Fig. 1: In vitro signaling potency of agonistic nanobody pN162 to melanocortin receptors.

a Dose-dependent human MC4R -induced cAMP signaling (GloSensor) of pN162 and control ligands (endogenous agonist α-MSH and clinical benchmark peptide agonist Setmelanotide). Each data point represents the mean of two replicates. Experiments were performed minimally twice (the number of experiment repeats is indicated in Table 1). Exemplary graphs are shown. Table 1 provides the average EC₅₀s of pN162, α-MSH, and setmelanotide. b Cell surface expression of human MC4R measured by flow cytometry. Overlays of fluorescence histograms obtained by anti-Flag mAb or isotype IgG control detection on Flag-tagged MC4R or mock GPCR transiently transfected HEK293T cells. Cell surface binding of 100 nM c-Myc-tagged pN162 to human MC4R (c) or melanocortin receptor subtypes (d) measured by flow cytometry. Overlays of fluorescence histogram obtained by pN162 or mock nanobody detection on human MC4R or mock GPCR transfected HEK293T cells (c). pN162 specific melanocortin receptor subtype staining, normalized for MC4R expressing HEK293 cells (d). Only intact cells were gated following Topro staining. MC2R was expressed in absence and presence of its expression chaperone MRAPα. e Dose dependent 125I-SHU9119 radioligand displacement with pN162. Within an experiment, each data point is an average of 2 repeats. Experiments were performed three times, and a representative graph is shown. Dose-dependent human MC1R (f), MC3R (g), and MC5R (h) induced cAMP signaling (GloSensor) of pN162 and control ligands (endogenous agonist α-MSH and clinical benchmark peptide agonist setmelanotide). Each data point represents the mean of two replicates. Experiments were performed minimally twice (the number of experiment repeats is indicated in Table 1). Exemplary graphs are shown. Table 1 provides the average EC₅₀s of pN162, α-MSH, and setmelanotide.