Journal: Molecular Pharmacology

Article Title: Small-Molecule Positive Allosteric Modulators of the β 2 -Adrenoceptor Isolated from DNA-Encoded Libraries

doi: 10.1124/mol.118.111948

Figure Lengend Snippet: Hit compounds from DEL screening with the agonist-occupied β 2 AR in HDL particles. (A) Cartoon for DEL screening. Purified human β 2 ARs were reconstituted in HDL particles ( β 2 AR Nanodiscs) and then occupied by BI-167107 (BI). DNA-encoded library molecules were mixed with the BI-occupied β 2 AR Nanodiscs immobilized on NeutrAvidin beads through biotin–avidin interaction of biotinylated membrane scaffolding protein ApoA1. Three rounds of iterative selection were performed with each library. (B) Structures of the Cmpd-6 and six other primary hits. These compounds have varied chemical scaffolds in a common region, designated as R1. The different chemical structures in the R1 region of each analog are illustrated. (C) Analysis of Cmpd-6 for its physical interaction with the agonist-bound, active β 2 AR by ITC. The thermogram (insert) and binding isotherm with the best titration curve fit are shown. One site model was used to fit the data. Data are representative of three independent experiments. The values summarizing binding affinity (K D ), stoichiometry (N), enthalpy (ΔH), and entropy (ΔS) are shown in box below the graph.

Article Snippet: Isothermal titration calorimetry (ITC) experiments were performed with maltose neopentyl glycol (MNG; Anatrace)–solubilized β 2 AR on a MicroCal Auto-iTC200 system (Malvern, Malvern, UK), according to the previously reported method ( ).

Techniques: Purification, Avidin-Biotin Assay, Membrane, Scaffolding, Selection, Binding Assay, Titration