Experimental confirmation of ZY001–ZY004 as highly potent EP4 antagonists. (A–D) Dose-response curves of ZY001 (A), ZY002 (B), ZY003 (C), and ZY004 (D) against EP4 in GloSensor cAMP assay, respectively (n = 3). (E) Dose-response curves of ZY001 against EP4 in CRE reporter assay (n = 3). (F) Dose-response curves of ZY001 against EP1 in NFAT reporter assay (n = 3). (G, H) Dose-response curves of ZY001 against EP2 (G) and EP3 (H) in CRE reporter assay (n = 3). (I) Schild plot analyses of ZY001 in the GloSensor cAMP assay. (J) The pA₂ and slope of this Schild plot (I) were 8.596 and 1.014, respectively. (K) Schild plot analyses of ZY003 in the GloSensor cAMP assay. (L) The pA₂ and slope of this Schild plot (K) were 8.239 and 0.985, respectively. Data were shown as the mean ± SEM.