Figure 1. The NTSR1 allosteric modulator SBI-553 exhibits transducer-specific efficacy.

Ligand-directed NTSR1 signaling was assessed in HEK293T cells transiently expressing NTSR1 and G protein or β-arrestin activation sensors. (A-E) NTSR1 ligand-induced G protein activation by TRUPATH. (A) Illustration of BRET2-based TRUPATH assay of G protein activation. (B) Depiction of TRUPATH data transformation. G protein activation results in reduced BRET as a BRET donor tagged-Gα and a BRET acceptor tagged-Gγ subunit dissociate. Curves were inverted such that G protein activation resulted in upward sloping curves. (C) G protein activation was assessed following treatment with the endogenous agonist NT, the NT peptide analog PD149163, the β-arrestin-biased ligand SBI-553, and the orthosteric antagonist SR142948A. (D) Maximal ligand-induced G protein activation. Colored asterisks over each bar indicate the treatments from which that compound significantly differed. Treatment vs NT (*), SR142948A (*), PD149163 (*), SBI-553(*). (E) Maximal SBI-553-induced G protein activation in cells transiently expressing NTSR1 or an empty control vector. (F-G) NTSR1 ligand-induced β-arrestin recruitment by BRET. (F) Illustration of BRET1-based assay for β-arrestin recruitment. (G) β-arrestin1/2 recruitment was assessed following treatment with NT, PD149163, SBI-553, or SR142948A. (H) Summary of compound efficacy and potency. Radar plots depict the extent of transducer activation relative to NT (i.e., fold change NT Emax for each transducer). Heat maps depict ligand potency. (I- K) NTSR1 ligand-induced G protein activation by TGFα shedding. (I) Illustration of TGFα shedding assay for G protein activation. (J) Ligand-induced G protein activation. (K) Maximal G protein activation following treatment with NT or SBI-553. Asterisks (*) indicate a change from the GΔC negative control, unless otherwise indicated. For curve parameters, sample size, and statistical comparisons, see Table S1.