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. 2013 Oct 10;4(10):1005-1010.
doi: 10.1021/ml400312j. Epub 2013 Sep 3.

Discovery of β2 Adrenergic Receptor Ligands Using Biosensor Fragment Screening of Tagged Wild-Type Receptor

Free PMC article

Discovery of β2 Adrenergic Receptor Ligands Using Biosensor Fragment Screening of Tagged Wild-Type Receptor

Tonia Aristotelous et al. ACS Med Chem Lett. .
Free PMC article


G-protein coupled receptors (GPCRs) are the primary target class of currently marketed drugs, accounting for about a quarter of all drug targets of approved medicines. However, almost all the screening efforts for novel ligand discovery rely exclusively on cellular systems overexpressing the receptors. An alternative ligand discovery strategy is a fragment-based drug discovery, where low molecular weight compounds, known as fragments, are screened as initial starting points for optimization. However, the screening of fragment libraries usually employs biophysical screening methods, and as such, it has not been routinely applied to membrane proteins. We present here a surface plasmon resonance biosensor approach that enables, cell-free, label-free, fragment screening that directly measures fragment interactions with wild-type GPCRs. We exemplify the method by the discovery of novel, selective, high affinity antagonists of human β2 adrenoceptor.

Keywords: Fragment screening; G-protein coupled receptors; surface plasmon resonance; β2 adrenoceptor.


Figure 1
Figure 1
SPR sensorgrams of (a) an antagonist alprenolol and an agonist fenoterol, and (b) fragments A to E interacting with β2 adrenoceptor. Red lines represent kinetic fit. The right-side inserted graphs for fragments D and E represent equilibrium fits.
Figure 2
Figure 2
Chemical structures of the validated β2 adrenoceptor fragment hits A to E and the synthesized fragment analogues F to L.
Figure 3
Figure 3
Radioligand competition binding and functional assays for the β2 adrenoceptor with fragments A to E. (a,b) Dose-dependent competition binding curves of the fragments for the β2 adrenoceptor (a) and β1 adrenoceptor (b). (c,d) Stimulatory dose response curves of the fragments obtained from cAMP production (c) and β-arrestin recruitment (d) assays. (e,f) Inhibitory effects of the fragments at 10 μM on isoproterenol-stimulated dose responses in cAMP production (e) and β-arrestin recruitment (f) assays. All of the data points represent mean ± SE obtained from three independent experiments done in duplicate. Dose response curves for each compound were obtained using the nonlinear iterative curve-fitting computer program Prism. ISO, isoproterenol; CYP, cyanopindolol; ICI, ICI-118,551 (a β2 adrenoceptor-specific antagonist); CGP, CGP-20712A (a β1 adrenoceptor-specific antagonist).

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