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 28heterologously 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–(N￾Ethylcarboxamido)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 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 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