Journal: Science Advances

Article Title: Discovery of a functionally selective serotonin receptor (5-HT 1A R) agonist for the treatment of pain

doi: 10.1126/sciadv.adv9267

Figure Lengend Snippet: ( A ) An inhibition of 5-HT 1A R–mediated cAMP accumulation is observed for serotonin, befiradol, and ST171. The stimulation of cAMP accumulation in the presence of PTX suggests an additional G s response for befiradol and serotonin (serotonin: EC 50 = 52 ± 6 nM; befiradol: EC 50 = 25 ± 11 nM) but not for ST171. Data were obtained in three to five independent experiments with a CAMYEL assay in CHO-K1 cells. ( B ) Inhibition of cAMP accumulation by serotonin, befiradol, and ST171 in HEK293A cells deficient of G s proteins and ( C ) accumulation of cAMP by serotonin and befiradol but not ST171 in HEK293A cells deficient of G i/o proteins. Data were obtained with a GloSensor in n = 6 experiments, each performed in triplicates. ( D ) ST171 inhibits cAMP accumulation caused by 1 μM serotonin in HEK293AΔG i/o cells and thus demonstrates functional antagonism for the activation of G s proteins (IC 50 = 3.9 ± 0.8 nM). Data were obtained with the GloSensor in n = 3 independent experiments, each performed as triplicates. ( E ) β-Arrestin 1 and ( F ) β-arrestin 2 recruitment is monitored by bystander BRET in HEK293T cells with elevated GRK2 levels [ n = 4 to 6 (β-arrestin 1) or 11 to 14 (β-arrestin 2) independent experiments]. ( G ) ST171 is a functional antagonist (IC 50 = 3.9 ± 0.5 nM) for 5-HT 1A R–mediated β-arrestin 2 recruitment stimulated with serotonin (1 μM, ~EC 80 ). Data were obtained with the PathHunter assay in the absence of GRK2 in three independent experiments, each performed in triplicates. All data were normalized to the maximum response of serotonin and are indicated as the means ± SEM of n independent experiments.

Article Snippet: After incubation, CP4 medium was exchanged with Hanks’ balanced salt solution [with added glucose (1 g/liter) and NaHCO 3 (0.35 g/liter)] containing GloSensor cAMP Reagent (3%, Promega) and plates were incubated for 60 min.

Techniques: Inhibition, Functional Assay, Activation Assay

Journal: Science Advances

Article Title: Discovery of a functionally selective serotonin receptor (5-HT 1A R) agonist for the treatment of pain

doi: 10.1126/sciadv.adv9267

Figure Lengend Snippet: ( A ) Top view of the befiradol binding pose within the G s protein–bound 5-HT 1A R complex and ( B ) comparison to the binding pose in the 5-HT 1A R-G i1 protein complex. ( C ) The extended binding pocket residues F112 3.28 and M92 2.60 exhibit different conformations in the cryo-EM structure with ST171 compared to the structures with befiradol bound to 5-HT 1A R in complex with the G i and G s proteins, respectively. ( D to G ) Mutation F112 3.28 W alone and in combination with M92 2.60 F substantially decreases befiradol’s potency for the activation of G i - and G s -mediated signaling and β-arrestin 2 recruitment at the 5-HT 1A R, while the signaling profile of ST171 is not affected. For both ligands, the relative efficacies remain unchanged compared to serotonin. Data were obtained with the cAMP GloSensor measuring the inhibition of forskolin-promoted cAMP accumulation in (D) parental HEK293A and (E) HEK cells deficient of G s proteins. (F) In HEK cells deficient in G i/o proteins, an accumulation of cAMP was observed with befiradol. (G) β-Arrestin 2 recruitment was monitored by BRET in HEK293T cells with elevated GRK2 levels. Data are normalized to the maximal response of serotonin (100%) and shown with ±SEM of n = 3 to 14 independent experiments.

Article Snippet: After incubation, CP4 medium was exchanged with Hanks’ balanced salt solution [with added glucose (1 g/liter) and NaHCO 3 (0.35 g/liter)] containing GloSensor cAMP Reagent (3%, Promega) and plates were incubated for 60 min.

Techniques: Binding Assay, Comparison, Cryo-EM Sample Prep, Mutagenesis, Activation Assay, Inhibition