Journal: bioRxiv

Article Title: Pharmacological proximities in the GPCR family discovered using contact-informed amino-acid and binding pocket similarities

doi: 10.64898/2026.05.02.720972

Figure Lengend Snippet: a , The pharmacological neighborhood of the β 2 adrenoceptor. The transmembrane sequence similarity of 285 Class A GPCRs to the β 2 adrenoceptor are plotted against their corresponding GPCR-CoINPocket v2 scores. GPCRs are coloured broadly by classification: aminergics (red), peptide (purple), orphans (gray), and others (navy). b , Chemical structures of micromolar affinity ligands assessed in this study. Observed K i (affinity) of experimental ligands (green) or propranolol (blue) from competitive radioligand binding are displayed above each structure along with the PSICHIC-predicted K i . c , Competitive radioligand binding assays using putative surrogate ligands. Binding assays used crude purified membranes of COS-1 cells transiently transfected with β 2 adrenoceptor. Unlabelled competitor ligands were titrated against 0.5 nM [ 3 H]DHA and their affinity derived. Points and bars represent means ± SEM of n=3 experiments performed in triplicate. Curves were fit to a logistic single site binding model using GraphPad Prism v9. d , Boltz-1 and AlphaFold 3-predicted binding poses of propranolol and the low micromolar affinity off-target ligands co-folded with the β 2 adrenoceptor and their cognate receptors. In blue, the binding pose of the β 2 antagonist propranolol is shown bound to the β 2 adrenoceptor.

Article Snippet: COS-1 cells were obtained from American Type Culture Collection (CRL-1650).

Techniques: Sequencing, Binding Assay, Purification, Transfection, Derivative Assay

Journal: bioRxiv

Article Title: Pharmacological proximities in the GPCR family discovered using contact-informed amino-acid and binding pocket similarities

doi: 10.64898/2026.05.02.720972

Figure Lengend Snippet: a , Competitive radioligand binding assay using putative surrogate ligands. Binding assays used crude purified membranes of COS-1 cells transiently transfected with β 2 adrenoceptor. Unlabelled competitor ligands were titrated against 0.5 nM [ 3 H]DHA and their affinity derived. Points and bars represent means ± SEMs of n=3 experiments performed in triplicate. Curves were fit to a logistic single site binding model using GraphPad Prism v9. b , Chemical structures of weakly-binding or inactive compounds assessed in this study. Observed K i (affinity) of experimental ligands from competitive radioligand binding > 1 μM (purple) or propranolol (blue) are displayed above each structure along with their PSICHIC-predicted K i . c , Radioligand dissociation assay of hit compounds in comparison to bona fide orthosteric antagonist propranolol. The rate at which [ 3 H]DHA dissociated from the β 2 adrenoceptor was measured over the course of 2 hrs at room temperature upon addition of 10 μM propranolol (blue) in the presence or absence of off-target compounds: 6 μM ATC 0175 (green) or 30 μM WAY 267464 (purple). Points and bars represent means ± SEMs of n=2-4 experiments performed in triplicate. Curves were fit to a logistic single site binding model using GraphPad Prism v9.

Article Snippet: COS-1 cells were obtained from American Type Culture Collection (CRL-1650).

Techniques: Radio Ligand Binding Assay, Binding Assay, Purification, Transfection, Derivative Assay, Comparison