Cohen et al High Throughput GPCR Screen Dose Response Curve N-acyl amide S1PR ° 0.01 concentration uM S1PR4 ⊥山uaLk 9 0.010.110100 c Concentration HM 120 9100/GPR119 GPR119 0 0.01 0.1 d GPCR Concentration HM GPR119 GPR119 GPCR 0.01 Concentration uM 120 GPR119 0010.1 10100 ConcentrationμM Extended Data Figure 3. Validation of hits from the high throughput GPCR screen When structural analogs were independently screened in the GPCr panel(e.g, N-oleoyl and palmitoyl serinol or N-3-hydroxypalmitoyl lysine and ornithine) they yielded the same both interact with SIPR4. Did not repeat. b, inset technical repeat of Nf panel. it also GPCR profile and when N-acyl serinol was re-assayed across all GPCRs in the yielded the same GPCR activity profile. a, b, N-3-hydroxypalmitoyl lysine and ornithine hydroxypalmitoyl ornithine dose response curve c, d technical repeats of N-palmitoyl serinol. e, d, e N-palmitoyl and oleoyl serinol both interact with GPr119 Screening data Nature. Author manuscript; available in PMC 2018 February 28Extended Data Figure 3. Validation of hits from the high throughput GPCR screen When structural analogs were independently screened in the GPCR panel (e.g., N-oleoyl and palmitoyl serinol or N-3-hydroxypalmitoyl lysine and ornithine) they yielded the same GPCR profile and when N-acyl serinol was re-assayed across all GPCRs in the panel, it also yielded the same GPCR activity profile. a, b, N-3-hydroxypalmitoyl lysine and ornithine both interact with S1PR4. * Did not repeat. b, inset technical repeat of N-3- hydroxypalmitoyl ornithine dose response curve. c, d technical repeats of N-palmitoyl serinol. c, d, e N-palmitoyl and oleoyl serinol both interact with GPR119. Screening data Cohen et al. Page 17 Nature. Author manuscript; available in PMC 2018 February 28. Author Manuscript Author Manuscript Author Manuscript Author Manuscript