In the sensitive GRK2-based BRET assay, YIL781 activated Gαq and Gα11 with reported EC50 values of 16 and 53 nM and efficacies of 45% and 43% relative to ghrelin. Its intrinsic activity was not detected by the alternative Gα/Gγ separation sensor, underscoring the dependence of the observed profile on assay configuration. [1]
Antagonism and intrinsic activity in calcium and IP assays
Cell-based study · HEK293 cells transiently expressing wild-type human ghrelin receptor [1]
YIL781 inhibited ghrelin-evoked calcium and inositol phosphate responses but also showed intrinsic partial agonism. Ghrelin and Abb13d supplied full-agonist and inverse-agonist comparisons. Lack of apparent agonism in the sequential calcium antagonism assay was attributed by the authors to the transient calcium response, rather than absence of intrinsic activity.
Literature-Reported Treatment Conditions
- Treatment concentration
- Concentration-response testing; ghrelin challenge 10 nM for antagonism.
- Exposure duration
- Antagonist preincubation 30 min; IP stimulation 45 min.
- Method
- Fluo-4 calcium mobilization and radiolabeled inositol phosphate accumulation.
- Co-treatment
- YIL781 alone for intrinsic activity; YIL781 followed by ghrelin for antagonism.
- Reported limits
- Exact tested salt, compound solvent and fitted YIL781 antagonism potencies are not established from the acquired main text; SI not retrieved.
Methods and source locations
Compound reported in the paper: YIL781; Compound label in the paper: Bayer YIL781
Evidence concerns the named YIL781 ligand in the reported assays; it does not validate an Adooq lot or establish a hydrochloride-specific potency.
Figure 1B–E; Results, Functional Evaluation, Second messenger for the Gαq/11 pathway; Methods, Cell Culture and Transfection, Ca2+ Mobilization and IP-Accumulation Assay
Intrinsic calcium mobilization relative to ghrelin: Fluo-4 calcium assay; Figure 1 caption: mean ± SEM of three to five independent experiments performed in triplicate. — Figure 1C and caption; Results, Functional Evaluation, Second messenger for the Gαq/11 pathway, paragraph beginning “When the compounds were tested”
Intrinsic inositol phosphate accumulation and antagonism of ghrelin: Radiolabeled IP accumulation; no YIL781-specific exact IP efficacy inferred from curves or a multi-compound range. — Figure 1D–E and caption; Methods, IP-Accumulation Assay
Pathway-dependent intrinsic activity in BRET biosensor assays
Cell-based study · HEK293 cells expressing human ghrelin receptor and G-protein or β-arrestin BRET sensors [1]
In the sensitive GRK2-based assay, YIL781 partially activated Gαq, Gα11 and Gα12, with no corresponding intrinsic activity detected for Gαi/o or Gα13. The alternative Gα/Gγ separation sensor did not detect YIL781 intrinsic activity. YIL781 antagonized ghrelin-induced β-arrestin recruitment and displayed little intrinsic efficacy, tending toward inverse agonism.
Literature-Reported Treatment Conditions
- Treatment concentration
- Concentration-response testing; β-arrestin antagonism assessed against 30 nM ghrelin.
- Exposure duration
- BRET methods: cells incubated with ligand for 10 min; luciferase substrate added 5 min before reading.
- Method
- Two G-protein BRET biosensor configurations and bystander BRET for β-arrestin membrane recruitment.
- Co-treatment
- YIL781 alone for intrinsic activity; YIL781 plus ghrelin in β-arrestin antagonism assays.
- Reported limits
- Salt form, compound solvent and complete sequential antagonism incubation schedule are not established in the acquired main text.
Methods and source locations
Compound reported in the paper: YIL781; Compound label in the paper: Bayer YIL781
Evidence concerns the named YIL781 ligand in the reported assays; it does not validate an Adooq lot or establish a hydrochloride-specific potency.
Figures 2A–B and 3A–D; Results, Monitoring G-protein activation and Monitoring β-arrestin recruitment; Methods, BRET Assay
Gαq activation by YIL781 in GRK2-based BRET sensor: GRK2-GFP10/RlucII-Gγ5 BRET; Figure 2 caption reports mean ± SEM, three to five independent experiments in triplicate. — Figure 2B; Results, Monitoring G-protein activation, paragraph beginning “Upon testing Abb13d and YIL781”
Gα11 activation by YIL781 in GRK2-based BRET sensor: GRK2-based BRET; values stated in main Results rather than estimated from curves. — Figure 2B; Results, Monitoring G-protein activation, paragraph beginning “Upon testing Abb13d and YIL781”
Inhibition of ghrelin-induced β-arrestin 1 and 2 recruitment: Bystander BRET with 30 nM ghrelin; Figure 3 caption: mean ± SEM of five to eight independent experiments in triplicate. Main text calls these potencies; no additional fitted-metric label imposed. — Figure 3A,C and caption; Results, Monitoring β-arrestin recruitment
