-phosphate.LC-MS/MS was performed on a TSQ Quantum Access triple quadrupole mass spectrometer in positive ion mode, coupled to an Accela UPLC system (Thermo Scientific). Extracts (20 μL) were separated on a 2 × 100 mm Eclipse XDB-C8 column with 1.8 μm particle size (Agilent Technologies), using a 7.5 min binary HPLC gradient: 0 min, 70% B (20% A); 1.5 min, 70% B; 3.5 min, 95% B; 4 min, 95% B; 4.5 min, 70% B; 7.5 min 70% B. Mobile phase A: 2 mM ammonium formate/1% formic acid in water; mobile phase B: 1 mM ammonium formate/1% formic acid in methanol. The flow rate was 0.4 mL min68. The flow rate was 260 µL/min and column oven 55 °C. Data was acquired in full scan/data-dependent MS2 mode . The ten most abundant ions in each cycle were subjected to MS2 using an isolation window of 1.4 m/z, collision energy 30 eV, resolution 17,500, maximum integration time 110 ms and dynamic exclusion window 10 s. An exclusion list of background ions was used based on a solvent blank. LipidSearch v4.1.30 software (ThermoFisher Scientific) was used for chromatogram alignment, peak identification and integration. Each lipid peak was manually verified for column elution time and characteristic fragment ions. Data were then exported to an Excel spreadsheet for normalisation of each lipid to its class-specific internal standard, and lipid concentrations were calculated relative to the internal standard.Lipidomic profiling was performed on a QExactive Plus mass spectrometer with heated electrospray ionization (HESI) probe and a Dionex UltiMate 2000 LC pump (Thermo Fisher Scientific). Extracts were resolved on a 2.1 × 100 mm Waters Acquity C18 UPLC column (1.7 µm pore size), using a binary gradient in which mobile phase A consisted of acetonitrile:water (60:40), 10 mM ammonium formate, 0.1% formic acid; and mobile phase B was isopropanol:acetonitrile (90:10), 10 mM ammonium formate, 0.1% formic acid69. Lipids were separated on a 3 × 150 mm Agilent XDB-C8 column (5 μM pore size) with a 10 min isocratic chromatography run using methanol containing 0.2% formic acid as the mobile phase. Precursor ions were the precise [M + H] species for each lipid. The m/z 184.1 product ion was used for SM species, and the 264.3 m/z product ion for ceramide and HexCer species. Deuterated (D7) ceramides were detected as the corresponding m/z + 7 ions.Targeted quantification of ceramide, HexCer, and SM species in primary neurons, HEK293 cells, mouse brain, and gastrocnemius muscle lipid extracts was performed by LC-MS/MS on a TSQ Access triple quadrupole mass spectrometer (Thermo Fisher Scientific)m/z values were: 300.3 and 264.3 for sphingosine; 302.3 and 266.3 for dihydrosphingosine.Targeted quantification of sphingosine and dihydrosphingosine used the HPLC column and chromatography conditions as described above for P053. Precursor/product ion Body composition (lean mass and fat mass) was determined by EchoMRI-900 Body Composition Analyser  in accordance with the manufacturer’s instructions.2H glucose, Sigma Aldrich) by oral gavage. Blood glucose was measured with a glucose meter  prior to and at 15, 30, 45, 60 and 90 min following gavage. Additional blood (~10 μL) was collected from the tail tip prior to and at 15 and 60 min following glucose gavage. These samples were used to perform mass isotopologue analysis of plasma glucose via gas chromatography–mass spectrometry (GC–MS)70. Specifically, plasma glucose was separated into the labelled glucose load and the endogenous (liver/kidney-derived) unlabelled glucose to permit differentiation of glucose disposal from endogenous glucose production  was commenced. Samples were collected at −30, −20, −10 and 0 min for basal glucose turnover (Rd) determination. At time 0, the rate of -glucose was increased (0.1 μCi/min) and mice received a primed (24 mU/kg)-continuous (6 mU/kg/min) infusion of insulin  with euglycemia maintained at ~8 mM during the clamp through variable infusion of glucose (25% solution). Once blood glucose was stable, four sequential samples were taken to determine insulin-stimulated glucose turnover. A bolus of 2[14C]deoxyglucose  was then administered and blood sampled at 2, 5, 10, 15, 20 and 30 min, prior to collection of muscles for determination of glucose uptake. Glucose uptake into muscle was determined via the following equation:14C]DGPtissue is the 2[14C]DGP radioactivity in the muscle (in dpm/g), AUC 2[14C]DGplasma is the area under the plasma 2[14C]DG disappearance curve (in dpm/min/ml), and [arterial glucose] is the average blood glucose (in mM). Animal numbers for clamps were n = 9 Chow vehicle, n = 8 Chow P053, n = 6 HFD vehicle and n = 6 HFD P053.At 5 weeks of diet/drug treatment, mice underwent surgery to implant catheters into the left carotid artery and right jugular vein. Catheters were flushed every 1–2 days with heparinised saline to maintain patency. Approximately 6–8 days post surgery, and after a ~5 h fast, a hyperinsulinemic-euglycemic clamp was conducted. Mice were conscious, unrestrained and were not handled during the procedure to minimize stress. At −90 min, a primed (5 μCi) continuous infusion (0.05 μCi/min) of -palmitate, pH7.4) for 90 min at 30 °C, after which reactions were stopped with addition of 100 µL of 1 M perchloric acid. 14C-labelled CO2 was collected in 100 µL of 1 M NaOH solution for 2 h. 14C-labelled CO2, as well as 14C-labelled ASM present in the acidified supernatant, were quantified by scintillation counting.Tibialis muscles were homogenized in 19 volumes of 250 mM sucrose, 10 mM Tris-HCl and 1 mM EDTA, pH 7.4. 50 µL of tissue homogenate was incubated with 450 µL reaction buffer  modified Krebs-Henseleit buffer . After a 30 min pre-incubation, muscles were transferred into vials containing 0.5 mCi/mL of [1-14C]-palmitate in modified Krebs-Henseleit buffer for another 1 h. The reaction was stopped by removal of the muscles and addition of 1 M perchloric acid to the reaction mixture. 14C-labelled CO2 was quantified as described above. The muscles were blotted dry and snap frozen in liquid nitrogen for subsequent lipid extraction and TLC. Muscles were homogenized using a Polytron  in 1.5 mL 2:1 chloroform-methanol (v/v). The homogenates were then sonicated in ice water for 15 min, after which 0.3 mL of distilled water was added, samples were vortexed, and centrifuged at 350 × g for 10 min to induce phase separation. The upper aqueous phase was used to quantify ASM, and the lower organic phase was collected and dried under stream of N2, then reconstituted in hexane and spotted on a Silica Gel 60 F254 TLC plate. The TLC plate was developed in hexane/diethyl ether/glacial acetic acid (85:15:1), then air dried. TAG bands were visualised using iodine vapour staining, using an external standard to mark their position, then scraped into vials and quantified by liquid scintillation counting.To quantify palmitate oxidation in intact SkM, whole soleus muscles were dissected tendon to tendon and placed into a vial containing pre-warmed (30 °C) and pre-gassed  and fibres were prepared immediately. Fibre bundles (~3 mg wet weight) were treated with saponin (50 μg/mL) for 20 min at 4 °C and subsequently washed in cold respiration medium B . Mitochondrial respiratory chain function was analyzed on a Clark-type electrode  in situ, in respiration medium B at 37 °C, with the sequential addition of glutamate (10 mM), malate (5 mM), ADP (2 mM), rotenone (0.5 μM), succinate (10 mM), antimycin A (5 μM), N,N,N′,N′-tetramethyl-p-phenylenediamine dihydrochloride (0.5 mM TMPD), ascorbate (2 mM), and cytochrome c (10 μM). Fibres were recovered after polarography and results were expressed as nmoles of O2/min/mg of tissue. Mitochondrial membrane integrity was verified by cytochrome c release test72.Permeabilized extensor digitorum longus (EDL) fibres were prepared and mitochondrial function analyzed according to published protocols with some modificationsTissues were homogenized in RIPA buffer  containing complete protease inhibitor cocktail (Roche) and phosphatase inhibitors  by bead-beating in a Precellys 24 at 4 °C. Extracts were centrifuged at 6500 rpm for 10 min at 4 °C. Supernatant was collected and protein concentrations determined with the Bicinchoninic acid (BCA) assay (Thermo Scientific). Protein lysates (15 μg) were resolved on SDS-PAGE, transferred to polyvinylidene difluoride (PVDF) membrane, and immunoblotted with antibodies against Complex I subunit NADH dehydrogenase , Complex II succinate dehydrogenase subunit B , Complex III subunit Core 2 Ubiquinol-cytochrome c reductase , Complex IV Cytochrome c oxidase , Complex V ATP synthase α subunit , PGC1α , AMPKα , phospho- AMPKα (Thr172) , ACC , phospho-ACC   and pan 14-3-3 . All antibodies were used at a dilution of 1:1000. A common loading control was included on every gel to account for variation in relative band intensities between different blots. Antibody-antigen binding was detected using ECL Reagent  and imaged on a Fujifilm Las-4000 CCD camera. Bands were quantified by densitometry with Fuji ImageQuantTL software. Full images of blots in Fig. g at 4 °C. Supernatants were used to determine the activity of CS and βHAD66. CS activity of extracts was measured spectrophotometrically in 100 mM Tris–HCl, pH 8.2, 300 mM acetyl CoA, 1 mM MgCl2, 1 mM EDTA, 0.1 mM 5,5′-dithio-bis (2-nitrobenzoic acid) (DTNB—ε 13.6 mM/cm at 412 nm) and 500 mM oxaloacetate, started by the addition of oxaloacetate. βHAD assays were conducted in 50 mM Imidazole buffer, pH 7.4, 1 mM EDTA, 0.15 mM NADH (ε 6.22 mM/cm at 340 nm) and 0.1 mM acetoacetyl-CoA, started by the addition of acetoacetyl-CoA.Liver and tibialis muscle were homogenized 1:19 (w/v) in 50 mM Tris-HCl, 1 mM EDTA, 0.1% Triton X-100, pH 7.4. The homogenates were subjected to three freeze-thaw cycles and centrifuged for 10 min at 7000 × PCR (40 cycles) was performed on a LightCycler 480 (Roche) using SensiFAST SYBR mastermix (Bioline). For quantification of CerS1 and CerS2 expression, total RNA was purified from mouse tissues with TRIzol reagent (Sigma-Aldrich), followed by DNase treatment (Promega), and reverse transcribed using random hexamer primers with FirstStrand cDNA synthesis kit (Roche), according to manufacturer’s instructions. CerS1 and CerS2 gene expression was determined using a standard curve of Ct against input cDNA, and normalised to 18s RNA. For mitochondrial genes and transcriptional regulators in SkM, total RNA was prepared with TRIzol, and oligo dT primer was used for reverse transcription. Relative gene expression was determined using the ΔΔCt method, with 36B4 and Rpl13 as the reference genes. Samples with excessively high Ct values (Ct > 30 for the housekeeping genes) were removed from data analysis  treatment. Relative levels of nuclear and mitochondrial DNA were determined by quantitative PCR on a LightCycler 480 (Roche) using SensiFAST SYBR mastermix (Bioline). Primers for quantification of genomic DNA were: 5′- GGCTGTATTCCCCTCCATCG-3′, and 5′- CCAGTTGGTAACAATGCCATGT-3′; and mitochondrial DNA: 5′- CCCAGCTACTACCATCATTCAAGT-3′, and 5′- GATGGTTTGGGAGATTGGTTGATGT-3′P values were adjusted for multiple comparisons in GraphPad PRISM.HEK293 cell culture experiment: Levels of each lipid in P053-treated HEK293 cells were normalised to the mean of the vehicle control group, and each concentration was compared to the control using ANOVA with Dunnett’s post-test. t-tests adjusted for multiple comparisons using GraphPad PRISM . A single outlier was identified using Grubbs test with high stringency (P < 0.001) and removed from the quadriceps sphingosine measurements.Lipidomic analysis of mouse tissue samples: Three comparisons were made for each lipid measured: chow control vs chow + P053, chow control vs HFD control, and HFD control vs HFD + P053. Data were log transformed, then subjected to two-tailed P values for the main effect of diet  and drug  are reported. Where post-tests were applied, Fisher’s Least Significant Difference test was used.Physiological and biochemical measures: Total ceramide, total sphingosine, and total TAG in SkM, western blotting and gene expression data, and physiological measures, were assessed by two-way ANOVA with diet as one variable and drug as the other. Correlations: Spearman analysis was used to assess correlations between SkM ceramides and adipose tissue mass or % body fat.Lipidomic datasets for SkM and liver are provided in Supplementary Data File 2. Data used for bar graphs is provided in Supplementary Data File 1. Other raw data files supporting the results and conclusions presented in this manuscript are available from the corresponding authors upon reasonable request.Supplementary InformationPeer Review FileDescription of Additional Supplementary InformationSupplementary Data File 1Supplementary Data File 2


This study provides a novel oxygen-sensing process that determines the substrate specificity of NAA10 depending on an ambient oxygen tension.The N-terminal acetyltransferase A (NatA) complex, which is composed of NAA10 and NAA15, catalyzes N-terminal acetylation of many proteins in a co-translational manner. Structurally, the catalytic subunit NAA10 was believed to have no activity toward an internal lysine residue because the gate of its catalytic pocket is too narrow. However, several studies have demonstrated that the monomeric NAA10 can acetylate the internal lysine residues of several substrates including hypoxia-inducible factor 1α (HIF-1α). How NAA10 acetylates lysine residues has been an unsolved question. We here found that human FIH (factor inhibiting HIF) hydroxylates human NAA10 at W38 oxygen-dependently and this permits NAA10 to express the lysyl-acetyltransferase activity. The hydroxylated W38 forms a new hydrogen-bond with A67 and widens the gate at the catalytic pocket, which allows the entrance of a lysine residue to the site. Since the FIH-dependent hydroxylation of NAA10 occurs oxygen-dependently, NAA10 acetylates HIF-1α under normoxia but does not under hypoxia. Consequently, the acetylation promotes the pVHL binding to HIF-1α, and in turn HIF-1α is destructed  The Asn803 hydroxylation blocks the p300/CBP recruitment to CAD, thereby repressing the HIF-1-driven gene expression 22.12 at 37 °C. The ambient oxygen level was 20% for normoxia, 0.5% for hypoxia, or near 0.1% for anoxia.Human embryonic kidney (HEK293 and HEK293T), human breast cancer (MDA-MB-231) cell lines were obtained from American Type Culture Collection . HEK293 and HEK293T cells were cultured in DMEM, MDA-MB-231 in RPMI1640, supplemented with 10% heat-inactivated fetal bovine serum (FBS). Cells were grown in a humidified atmosphere containing 5% CO2.2Culture media, FBS, dimethyloxalylglycine (DMOG), and antibodies against FLAG-tag , and HA-tag  were purchased from Sigma-Aldrich . Antibodies against FIH , GST , β-tubulin , Lamin-B , VHL  and HRP-conjugated goat anti-rat secondary antibody  were from Santa Cruz Biotechnology . HRP-conjugated goat anti-rabbit , HRP-conjugated goat anti-mouse , Alexa Fluor 488 rabbit anti-goat  and Alexa Fluor 594 goat anti-rat  secondary antibodies were purchased from Invitrogen ; anti-acetylated lysine antibody  from Cell Signaling Technology ; anti-His(6)-tag antibody  from MBL . Anti-HIF-1α (1:1000) and anti-NAA10 antibodies  were generated in rabbits, as previously described 2.3The NAA10, FIH and HIF-1α plasmids were constructed, as previously described 2.4Proteins were separated on SDS-polyacrylamide gels and transferred to Immobilon-P membranes . The membranes were blocked with 5% skim milk, incubated overnight at 4 °C with a primary antibody, and incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h, and visualized using the ECL-plus kit . To analyze protein interactions, cell lysates were incubated with anti-FIH, anti-NAA10, or IgG overnight at 4 °C, and the immune-complexes were pulled down with protein A/G beads (Santa Cruz Biotechnology). Otherwise, cell lysates were incubated with EZview Red anti-HA or anti-FLAG affinity gel (Sigma-Aldrich) at 4 °C for 4 h. The bound proteins were eluted in a denaturing SDS sample buffer or HA/FLAG-tag peptides, and loaded on SDS-PAGE. All Western blotting experiments were performed three or more times.2.5g for 5 min and the pellet was lysed in a buffer containing 10 mM Tris-HCl/pH 7.4, 10 mM KCl, 1 mM EDTA, 1.5 mM MgCl2, 0.2% NP-40, 0.5 mM dithiotheritol, 1 mM sodium orthovanadate, and 400 μM PMSF. The cell lysates were separated into pellet (for nuclear fraction) and supernatant (for cytosolic fraction) using centrifugation at 1000 ×g for 5 min. One packed volume of a nuclear extraction buffer  was added to the pellet, and vortexed intermittently at low speed on ice for 30 min. The nuclear and cytosolic factions were spun at 20,000 ×g for 10 min and stored at −70 °C.Cells were centrifuged at 1000 ×2.6Cells were fixed with 3.7% formaldehyde for 10 min and permeabilized with 0.1% Triton X-100 for 30 min. Cells were incubated in PBS containing 0.05% Tween‐20 and 3% bovine serum albumin for 1 h, and further incubated overnight at 4 °C with a primary antibody. Cells were incubated with Alexa Fluor 488 or Alexa Fluor 594-conjugated secondary antibodies for 1 h. To stain nuclei, cells were incubated with DAPI (Sigma-Aldrich) for 30 min, and mounted in Faramount aqueous mounting medium . Immunostained cells were observed under Carl Zeiss LSM510 META confocal microscope.2.7Escherichia coli BL21 cells, pulled down using glutathione-affinity beads  at 4 °C for 1 h, and eluted with 10 mM reduced glutathione (Sigma-Aldrich). Recombinant His(6)-NAA10 WT and W38F proteins were expressed in BL21 cells, bound to Nickel-NTA affinity beads  at 4 °C for 1 h, and eluted with 250 mM imidazole. The amounts and purities of extracted proteins were checked by SDS-PAGE and Coomassie Brilliant Blue R-250 staining.Recombinant GST-FIH, GST-ODDD, GST-CT, and free GST proteins were expressed in 2.82, 0.5 mM dithiotheritol, 0.1% NP‐40, and 10% glycerol) at 4 °C for 1 h, and further incubated with glutathione or nickel affinity beads at 4 °C for 1 h. After the beads were washed with the binding buffer, bound proteins were eluted in the denaturing SDS sample buffer and subjected to Western blotting.The mixtures of GST-FIH and His-NAA10 WT (or W38F) were incubated in a binding buffer  in a hydroxylation buffer  at 37 °C for 1 h. His-NAA10 was electrophoresed, digested in gel, and analyzed by LC-MS/MS. For hydroxylation assay of HIF-1α CT, an acetyltransferase reaction was performed with GST-FIH and His-NAA10 at 37 °C for 1 h, followed by in vitro hydroxylase reaction with GST-HIF-1α CT and hydroxylation cofactors at 37 °C for 1 h. GST-HIF-1α CT was electrophoresed and immunoblotted with anti-N803(OH) HIF-1α antibody 2.10in vitro hydroxylated NAA10, recombinant His-NAA10, which was reacted as described in the previous paragraph, was separated by SDS-PAGE. Proteins in gel slices were digested with trypsin and loaded to Easy n-LC . Samples were separated on a C18 nanobore column . The mobile phase A for LC separation was 0.1% formic acid, 3% acetonitrile in deionized water and the mobile phase B was 0.1% formic acid in acetonitrile. The chromatography gradient was designed for a linear increase from 5% B to 55% B in 40 min, 52% B to 75% B in 4 min, 95% B in 4 min, and 3% B in 6 min. The flow rate was maintained at 1500 nL/min. Mass spectra were analyzed using LTQ Orbitrap XL mass spectrometer (Thermo Fisher) equipped with a nano-electrospray source. Mass spectra were acquired using data-dependent acquisition with a full mass scan (350–1200 m/z) followed by 10 MS/MS scans. For MS1 full scans, the orbitrap resolution was 30,000 and the AGC was 2 × 105. For MS/MS in the LTQ, the AGC was 1 × 104. The hydroxylation was identified by the additional mass of 16 Da on digested peptides.For LC-MS/MS analysis of intracellular NAA10 protein, HEK293 cells, which had been transfected with the FLAG/SBP-NAA10 plasmid, were treated with PBS or 500 μM DMOG for 24 h. FLAG/SBP-NAA10 was purified using FLAG-affinity beads and separated by SDS-PAGE. For LC-MS/MS analysis of https://data.mendeley.com/datasets/9b6xh36f3p/draft?a=f7ec8dac-5b5d-407b-b980-af2e78ab57f1LC-MS/MS data files have been published in Mendeley Data and are available at 2.112 capture assay, as previously described 4, and 5 μl of [1–14C]-labeled 2-oxoglutarate with a specific activity of 58.7 mCi/mmole  in a reaction buffer which contains 20 mM Tris-HCl/pH7.5, 0.5 mM dithiotheritol, 0.1% BSA, and 150 mM NaCl. A filter paper soaked in 30 mM Ca(OH)2 solution was immediately placed in the airtight screw-cap microcentrifuge tube. Each reaction was timed individually at 37 °C for 20 min and terminated by removal of filters. The hydroxylated level of NAA10 was measured by quantifying [14C]-labeled CO2 captured by the air dried filter paper using a liquid scintillation counter.The dioxygenase activity of FIH was measured using the CO2.124 in the hydroxylation buffer at 37 °C for 30 min. After the reaction, GST-HIF-1α ODDD was added to the reaction mixture with the acetylation buffer as total volume of 60 μl, and further reacted with 200 μM [14C]-labeled acetyl CoA (60 mCi/mmole) and 200 μM unlabeled acetyl CoA at 37 °C for 2 h. The acetylated GST-HIF-1α ODDD was pulled down using glutathione affinity beads and its radioactivity was measured by a liquid scintillation counter.GST-FIH (0.5 μg) was incubated with His-NAA10 (0.5 μg) in an acetylation buffer  at 37 °C for 2 h. GST-FIH was pulled down using glutathione affinity beads, and subjected to Western blotting using anti-acetyl lysine antibody. For radio-isotopic assay, the mixture (20 μl) of 0.5 μg of His-NAA10 WT (or W38F) and 0.5 μg of GST-FIH were incubated with 1 mM α-ketoglutarate, 1 mM ascorbate, and 100 μM FeSO2.13All-atom atomistic MD models were used to simulate heterodimeric NAA10/NAA15 and monomeric NAA10 systems. The initial protein structures were obtained from the protein data bank (PDB ID code: 5nnp) 2.14BNIP3; CCTTTGAGGAAAATTGACAG and AACCCTCTAGGGAATACAGC for human PDK1; GGAAGAAAACAGTGCCTATG and GTGTAGTCAGAGACCCCTCA for human CA9; GAGTCAACGGATTTGGTCGT and TTGATTTTGGAGGGATCTCG for human GAPDH.Total RNAs were extracted using TRIZOL , and reverse-transcribed in a reaction mixture containing M-MLV Reverse Transcriptase , RNase inhibitor, dNTPs, and random primers at 42 °C for 60 min. Quantitative real-time PCR on 96-well optical plates was performed in the qPCR Mastermix , and fluorescence emitting from dye-DNA complex was monitored in CFX Connect Real-Time Cycler . The mRNA values of targeted genes were calculated relative to GAPDH expression for each sample. All reactions were performed as triplicates. The nucleotide sequences (5′ to 3′) of PCR primers are; TTCTGAAAGTTTTCCTTCCA and TGTTGCAAGCTCAGAAGTAA for human 2.15t-test was used to compare the results from in vitro assays, densitometry analyses of immunoblots, and quantitative RT-PCR. All statistical significances were considered when P values were less than 0.05.All data were analyzed using the Microsoft Excel 2013 or GraphPad Prism 5.0 software, and results were expressed as the means and standard deviation. The unpaired, two-sided Student's 33.1in vitro binding analysis, recombinant His-NAA10 and GST-FIH proteins were pulled down together by glutathione affinity beads  residue alone was identified to be specifically hydroxylated by FIH , and a loop between β6 and β7 (Lβ6-β7). The cavity-like shape is optimized for a specific accommodation of the N-terminal end of its substrates amino acids. In addition, Lβ6-β7 and Lα1-α2 are placed at the entrance gate for target amino acids. Because the interloop distance is as narrow as 4 Å, NAA10 is not expected to catalyze lysine having a long side chain α1-α2, we tested the possibility that the substrate-binding site gets changed in shape by the W38 hydroxylation. We employed an atomistic molecular dynamics (MD) model for simulating NAA10/NAA15 dimer and NAA10 monomer. The analyses of the dictionary of protein secondary structure (DSSP)    and the e (DSSP)  and F reon (RDF) B. The suo Lα1-α2  and H. T± 1.2 Å) C. Upon c± 1.2 Å) D, the hyoove one E. It is doi:10.1016/j.redox.2018.09.002.Supplementary material related to this article can be found online at Molecular dynamics (MD) simulation of W38-hydroxylated NAA10. The substrate gate opening by W38 hydroxylation was simulated using GROMACS program. Acetyl CoA was presented as skeletal molecular model and catalytic pocket of NAA10 as ribbon diagram. Lα1-α2 and Lβ6-β7 were presented as green and blue loops, respectively. α2 helix was presented as yellow ribbon and β3 sheet was presented as orange ribbon. The interloop distance between Lα1-α2 and Lβ6-β7 becomes larger when W38 is hydroxylated. The movie is related to main Molecular dynamics simulation of non-hydroxylated NAA10. The substrate gate of non-hydroxylated NAA10 was simulated using GROMACS program. Acetyl CoA was presented as skeletal molecular model and catalytic pocket of NAA10 as ribbon diagram. Lα1-α2 and Lβ6-β7 were presented as green and blue loops, respectively. α2 helix was presented as yellow ribbon and β3 sheet was presented as orange ribbon. The interloop distance between Lα1-α2 and Lβ6-β7 of non-hydroxylated NAA10 is narrow than of W38-hydroxylated NAA10. The movie is related to main The following is the Supplementary material related to this article 3.5in vitro only by FIH-hydroxylated NAA10, but not by the NAA10 W38F mutant  of proteins plays a significant role in regulating cellular processes. Several reports showed that PTM of proteins are largely regulated in response to chronic sustained and intermittent forms of hypoxic stress in vitro, several research groups refuted this hypothesis in vitro assay, it is obvious that NAA10 did not acetylate HIF-1α. After NAA10 is hydroxylated by FIH, it gets a new ability to acetylate HIF-1α even in vitro, which may be the answer to this question. The other is how the HIF-1α acetylation is regulated in an oxygen-dependent fashion. As a mechanism underlying this regulation, the downregulation of NAA10 under hypoxia was suggested In respect of the oxygen-dependent regulation of HIF-1α stability by NAA10, two questions have not been answered for two last decades. One is the acetyltransferase activity of NAA10 for HIF-1α. Based on biochemical data showing that NAA10 failed to acetylate HIF-1α We here propose that the FIH-mediated W38 hydroxylation widely opens the entrance gate to admit the lysine into the pocket. This molecular dynamics model was also verified by biochemistry- and cell biology-based experiments for HIF-1α. In view of molecular dynamics, other substrates, which were previously known to be lysyl-acetylated by NAA10 FIH was originally identified to hydroxylate HIF-1α at N803 In conclusion, our results suggest that the FIH-mediated hydroxylation of NAA10 at W38 is a critical step in the oxygen-dependent regulation of HIF-1α stability through lysyl acetylation. This is a novel oxygen-sensing process that modulates the substrate specificity of enzyme depending on ambient oxygen tension.


Scientific Reports 10.1038/s41598-018-30559-7, published online 13 August 2018Correction to: This Article contains an error in the Methods section.3 and protein concentration less than 40 mg/dl);”“(2) an abnormal CSF (i.e. reactive VDRL and/or greater than 4 leukocytes/mmshould read:3 and protein concentration greater than 40 mg/dl);”“(2) an abnormal CSF (i.e reactive VDRL and/or greater than 4 leukocytes/mm


N,N-diethylformamide)dizinc(II)], built from windmill-type secondary building units and forming zigzag shaped two-dimensional stacked layers, has been solvothermally synthesized from naphthalene-2,6-dicarboxylic acid and zinc(II) acetate as the metal source in N,N-diethylformamide containing small amounts of formic acid.The zinc metal organic framework poly[bisdizinc(II)], [Zn(C12H6O4)(C15H11NO)]n, built from windmill-type secondary building units and forming zigzag shaped two-dimensional stacked layers, has been solvothermally synthesized from naphthalene-2,6-dicarboxylic acid and zinc(II) acetate as the metal source in N,N-diethylformamide containing small amounts of formic acid.A zinc metal–organic framework, namely poly[bis( N,N-diethylformamide (DEF), under solvothermal conditions, can be crucial in steering the production of new MOF  structures, as exemplified by the formation of two new zinc–terephthalate MOFs based on the trinuclear Zn3(O2CR)6 secondary building unit (SBU) and containing the formate anion, solvothermally obtained from the well-studied MOF-5 system Zn/H2BDC/DEF  in the presence of small amounts of added formic acid  is considered to further study the influence of added formic acid in DEF in MOF synthesis. The NDC2− ligand has been widely used previously to prepare a number of MOFs 2·6H2O in DEF using a common solvothermal route  acetate as the metal source instead of zinc(II) nitrate, we successfully isolated a new 2D coordination network, [Zn(NDC)(DEF)]n (1), identified by satisfactory elemental analysis and single-crystal X-ray diffraction.In a preceding study, we showed how the presence of a small amount of added organic acids in the solvent 1 crystallizes in the triclinic P2+ cation, one fully deprotonated NDC2− ligand and a Zn-coordinated DEF molecule. Each ZnII ion is pentacoordinated by five O atoms  from three individual NDC2− anions and one DEF molecule in a tetragonal pyramidal configuration. The SBU consists of doubly-bridged dinuclear units of ZnII atoms in a ‘windmill’ fashion  or a μ2-η1:η1 (O3 and O5) coordination mode.Complex on Fig. 1, with a le Fig. 2. The two1 shows a three-dimensional (3D) supramolecular framework built of zigzag-shaped two-dimensional (2D) stacked layers. Neighbouring 2D layers are interconnected through nonclassical hydrogen-bonding interactions between carboxylate O atoms (O1 and O3) and β-H atoms of NDC2− ligands with COO⋯H—Cβ—NDC distances of 3.307 (4) (O1—C4) and 3.548 (4) Å (O3—C12). Other interactions contributing to the stability of the framework involve Hcentroid–π interactions of H16—C16 (DEF hydrogens) and the centroids  of the aromatic rings of the NDC2− ligands, with Cg⋯H distances of 2.99 Å , including 1,4-, 1,8- and 2,6-NDC, have been, due to their stability, richness in coordination modes and structural rigidity, widely used as organic molecules in the synthesis of novel MOF structures with a variety of metal ions, such as ZnII, CdII, CoII, NiII, MnII or AgI. Among all the 2,6-NDC/Zn-based MOFs, two are closely related to MOF 1, i.e. a MOF of formula [Zn22(DMF)2]n , and MOF-105 and its derivatives of generic formula [Zn22(DMF)2] 4]. For MOF 1, the two carboxylate groups of the same NDC2− ligand adopt either a μ1-η1:η1 (O1 and O2) or a μ2-η1:η1 (O3 and O5) coordination mode, giving an uncommon pw2 paddle-wheel (‘windmill’) structural feature, [M2(CO2)2].Naphthalene dicarboxylic acid derivatives  acetate. 2,6-H2NDC  and Zn(OAc)2·2H2O  were dissolved in DEF (10 ml) containing formic acid  and sealed in a glass vial. The vial was heated in an oven to 110 °C for 17 h. After cooling to room temperature, the reaction was allowed to stand until colorless crystals suitable for X-ray diffraction formed. For further characterizations, the crystals were collected by filtration, washed with DEF several times, and dried at 373 K under vacuum. Elemental analysis (%) for C17H17NO5Zn based on the formula [Zn(NDC)(DEF)] found : C 53.00 (53.63), H 4.47 (4.50), N 3.39 (3.68), Zn 17.51 (17.17). FT–IR (cm−1): 2979, 2938, 1647, 1602, 1586, 1557, 1494, 1460, 1406, 1385, 1361, 1348. The identity of the as-synthesized bulk material was confirmed by comparing the powder X-ray diffraction (PXRD) pattern with that simulated from the crystal structure . Elemental analysis (%) for C12H6O4Zn based on the formula [Zn(NDC)] found : C 48.85 (51.56), H 2.75 (2.16), N 0.22 (0.00), Zn 21.47 (23.39). It should be noted that after removal of DEF, MOF 1 lost its crystallinity, as evidenced by the PXRD pattern.MOF re Fig. 6. After h3), 0.99 (CH2) or 1.0 Å (CH), with Uiso(H) = 1.5Ueq(C) for methyl H atoms or 1.2Ueq(C) otherwise. Crystal data, data collection and structure refinement details are summarized in Table 2The ethyl groups of DEF were disordered over two positions, for which the occupancies were refined, converging to 0.51 and 0.49. The SAME, DELU and SIMU restraints were applied to model the disorder  global. DOI: 1959604, 1959604CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Scoparia dulcis showing symptoms caused by root-knot nematodes were detected in the municipality of Cachoeira do Sul, Rio Grande do Sul State, Brazil. Based on the morphological, esterase phenotypes, and molecular analyses of the mitochondrial DNA region between the cytochome oxidase subunit II and 16S rRNA genes (mtDNA) and species-specific sequence characterized amplified region (SCAR), the causal agent of the observed symptoms was identified as Meloidogyne javanica. Pathogenicity was confirmed by fulfilling a modified version of Koch’s postulates. To our knowledge, this is the first report of M. javanica infecting S. dulcis in Brazil.Medicinal plants  Scoparia dulcis L. (Scrophulariaceae), commonly known as sweet broom, is a perennial plant, widely distributed in tropical and subtropical regions, especially in flooded environments  (n = 40) and females (n = 20), and perineal patterns (n = 20); and molecular characterization of the mitochondrial DNA region between the cytochome oxidase subunit II (COII) and 16S rRNA genes (mtDNA) using the primers C2F3 (5´-GGTCAATGTTCAGAAATTTGTGG-3´) and 1108 (5´-TACCTTTGACCAATCACGCT-3´)  ; morpholCGCT-3´)  along wiCGCT-3´) .M. javanica  Chitwood, 1949 are in agreement with the original description  = 414.5 ± 36.1 (345.0–470.5) µm, a = 33.7 ± 2.8 (28.1–32.5), c = 8.4 ± 0.9 (6.3–10.1), stylet length = 11.5 ± 1.2 (10.1–14.2) µm, dorsal esophageal gland orifice (DGO)  = 3.5 ±  0.4 (3.0–4.6) µm, tail length = 51.1 ± 3.3 (45.8–54.4) µm and hyaline tail terminus = 11.5 ± 1.7 (9.1–14.7) µm; and females L = 850.5 ± 90.5 (640.5–995.5) µm, stylet length = 14.5 ± 0.8 (12.5–15.8) µm, and DGO = 3.4 ± 0.7 (2.8–4.2) µm. The perineal patterns showed a low dorsal arch, trapezoid shape, smooth striae interrupted by a pair of incisures on both sides corresponding to lateral fields, clearly demarcated from striae by parallel lines, tail whorl often distinct. The overall morphology and morphometrics of the population of cription .M. javanica (M. javanica isolates from South Africa (GenBank JX987326), Taiwan (GenBank JX100439), Turkey (GenBank LT602894), and Vietnam (GenBank MK033441). The PCR amplification using SCAR technique produced a specific fragment of expected size (∼670 bp) for M. javanica  typical of javanica . The mtDjavanica .S. dulcis plantlets were maintained in pots with 2,000 dm3 sterilized soil. In total, 10 replicates were inoculated with 5,000 eggs and J2s from the original population of M. javanica, in addition to a non-inoculated control. Plants were well maintained under greenhouse conditions at 25 to 27°C. After 55 days, the inoculated plants exhibited galled root systems similar to plants observed in the field, with a nematode reproduction factor  of 24.5. The non-inoculated plants did not exhibit any galls. The morphological and molecular characterization of this re-isolated root-knot nematode were identical those of M. javanica.In greenhouse tests, M. javanica parasitising S. dulcis in Brazil. Globally, M. javanica is considered an economically important agricultural nematode reported over 770 plant species, including tea [(Camellia sinensis (L.) Kuntze], grape (Vitis sp. L.), many vegetables, fruit trees, weeds, cereals, and ornamentals, causing severe yield losses (S. dulcis, grown in tropical, subtropical, and temperate regions.This is the first report of d losses . Hence,


The title compound, an example of a stable 1,2-naphthoquinone, illustrates steric buttressing of the adamantanyl group. 20H21NO2, an example of a stable 1,2-naphthoquinone, was determined by single-crystal X-ray diffraction analysis at 100 K. This structure illustrates steric buttressing of the adamantanyl group, forcing the N—H group into the coplanar aromatic C—H. The presence of strong delocalization between the planar N atom at the 4-position and the carbonyl group at the 2-position is indicated. In the crystal, C—H⋯O and C—H⋯π interactions link the molecules into a three-dimensional network.The title compound, C The C10—C11—C2 angle of 117.8 (2)°, C10—C11—C2 angle of 117.9 (2)° and C2—C3—C4 angle of 123.5 (2)° show the largest deviations from the ideal value of 120°. The aromatic ring is planar, as expected, and has internal bond angles that range from 117.9 (2) to 120.9 (2)°.In the crystal structure of the title compound Fig. 2, molecuet al., 2016et al., 2017et al., 2017N-methylaniline), which has a completely different structure from ZARNOY: the nitrogen is not planar, the amino moiety is twisted with respect to the naphthoquinone plane and the C4—N bond distance is greater in the case of the secondary amine. The authors summarize the differences between the structures and rationalize these differences using the concept of tautomerization  in the structure with the primary amine.A search of the Cambridge Structural Database . A cellulose extraction thimble with Li2CO3 was placed above the reaction mixture. This solution was stirred at 393 K under a nitrogen atmosphere for 5 h. After being allowed to cool to room temperature, the dark-brown solution was diluted with toluene (30 mL), filtered and concentrated under reduced pressure. A brownish-red solid (503 mg) of the crude product was obtained. The crude product was further purified by column chromatography using silica gel with a gradient solvent elution [100% dichloromethane (CH2Cl2) and then dichloromethane/2-propanol ]; the fractions were dried under vacuum to yield 72 mg of a dark-orange solid product (47% yield), determined pure by NMR analysis. Part of the purified product was re-dissolved in heptane and cooled to 203 K for crystallization. Red crystalline blocks suitable for X-ray analysis were obtained, m.p. 522 K (decomposition) determined using a Fisher–Johns melting-point apparatus with calibrated thermometer. 1H NMR  δ 8.20–8.21 , 7.66–7.69 , 7.58–7.61 , 7.39–7.40 , 6.20 , 5.47 , 2.22 , 2.16 , 1.73–1.79 .The synthesis of 4-[(adamantan-1-yl)amino]naphthalene-1,2-dione is based on a new procedure (complete publication in progress). In a reaction tube were mixed 740 mg (2.00 mmol) of 1,2-naphthoquinone-4-sulfonic acid cesium salt, 76 mg  of adamantan-1-amine, and 302 mg  of tetrabutylammonium acetate. The solids were dissolved in Uiso(H) = 1.2 Ueq(N). All other H atoms were placed geometrically and refined using a riding-atom approximation, with C—H = 0.95–1.00 Å, and with Uiso(H) = 1.2Ueq(C).Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989018017917/rz5250sup1.cifCrystal structure: contains datablock(s) global, I. DOI: 10.1107/S2056989018017917/rz5250Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989018017917/rz5250Isup3.cmlSupporting information file. DOI: 1876987CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Capable of broad polyclonal responses to pyrophosphate antigens (pAg), they are implicated in immunity to a diverse range of infections. Previously Vγ9Vδ2+ development was thought to involve postnatal selection and amplification of public Vγ9 clonotypes in response to microbial stimuli. However, recent data indicate the Vγ9Vδ2+ T-cell receptor (TCR) repertoire, which is generated early in gestation, is dominated by public Vγ9 clonotypes from birth. These chains bear highly distinct features compared to Vγ9 chains from Vδ1+ T-cells, due either to temporal differences in recombination of each subset and/or potentially prenatal selection of pAg-reactive clonotypes. While these processes result in a semi-invariant repertoire featuring Vγ9 sequences preconfigured for pAg recognition, alterations in TCRδ repertoires between neonate and adult suggest either peripheral selection of clonotypes responsive to microbial antigens or altered postnatal thymic output of Vγ9Vδ2+ T-cells. Interestingly, some individuals demonstrate private Vγ9Vδ2+ expansions with distinct effector phenotypes, suggestive of selective expansion in response to microbial stimulation. The Vγ9Vδ2+ T-cell subset, therefore, exhibits many features common to mouse γδ T-cell subsets, including early development, a semi-invariant TCR repertoire, and a reliance on butyrophilin-like molecules in antigen recognition. However, importantly Vγ9Vδ2+ T-cells retain TCR sensitivity after acquiring an effector phenotype. We outline a model for Vγ9Vδ2+ T-cell development and selection involving innate prenatal repertoire focusing, followed by postnatal repertoire shifts driven by microbial infection and/or altered thymic output.Vγ9Vδ2 Essentially all Vγ9Vδ2+ T-cells respond to small pyrophosphate antigens (pAg) (Vγ9Vδ2ns (pAg)  in a T-cns (pAg) . The popns (pAg) , typical+ T-cells dominate the γδ repertoire  gene segments are the first γ/δ chains to undergo rearrangement in development, detected in fetal liver from as early as 5–6 weeks gestation  diversity (D) segments , and typically used joining (J) segment Jδ1 , generated by germline Vγ9-JγP recombination with minimal nucleotide trimming and no N-nucleotide addition, was present in many healthy donors  do not reflect clonal expansion , but are likely recombined independently multiple times and pair with distinct Vδ2 chains. Single cell PCR in several individuals has substantiated the feasibility of this hypothesis, establishing unequivocally that public Vγ9 CDR3 clonotypes each paired with multiple Vδ2 clonotypes  in the fetus  , those present often have shorter CDR3s, incorporating fewer N-nucleotides [as observed in fetal liver   .+ compartment suggest similarities to mouse γδ T-cell subsets (+ production, combined with the semi-invariant Vγ9Vδ2+ TCR repertoire, mirrors early waves of semi-invariant mouse γδ T-cells. The first T-cells to develop in mouse fetal thymus are Vγ5Vδ1+ dendritic epidermal T-cells, which have limited junctional diversity in both TCR chains (+ T-cell recognition (+ T-cells. Notably Vγ9Vδ2+ T-cells remain responsive to both pAg and anti-CD3 stimulation, a feature which underlies their potential use in several cancer immunotherapy applications (Several features of the Vγ9Vδ2 subsets . The earR chains . This isR chains . Some ofR chains , 34; theR chains . AnotherR chains , 37, altognition . Howeverognition , this doications , and theications –44.+ T-cell response to pAg, the extent to which clonotype-specific responses occur remains unclear. Vγ9Vδ2+ T-cells expand in various infections (+ T-cell phenotype to CD45RAnegCD27neg (negCD45RO+CD27+ phenotype observed in most healthy donors (+ T-cells of differing effector phenotypes, although the clonality of Vγ9Vδ2+ T-cells was not examined. Expansion of particular Vδ2 clonotypes has also been noted in tuberculosis (+ clonotypes helps protect the host, given the polyclonal response of Vγ9Vδ2+ T-cells to pAg. Conceivably expanded clones could respond with higher avidity, or alternatively could be reactive to different pathogen-specific stimuli, such as chemically diverse antigens. Additional work will no doubt address these questions.Although clear evidence supports a broad polyclonal Vγ9Vδ2fections  but TCR fections , a minorfections , with thfections . These cgCD27neg , distincy donors  have alsrculosis , 48, humrculosis , and in rculosis . Public rculosis , however+ T-cell development is shaped by both prenatal and postnatal events (Figure + TCR repertoire is composed of highly public Vγ9 chains produced by frequent recombination events that occur in every individual, resulting in a semi-invariant repertoire largely preconfigured from birth for pAg reactivity. These Vγ9 chains may undergo prenatal selection based on pAg reactivity, or unknown factors may constrain Vγ9-JγP rearrangements. Alongside public Vγ9 sequences, the Vδ2 repertoire is very diverse and private, and changes between neonatal and adult Vδ2 TCR repertoires suggest several selection events throughout life. Vδ2-Jδ3 TCRs are prevalent in cord blood and these may be positively selected in fetal development for recognition of host pAgs, or these rearrangements may be preferentially generated in early gestation. Vδ2-Jδ1 chains with longer CDR3 and hydrophobic amino acids at position 5 ultimately dominate the Vδ2 repertoire in adults, and these may be selected from rare rearrangements in cord blood following microbial pAg exposure, or further Vγ9Vδ2+ T-cell generation may occur in the postnatal thymus. Nevertheless, these selection events produce a repertoire that exploits the somatically recombined Vγ9Vδ2+ TCR as a surrogate pattern recognition receptor to sense pAgs. Further clonal selection appears to occur in some healthy adults and during some infections, however, exactly what protection such favored clonotypes provide that are not provided already by the broad Vγ9Vδ2+ TCR repertoire is an intriguing question future studies can address.In summary, we suggest Vγ9Vδ2s Figure , which iCW, MD, and BW jointly conceived the concepts presented in this review. CW analyzed data, prepared figures, and wrote the first draft; MD prepared figures and helped finalize the manuscript; BW helped plan and write the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Context:Ferulago (Apiaceae) species have been used since ancient times for the treatment of intestinal worms, hemorrhoids, and as a tonic, digestive, aphrodisiac, or sedative, as well as in salads or as a spice due to their special odors.Objectives: This study reports the α-amylase and α-glucosidase inhibitory activities of dichloromethane extract and bioactive compounds isolated from Ferulago bracteata Boiss. & Hausskn. roots.Materials and methods: The isolated compounds obtained from dichloromethane extract of Ferulago bracteata roots through bioassay-guided fractionation and isolation process were evaluated for their in vitro α-amylase and α-glucosidase inhibitory activities at 5000–400 µg/mL concentrations. Compound structures were elucidated by detailed analyses (NMR and MS).Results: A new coumarin, peucedanol-2′-benzoate (1), along with nine known ones, osthole (2), imperatorin (3), bergapten (4), prantschimgin (5), grandivitinol (6), suberosin (7), xanthotoxin (8), felamidin (9), umbelliferone (10), and a sterol mixture consisted of stigmasterol (11), β-sitosterol (12) was isolated from the roots of F. bracteata. Felamidin and suberosin showed significant α-glucosidase inhibitory activity  when compared to the reference standard acarbose (IC50 4.95 mg/mL). However, none of the tested extracts were found to be active on α-amylase inhibition.Discussion and conclusions: The present study demonstrated that among the compounds isolated from CH2Cl2 fraction of F. bracteata roots, coumarins were determined as the main chemical constituents of this fraction. This is the first report on isolation and characterization of the bioactive compounds from root extracts of F. bracteata and on their α-amylase and α-glucosidase inhibitory activities. Ferulago W. Koch. (Apiaceae) is represented by approximately 50 taxa throughout the world and 35 taxa in Turkey, 18 of which are endemic. Therefore, Anatolia is considered to be the gene center of this genus , along with nine known ones (2–10) and a sterol mixture (11–12). The α-amylase and α-glucosidase inhibitory activities of isolated coumarins were also evaluated. Coumarins may be a potential source of new antidiabetic agents and may also be used by peripheral tissues by improving insulin resistance and increasing glucose uptake  and Sephadex LH-20 (Fluka). TLC was carried out on pre-coated Kieselgel 60 F254 aluminum sheets (Merck).NMR spectra were recorded on a Varian Mercury Plus at 400 MHz for F. bracteata were collected in July 2014 from Gaziantep  and identified by Prof. Dr. Hayri Duman, a plant taxonomist in the Department of Biological Sciences, Faculty of Art and Sciences, Gazi University. A voucher specimen (No AEF 26676) is stored in the Herbarium of Faculty of Pharmacy, Ankara University, Turkey.The roots of F. bracteata (450 g) were powdered and macerated three times with methanol for 8 h in a water bath not exceeding 45 °C (4 × 2 L) using a mechanical mixer at 300 rpm. Combined extracts were filtered and concentrated to dryness (60.94 g), then dispersed in methanol:water (1:9) and fractionated four times with 400 mL of dichloromethane, ethyl acetate, and n-butanol, respectively. Each fraction was concentrated to dryness, and 17.96 g dichloromethane, 2.44 g ethyl acetate, and 14.98 g of n-butanol fractions were obtained. Finally, 23.55 g of aqueous fraction remained. The roots (50 g) were grounded and macerated with 500 mL of distilled water for 8 h/3 days at 30 to 35 °C. This material was filtered and lyophilized to give aqueous extracts of 3.99 g roots.Air-dried roots of n-hexane:ethyl acetate  and ethyl acetate:methanol , and eight fractions (Fr. A–H) were obtained. Fr. A was subjected to a silica gel column, which was eluted with a mixture of n-hexane:ethyl acetate (95:5) and compounds 11 and 12 were obtained as a mixture (217 mg). Repetitive silica gel column chromatography with n-hexane:ethyl acetate (90:10 and 95:5) solvent system on Fr. B gave compound 2 (220 mg). Fr. C was applied to silica gel column eluting with n-hexane:ethyl acetate (85:15) and Sephadex LH-20 column eluting with ethyl acetate to give compounds 3 (125 mg) and 4 (130 mg). Eluting with n-hexane-ethyl acetate (90:10) over silica gel column of Fr. D gave compound 5 (400 mg) and Fr. E gave compounds 1 (320 mg), 6 (150 mg), and 7 (330 mg). Fr. F was eluted with 25% ethyl acetate in n-hexane and rechromatographed with 25% ethyl acetate in n-hexane on silica gel column to obtain compound 8 (110 mg). Fr. G was fractioned by column chromatography over silica gel using n-hexane:ethyl acetate mixtures (70:30 and 90:10) consecutively to obtain compound 9 (325 mg). Fr. H was submitted on a silica gel column using n-hexane:ethyl acetate (65:35) and the resulting fraction was chromatographed on silica gel column using n-hexane:ethyl acetate (90:10) to obtain compound 10.As a result of the bioguided fractionation study, the effective dichloromethane extract was first submitted to a silica gel column  and eluted with a gradient of νmax (KBr) cm−1: 1702, 1623, 1565; UV λmax (CH2Cl2) nm (log ɛ): 350 (4.20); 1 H NMR  and 13 C NMR  spectroscopic data, see m/z 367.1999 [M-H]+ .White powder; IR α-Amylase inhibitory activity was established in accordance with the reported method  was used as the substrate and was prepared in 0.1 M potassium phosphate buffer (pH 6.8). α-Glucosidase  was dissolved in the same buffer. Samples were dissolved in dimethyl sulfoxide (DMSO) and all samples (20 µL) together with the enzyme solution (20 µL) were mixed in the plate. Afterwards, the substrate (40 µL) was added for initiation of the reaction and the mixture was incubated at 37 °C for 40 min. After incubation is complete, 0.2 M sodium carbonate (80 µL) in phosphate buffer (pH 6.8) was added to all wells in order to quench the reaction. The amount of released p-nitrophenol (pNP) was measured at 405 nm using a 96-well microplate reader. Each assay for all samples was carried out in triplicate. Percentage inhibition of all samples was calculated using the following equation:α-Glucosidase inhibitory activity was established by using a 96-well microtiter plate in accordance with the described method  and the obtained fractions were evaluated for their α-amylase and α-glucosidase inhibitory activities. The active dichloromethane extract was subjected to column chromatography over silica gel and Sephadex LH-20. As a result, a new coumarin, peucedanol-2′-benzoate (1), together with nine known ones, osthole (2)      (8) , umbelliferone (10) , β-sitosterol (12)  , xanthot1) was isolated as a white powder with the molecular formula of C21H20O6 as determined by the HR-ESI-MS at m/z 367.1999 [M − H]+ . The IR spectrum of 1 showed absorption bands for C = O groups (1702 cm−1) and -CH = CH- bonds . The 1 H NMR spectrum of compound 1 displayed two AB type system protons at δH 6.26 and 7.64  which was attributed to the H-3 and H-4 protons of the coumarin nucleus. The two single aromatic proton signals at δH 7.28 and 6.80 were assigned to H-5 and H-8 protons. The 13 C NMR spectrum revealed the presence of 9 carbons resonances including four methine , three oxygenated quaternary , and two non-oxygenated quaternary carbons  for coumarin skeleton. Two tertiary methyl groups at δH 1.72 , 1.71  and at δC 22.16 (C-4′), 21.38 (C-5′) with the hydroxyl group; an oxygenated methine at δH 5.16  and at δC 89.12 (C-2′); and a methylene at δH 3.38  and at δC 29.67 (C-1′) confirmed the 2′,3′-dihydroxy-3′-methyl butyl moiety. HMBC correlation (δH 3.38) and C-6 (δC 124.55) suggested that it was attached to C-6 position. Characteristic signals of a benzoyl moiety were also exhibited, including a pair of 2 H at δH 7.73  and 7.32  and 1 H at 7.51 (H-5″) in the 1 H NMR spectrum and aromatic carbons at δC 131.04 (C-2″), 129.39 , 128.27 , 132.86 (C-5″) with a carbonyl carbon at δC 165.40 (C-1″) in the 13 C NMR spectrum. The linkage of the benzoyl group to the 2′,3′-dihydroxy-3′-methyl butyl moiety was deduced from the downfield shifted signal of H-2′ (δH 5.16) and C-2′ (δC 89.12). Thus, the structure of the compound 1 was characterized as peucedanol-2′-benzoate.Peucedanol-2′-benzoate  were given in 50 0.95 mg/mL) and among the tested compounds felamidin (IC50 0.42 mg/mL) possessed the best inhibitory activity which were more potent than acarbose (IC50 4.95 mg/mL). Suberosin, osthole, imperatorin, prantschimgin, peucedanol-2′ benzoate also showed α-glucosidase inhibitory activity  which had lower effect than felamidin but stronger than acarbose. Among the tested compounds bergapten, xanthotoxin, and umbelliferone  showed lower activity than reference compound acarbose. Grandivitinol (IC50 20.01 mg/mL) possessed the worst inhibitory activity. However, none of the extracts showed meaningful α-amylase inhibitory activity, while acarbose indicated 82.28% inhibition at a concentration of 1 mg/mL. These results indicate that felamidin was 11 times more effective than acarbose against α-glucosidase.The extracts and compounds F. bracteata and the isolated coumarins prantschimgin, peucedanol-2′-benzoate, felamidin, grandivitinol, and suberosin. Also, this is the first report on the phytochemical analysis of F. bracteata.To our knowledge, no previous studies have been reported on α-glucosidase and α-amylase inhibitory activities of 50 value of umbelliferone was found to be 7.79 ± 0.11 μg/mL, we have found a higher inhibitory activity with 9.32 mg/mL . Luo et al.  of action. Natural products are still considered as potential sources for drug exploration and play a significant role in drug development programs. Furthermore, many medicinal plants could be rich sources of bioactive chemicals that are importantly free from undesired adverse effects and show powerful pharmacological activities.Our results are similar to the previous studies performed on related coumarins. Shalaby et al.  found tho et al.  reportedo et al. . Medicalo et al. . One of o et al. . The tes2Cl2 fraction of Ferulago bracteata roots, coumarins were determined the main chemical constituents of this fraction. The most potent compounds found were felamidin and suberosin, respectively. Thus, F. bracteata along with its isolated compounds could be used for further studies on the development of novel preventive or therapeutic agents for the treatment of diabetes.In conclusion, the present study demonstrated that among the compounds isolated from CH