Figure 1: Strategies for the development of a testing funnel to detect PAMs targeting SCTRs:

(A) Advantages of allosteric modulator signaling and display of SCTR signaling pathways (adopted from ref.⁵¹). (B) Development of biological assays for the establishment of a testing funnel to identify biologically relevant SCTR small molecule modulators consisting of four levels: HTS and hit confirmation, hit validation, MOA and G-protein bias as well as scaffold validation and SAR studies; Check marks indicate stages that have been completed successfully. (C) GLP-1R PAM BETP^{18, 34} as a model demonstrating substantial probe-dependency and corresponding effect on cAMP accumulation assay sensitivity; left panel: DMSO (black) vs BETP (12.5 μM, red) treated GLP-1 full-length (full agonist) predicting primary screening response around 35%; right panel: DMSO (black) vs BETP (12.5 μM, red) treated GLP-1(9–36) (partial agonist) predicting primary screening response well beyond 100%; TR-FRET ratios (relative fluorescence units (RFU)) normalized to corresponding peptide agonist and plotted using GraphPad Prism; data points shown as mean ± SEM. Depiction of assay windows of PAM mode (neg. ctrl: EC₂₀, pos. ctrl: EC₉₅ of ligand) and agonist mode (neg. ctrl: no ligand, pos. ctrl: EC₉₅ of ligand), which served as the basis of SCTR screening assays. (D) Secretin and its truncated analogs mimicking potential metabolites of secretin (from top down): Sec(1–23), product of C-terminal truncation (carboxylic acid (-OH)); Sec-FL (full-length, amide (-NH₂)), endogenous peptide acting on SCTRs; Sec(3–27) amide, product of N-terminal truncation.