heterologously expressed could be identified in the culture broth extracts from the bacteria that harbored that hm-NAS gene(Extended Data Fig. 1) GPCR screen of N-acyl amide small molecules For each of the 6 N-acyl amide families(1-6) the analog produced at the highest level in our heterologous expression experiments was assayed for GCPR activity In the case of family 4 the major lysine analog(N-3-hydroxyoleoyl lysine) was also screened. For family I the major glycine analog(N-3-hydroxypalmitoyl glycine)was previously screened. Using B- arrestin cell-based assays at 10 uM ligand concentration, agonist and antagonist activity was assessed by DiscoveRx Corporation against 168 GPCRs with known ligands as well as 72 orphan GPCRs. The most potent interactions between N-acyl amides and GPCRs were validated by repeating the assay in duplicate and generating dose response curves. Synthetic N-acyl amides were assayed in the same fashion. HEK293 cells expressing GPr119 were exposed to equimolar concentrations of N-palmitoyl serinol or OEA. CAMP was used as an 于9之 orthogonal assay to measure GPR119 activation. cAMP was measured in HEK293 cells engineered to express a cAMP sensitive ion channel that permits cAMP measurement in live cells by monitoring calcium flux(ACTOne cells, Codex Biosolutions ). +I Calcium eflux was measured using the ACTone membrane potential kit( Codex Biosolutions CB-80500-201) For our analysis ACTOne HEK293 cells transfected with GPR119 (ACTOne-GCPR 119)were compared to cells not transfected with GPr119. Each cell line was exposed to equimolar concentrations of OEA, N-oleoyl serinol or [5-(N- Ethy lcarboxamido )adenosine] which stimulates GPCR ADORA2B in the parental HEK293 cell line. For a reference to the quality control of each GPCR reporter cell line from Discoverxpleaserefertohttps://www.discoverx.com/targets/ class=gpcr&pcat=stable%20cell%20lines&readout=arrestinorCodexBiosolutionshttp:// codexbiosolutions. com/actone cell lines. php Synthesis of proteinogenic amino acid containing N-acyl-palmitoyl analogs Wang resins with preloaded amino acids were purchased from Matrix Innovation. Coupling reagents(Py BOP and Cl-HOBt)were purchased from P3 BioSystems Palmitoyl chloride and all other reagents were purchased from Sigma-Aldrich. Dimethylformamide(dmf )was added to preloaded Wang resins(-80 mg)and incubated for 30 minutes. Removal of N Fmoc from swollen resins was accomplished by two rounds of piperidine treatment [20% solution in DMF(V/v), 3 ml] for 3 and 10 minutes, followed by several washes with DME Palmitoyl chloride(l equivalent)in DMf was then added and the resin suspension was shaken for 2 hours at room temperature. The N-acylated amino acid product was cleaved from the resins by treatment with trifluoroacetic acid (TFA)supplemented with 2.5%(v/ water and 2.5%(v/v) triisopropylsilane(TIPs). After evaporation of TfA the crude product was purified by automated reversed phase flash chromatography (Teledyne Isco, C18 RediSep RF Gold m 15 g), binary solvent system: water and acetonitrile supplemented with 0. 1% acetic acid. All final products were verified by MS (Supplementary Table 3) In-vitro study of GLP-1 release from GLUTag cells Oleoylethanolamide(oEa)and 2-oleoyl glycerol (2-OG)were purchased from Cayman Chemical Company and resuspended in DMsO to a concentration of 10 mM. N-oleoyl Nature. Author manuscript; available in PMC 2018 February 28
heterologously expressed could be identified in the culture broth extracts from the bacteria that harbored that hm-NAS gene (Extended Data Fig. 1). GPCR screen of N-acyl amide small molecules For each of the 6 N-acyl amide families (1–6) the analog produced at the highest level in our heterologous expression experiments was assayed for GCPR activity. In the case of family 4 the major lysine analog (N-3-hydroxyoleoyl lysine) was also screened. For family 1 the major glycine analog (N-3-hydroxypalmitoyl glycine) was previously screened.3 Using β- arrestin cell-based assays at 10 µM ligand concentration, agonist and antagonist activity was assessed by DiscoveRx Corporation against 168 GPCRs with known ligands as well as 72 orphan GPCRs. The most potent interactions between N-acyl amides and GPCRs were validated by repeating the assay in duplicate and generating dose response curves. Synthetic N-acyl amides were assayed in the same fashion. HEK293 cells expressing GPR119 were exposed to equimolar concentrations of N-palmitoyl serinol or OEA. cAMP was used as an orthogonal assay to measure GPR119 activation. cAMP was measured in HEK293 cells engineered to express a cAMP sensitive ion channel that permits cAMP measurement in live cells by monitoring calcium eflux (ACTOne cells, Codex Biosolutions).41 Calcium eflux was measured using the ACTone membrane potential kit (Codex Biosolutions CB-80500-201). For our analysis ACTOne HEK293 cells transfected with GPR119 (ACTOne-GCPR 119) were compared to cells not transfected with GPR119. Each cell line was exposed to equimolar concentrations of OEA, N-oleoyl serinol or [5–(NEthylcarboxamido)adenosine] which stimulates GPCR ADORA2B in the parental HEK293 cell line. For a reference to the quality control of each GPCR reporter cell line from DiscoveRx please refer to https://www.discoverx.com/targets/cell-based-assay-list? class=gpcr&pcat=stable%20cell%20lines&readout=arrestin or Codex Biosolutions http:// codexbiosolutions.com/actone_cell_lines.php. Synthesis of proteinogenic amino acid containing N-acyl-palmitoyl analogs Wang resins with preloaded amino acids were purchased from Matrix Innovation. Coupling reagents (PyBOP and Cl-HOBt) were purchased from P3 BioSystems. Palmitoyl chloride and all other reagents were purchased from Sigma-Aldrich. Dimethylformamide (DMF) was added to preloaded Wang resins (~80 mg) and incubated for 30 minutes. Removal of NFmoc from swollen resins was accomplished by two rounds of piperidine treatment [20% solution in DMF (v/v), 3 ml] for 3 and 10 minutes, followed by several washes with DMF. Palmitoyl chloride (1 equivalent) in DMF was then added and the resin suspension was shaken for 2 hours at room temperature. The N-acylated amino acid product was cleaved from the resins by treatment with trifluoroacetic acid (TFA) supplemented with 2.5% (v/v) water and 2.5% (v/v) triisopropylsilane (TIPS). After evaporation of TFA the crude product was purified by automated reversed phase flash chromatography (Teledyne Isco, C18 RediSep RF GoldTm 15 g), binary solvent system: water and acetonitrile supplemented with 0.1% acetic acid. All final products were verified by MS (Supplementary Table 3). In-vitro study of GLP-1 release from GLUTag cells Oleoylethanolamide (OEA) and 2-oleoyl glycerol (2-OG) were purchased from Cayman Chemical Company and resuspended in DMSO to a concentration of 10 mM. N-oleoyl Cohen et al. Page 11 Nature. Author manuscript; available in PMC 2018 February 28. Author Manuscript Author Manuscript Author Manuscript Author Manuscript
serinol was isolated and purified in the same manner as N-palmitoyl serinol described above and its identity was confirmed by H NMR and HRMS. N-oleoyl serinol was resuspended at 10 mM concentration in DMSO. GLUTag cells were obtained from the Mangelsdorf Lab (University of Texas Southwestern) with permission from Daniel Drucker(Mount SinaI Hospital Toronto). GLUTag cells were grown in DMEM, low glucose, GlutaMAX (ThermoFisher) supplemented with 10% FBS and 1% Penicillin/Streptomycin. Once cells grew to 80% confluence they were harvested and plated 1: I into 24 well culture plates in fresh culture media at 50,000 cells per well. After overnight growth in culture plates, cells were washed twice with Krebs buffer supplemented with 20 HL per ml of DPP4 inhibitor (Millipore). GLUTag cells were incubated for 30 minutes in supplemented Krebs buffer and ompounds were added at 1 uM and 100 uM. Cells were incubated with compounds for 2 hours. Media was then collected, centrifuged at 500xg(4C)for 5 minutes and cell free supernatant was analyzed for GLP-1 level using the Active GLP-1 V2 kit(Mesoscale Discovery ) Data from 2 independent experiments were analyzed together for Figure 5c(N 于9之 6 wells for OEA, N-oleoyl serinol andN=4 wells for 2-OG and DMsO) Colonization of germ-free and antibiotic treated mice with N-acyl serinol producing E coli All experimental procedures were approved by the Animal Care and Use Committee of The Rockefeller University. Germ free C57BL/6 mice were maintained in sterile isolators with Itoclaved food and water in the gnotobiotic Facility of the Mucida laboratory at The Rockefeller University. Wild type C57BL/6 mice were purchased from Jackson Labs. 8- week-old mice were used for all experiments For colonization studies 5 ml of an overnight culture(LB with 50 ug/ml kanamycin)of E coli transformed with pET28c hm-NAS N-acyl serinol synthase( treatment group)or E coli transformed with the empty pET28e vector (control group) was centrifuged at 500 x g for 2 minutes, the supernatant was decanted and the cells were resuspended in 2 ml of sterile PBS. Germ free mice were gavaged with 100 uL of bacterial culture immediately upon removal from sterile isolators. Antibiotic treated mice were given water supplemented with 500 ug/ml ampicillin for 1 week followed by gavage with E. coli clones. Suppression of endogenous microbiota was confirmed prior to gavage by culturing stool on non-selective LB agar and demonstrating no colony formation After colonization mice were housed in specific-pathogen-free conditions and fed with autoclaved water and food. Water was supplemented with 35 ug/ml kanamycin and 25 mM PTG. Fecal pellets from mice were analyzed each week for 3 weeks to confirm colonization by the appropriate bacteria and to check for contamination by plating on LB agar with and without kanamycin 50 ug/ml(Extended Data Fig 7). A single fresh mouse fecal pellet was weighed and resuspended in PBS at a fixed concentration (400 HLper mg). I HL of this suspension was diluted 1: 100 in PBS and plated in duplicate, the average number of colonies per plate(2 plates)was recorded. Plasmids were isolated and restriction mapped from these colonies to confirm the presence of the correct hm-NAS gene insert or lack thereof. Ethyl acetate extracts from broth cultures were also examined as previously and 4(2M, 2F)in the control group. The independent replicate experiment of germ fre 3 5 ssed to confirm the production of N-acyl serinols by bacteria in the tre the first experiment of germ free, 7 mice were studied -3(IM, 2F)in the treatment grou mice also consisted of 7 mice(all female, 3 in the treatment group and 4 in the control group). Mice were all individually caged and at the end of each week food consumption and Nature. Author manuscript; available in PMC 2018 February 28
serinol was isolated and purified in the same manner as N-palmitoyl serinol described above and its identity was confirmed by 1H NMR and HRMS. N-oleoyl serinol was resuspended at 10 mM concentration in DMSO. GLUTag cells were obtained from the Mangelsdorf Lab (University of Texas Southwestern) with permission from Daniel Drucker (Mount Sinai Hospital Toronto). GLUTag cells were grown in DMEM, low glucose, GlutaMAX (ThermoFisher) supplemented with 10% FBS and 1% Penicillin/Streptomycin. Once cells grew to 80% confluence they were harvested and plated 1:1 into 24 well culture plates in fresh culture media at 50,000 cells per well. After overnight growth in culture plates, cells were washed twice with Krebs buffer supplemented with 20 µL per ml of DPP4 inhibitor (Millipore). GLUTag cells were incubated for 30 minutes in supplemented Krebs buffer and compounds were added at 1 µM and 100 µM. Cells were incubated with compounds for 2 hours. Media was then collected, centrifuged at 500×g (4 °C) for 5 minutes and cell free supernatant was analyzed for GLP-1 level using the Active GLP-1 V2 kit (Mesoscale Discovery). Data from 2 independent experiments were analyzed together for Figure 5c (N = 6 wells for OEA, N-oleoyl serinol and N = 4 wells for 2-OG and DMSO). Colonization of germ-free and antibiotic treated mice with N-acyl serinol producing E. coli All experimental procedures were approved by the Animal Care and Use Committee of The Rockefeller University. Germ free C57BL/6 mice were maintained in sterile isolators with autoclaved food and water in the Gnotobiotic Facility of the Mucida Laboratory at The Rockefeller University. Wild type C57BL/6 mice were purchased from Jackson Labs. 8- week-old mice were used for all experiments. For colonization studies 5 ml of an overnight culture (LB with 50 µg/ml kanamycin) of E. coli transformed with pET28c:hm-NAS N-acyl serinol synthase (treatment group) or E. coli transformed with the empty pET28c vector (control group) was centrifuged at 500 × g for 2 minutes, the supernatant was decanted and the cells were resuspended in 2 ml of sterile PBS. Germ free mice were gavaged with 100 µL of bacterial culture immediately upon removal from sterile isolators. Antibiotic treated mice were given water supplemented with 500 µg/ml ampicillin for 1 week followed by gavage with E. coli clones. Suppression of endogenous microbiota was confirmed prior to gavage by culturing stool on non-selective LB agar and demonstrating no colony formation. After colonization mice were housed in specific-pathogen-free conditions and fed with autoclaved water and food. Water was supplemented with 35 µg/ml kanamycin and 25 mM IPTG.32 Fecal pellets from mice were analyzed each week for 3 weeks to confirm colonization by the appropriate bacteria and to check for contamination by plating on LB agar with and without kanamycin 50 µg/ml (Extended Data Fig 7). A single fresh mouse fecal pellet was weighed and resuspended in PBS at a fixed concentration (400 µL per 40 mg). 1 µL of this suspension was diluted 1:100 in PBS and plated in duplicate, the average number of colonies per plate (2 plates) was recorded. Plasmids were isolated and restriction mapped from these colonies to confirm the presence of the correct hm-NAS gene insert or lack thereof. Ethyl acetate extracts from broth cultures were also examined as previously discussed to confirm the production of N-acyl serinols by bacteria in the treatment group. In the first experiment of germ free, 7 mice were studied - 3 (1M, 2F) in the treatment group and 4 (2M, 2F) in the control group. The independent replicate experiment of germ free mice also consisted of 7 mice (all female, 3 in the treatment group and 4 in the control group). Mice were all individually caged and at the end of each week food consumption and Cohen et al. Page 12 Nature. Author manuscript; available in PMC 2018 February 28. Author Manuscript Author Manuscript Author Manuscript Author Manuscript