The conformational complexity of transmembrane signaling of G‐protein‐coupled receptors (GPCRs) is a central hurdle for the design of screens for receptor agonists. In their basal states, GPCRs have lower affinities for agonists compared to their G‐protein‐bound active state conformations. Moreover, different agonists can stabilize distinct active receptor conformations and do not uniformly activate all cellular signaling pathways linked to a given receptor (agonist bias). Comparative fragment screens were performed on a β2‐adrenoreceptor–nanobody fusion locked in its active‐state conformation by a G‐protein‐mimicking nanobody, and the same receptor in its basal‐state conformation. This simple biophysical assay allowed the identification and ranking of multiple novel agonists and permitted classification of the efficacy of each hit in agonist, antagonist, or inverse agonist categories, thereby opening doors to nanobody‐enabled reverse pharmacology.
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