A New Molecular Mechanism To Engineer Protean Agonism at a G Protein-Coupled Receptor.
Protean agonists are of great pharmacological interest as their behavior may change in magnitude and direction depending on the constitutive activity of a receptor. Yet, this intriguing phenomenon has been poorly described and understood, due to the lack of stable experimental systems and design strategies. In this study, we overcome both limitations: First, we demonstrate that modulation of the ionic strength in a defined experimental set-up allows for analysis of G protein-coupled receptor activation in the absence and presence of a specific amount of spontaneous receptor activity using the muscarinic M2acetylcholine receptor as a model. Second, we employ this assay system to show that a dualsteric design principle, that is, molecular probes, carrying two pharmacophores to simultaneously adopt orthosteric and allosteric topography within a G protein-coupled receptor, may represent a novel approach to achieve protean agonism. We pinpoint three molecular requirements within dualsteric compounds that elicit protean agonism at the muscarinic M2acetylcholine receptor. Using radioligand-binding and functional assays, we posit that dynamic ligand binding may be the mechanism underlying protean agonism of dualsteric ligands. Our findings provide both new mechanistic insights into the still enigmatic phenomenon of protean agonism and a rationale for the design of such compounds for a G protein-coupled receptor.
Pharmacology and Toxicology Section, Institute of Pharmacy (A.D.M., J.H., C.T., K.M., R.S.), Research Training Group 1873 (A.D.M., E.K., K.M.), and Molecular-, Cellular-, and Pharmacobiology Section, Institute of Pharmaceutical Biology (E.K.), University of Bonn, Bonn, Germany; Dipartimento di Scienze Farmaceutiche, Sezione di Chimica Farmaceutica 'Pietro Pratesi,' Università degli Studi di Milano, Milano, Italy (C.M., M.D.A., C.D.); Institute of Pharmacology and Toxicology, University of Würzburg, Würzburg, Germany (A.B.); Department of Pharmaceutical and Medicinal Chemistry, Institute of Pharmacy, University of Würzburg, Würzburg, Germany (J.K., M.M., U.H.); and Department of Pharmacology, University of North Carolina School of Medicine, Chapel Hill, North Carolina (T.K.).
Anna
A
De Min
Pharmacology and Toxicology Section, Institute of Pharmacy (A.D.M., J.H., C.T., K.M., R.S.), Research Training Group 1873 (A.D.M., E.K., K.M.), and Molecular-, Cellular-, and Pharmacobiology Section, Institute of Pharmaceutical Biology (E.K.), University of Bonn, Bonn, Germany; Dipartimento di Scienze Farmaceutiche, Sezione di Chimica Farmaceutica 'Pietro Pratesi,' Università degli Studi di Milano, Milano, Italy (C.M., M.D.A., C.D.); Institute of Pharmacology and Toxicology, University of Würzburg, Würzburg, Germany (A.B.); Department of Pharmaceutical and Medicinal Chemistry, Institute of Pharmacy, University of Würzburg, Würzburg, Germany (J.K., M.M., U.H.); and Department of Pharmacology, University of North Carolina School of Medicine, Chapel Hill, North Carolina (T.K.).
Carlo
C
Matera
Pharmacology and Toxicology Section, Institute of Pharmacy (A.D.M., J.H., C.T., K.M., R.S.), Research Training Group 1873 (A.D.M., E.K., K.M.), and Molecular-, Cellular-, and Pharmacobiology Section, Institute of Pharmaceutical Biology (E.K.), University of Bonn, Bonn, Germany; Dipartimento di Scienze Farmaceutiche, Sezione di Chimica Farmaceutica 'Pietro Pratesi,' Università degli Studi di Milano, Milano, Italy (C.M., M.D.A., C.D.); Institute of Pharmacology and Toxicology, University of Würzburg, Würzburg, Germany (A.B.); Department of Pharmaceutical and Medicinal Chemistry, Institute of Pharmacy, University of Würzburg, Würzburg, Germany (J.K., M.M., U.H.); and Department of Pharmacology, University of North Carolina School of Medicine, Chapel Hill, North Carolina (T.K.).
Andreas
A
Bock
Pharmacology and Toxicology Section, Institute of Pharmacy (A.D.M., J.H., C.T., K.M., R.S.), Research Training Group 1873 (A.D.M., E.K., K.M.), and Molecular-, Cellular-, and Pharmacobiology Section, Institute of Pharmaceutical Biology (E.K.), University of Bonn, Bonn, Germany; Dipartimento di Scienze Farmaceutiche, Sezione di Chimica Farmaceutica 'Pietro Pratesi,' Università degli Studi di Milano, Milano, Italy (C.M., M.D.A., C.D.); Institute of Pharmacology and Toxicology, University of Würzburg, Würzburg, Germany (A.B.); Department of Pharmaceutical and Medicinal Chemistry, Institute of Pharmacy, University of Würzburg, Würzburg, Germany (J.K., M.M., U.H.); and Department of Pharmacology, University of North Carolina School of Medicine, Chapel Hill, North Carolina (T.K.).
Janine
J
Holze
Pharmacology and Toxicology Section, Institute of Pharmacy (A.D.M., J.H., C.T., K.M., R.S.), Research Training Group 1873 (A.D.M., E.K., K.M.), and Molecular-, Cellular-, and Pharmacobiology Section, Institute of Pharmaceutical Biology (E.K.), University of Bonn, Bonn, Germany; Dipartimento di Scienze Farmaceutiche, Sezione di Chimica Farmaceutica 'Pietro Pratesi,' Università degli Studi di Milano, Milano, Italy (C.M., M.D.A., C.D.); Institute of Pharmacology and Toxicology, University of Würzburg, Würzburg, Germany (A.B.); Department of Pharmaceutical and Medicinal Chemistry, Institute of Pharmacy, University of Würzburg, Würzburg, Germany (J.K., M.M., U.H.); and Department of Pharmacology, University of North Carolina School of Medicine, Chapel Hill, North Carolina (T.K.).
Jessica
J
Kloeckner
Pharmacology and Toxicology Section, Institute of Pharmacy (A.D.M., J.H., C.T., K.M., R.S.), Research Training Group 1873 (A.D.M., E.K., K.M.), and Molecular-, Cellular-, and Pharmacobiology Section, Institute of Pharmaceutical Biology (E.K.), University of Bonn, Bonn, Germany; Dipartimento di Scienze Farmaceutiche, Sezione di Chimica Farmaceutica 'Pietro Pratesi,' Università degli Studi di Milano, Milano, Italy (C.M., M.D.A., C.D.); Institute of Pharmacology and Toxicology, University of Würzburg, Würzburg, Germany (A.B.); Department of Pharmaceutical and Medicinal Chemistry, Institute of Pharmacy, University of Würzburg, Würzburg, Germany (J.K., M.M., U.H.); and Department of Pharmacology, University of North Carolina School of Medicine, Chapel Hill, North Carolina (T.K.).
Mathias
M
Muth
Pharmacology and Toxicology Section, Institute of Pharmacy (A.D.M., J.H., C.T., K.M., R.S.), Research Training Group 1873 (A.D.M., E.K., K.M.), and Molecular-, Cellular-, and Pharmacobiology Section, Institute of Pharmaceutical Biology (E.K.), University of Bonn, Bonn, Germany; Dipartimento di Scienze Farmaceutiche, Sezione di Chimica Farmaceutica 'Pietro Pratesi,' Università degli Studi di Milano, Milano, Italy (C.M., M.D.A., C.D.); Institute of Pharmacology and Toxicology, University of Würzburg, Würzburg, Germany (A.B.); Department of Pharmaceutical and Medicinal Chemistry, Institute of Pharmacy, University of Würzburg, Würzburg, Germany (J.K., M.M., U.H.); and Department of Pharmacology, University of North Carolina School of Medicine, Chapel Hill, North Carolina (T.K.).
Christian
C
Traenkle
Pharmacology and Toxicology Section, Institute of Pharmacy (A.D.M., J.H., C.T., K.M., R.S.), Research Training Group 1873 (A.D.M., E.K., K.M.), and Molecular-, Cellular-, and Pharmacobiology Section, Institute of Pharmaceutical Biology (E.K.), University of Bonn, Bonn, Germany; Dipartimento di Scienze Farmaceutiche, Sezione di Chimica Farmaceutica 'Pietro Pratesi,' Università degli Studi di Milano, Milano, Italy (C.M., M.D.A., C.D.); Institute of Pharmacology and Toxicology, University of Würzburg, Würzburg, Germany (A.B.); Department of Pharmaceutical and Medicinal Chemistry, Institute of Pharmacy, University of Würzburg, Würzburg, Germany (J.K., M.M., U.H.); and Department of Pharmacology, University of North Carolina School of Medicine, Chapel Hill, North Carolina (T.K.).
Marco
M
De Amici
Pharmacology and Toxicology Section, Institute of Pharmacy (A.D.M., J.H., C.T., K.M., R.S.), Research Training Group 1873 (A.D.M., E.K., K.M.), and Molecular-, Cellular-, and Pharmacobiology Section, Institute of Pharmaceutical Biology (E.K.), University of Bonn, Bonn, Germany; Dipartimento di Scienze Farmaceutiche, Sezione di Chimica Farmaceutica 'Pietro Pratesi,' Università degli Studi di Milano, Milano, Italy (C.M., M.D.A., C.D.); Institute of Pharmacology and Toxicology, University of Würzburg, Würzburg, Germany (A.B.); Department of Pharmaceutical and Medicinal Chemistry, Institute of Pharmacy, University of Würzburg, Würzburg, Germany (J.K., M.M., U.H.); and Department of Pharmacology, University of North Carolina School of Medicine, Chapel Hill, North Carolina (T.K.).
Terry
T
Kenakin
Pharmacology and Toxicology Section, Institute of Pharmacy (A.D.M., J.H., C.T., K.M., R.S.), Research Training Group 1873 (A.D.M., E.K., K.M.), and Molecular-, Cellular-, and Pharmacobiology Section, Institute of Pharmaceutical Biology (E.K.), University of Bonn, Bonn, Germany; Dipartimento di Scienze Farmaceutiche, Sezione di Chimica Farmaceutica 'Pietro Pratesi,' Università degli Studi di Milano, Milano, Italy (C.M., M.D.A., C.D.); Institute of Pharmacology and Toxicology, University of Würzburg, Würzburg, Germany (A.B.); Department of Pharmaceutical and Medicinal Chemistry, Institute of Pharmacy, University of Würzburg, Würzburg, Germany (J.K., M.M., U.H.); and Department of Pharmacology, University of North Carolina School of Medicine, Chapel Hill, North Carolina (T.K.).
Ulrike
U
Holzgrabe
Pharmacology and Toxicology Section, Institute of Pharmacy (A.D.M., J.H., C.T., K.M., R.S.), Research Training Group 1873 (A.D.M., E.K., K.M.), and Molecular-, Cellular-, and Pharmacobiology Section, Institute of Pharmaceutical Biology (E.K.), University of Bonn, Bonn, Germany; Dipartimento di Scienze Farmaceutiche, Sezione di Chimica Farmaceutica 'Pietro Pratesi,' Università degli Studi di Milano, Milano, Italy (C.M., M.D.A., C.D.); Institute of Pharmacology and Toxicology, University of Würzburg, Würzburg, Germany (A.B.); Department of Pharmaceutical and Medicinal Chemistry, Institute of Pharmacy, University of Würzburg, Würzburg, Germany (J.K., M.M., U.H.); and Department of Pharmacology, University of North Carolina School of Medicine, Chapel Hill, North Carolina (T.K.).
Clelia
C
Dallanoce
Pharmacology and Toxicology Section, Institute of Pharmacy (A.D.M., J.H., C.T., K.M., R.S.), Research Training Group 1873 (A.D.M., E.K., K.M.), and Molecular-, Cellular-, and Pharmacobiology Section, Institute of Pharmaceutical Biology (E.K.), University of Bonn, Bonn, Germany; Dipartimento di Scienze Farmaceutiche, Sezione di Chimica Farmaceutica 'Pietro Pratesi,' Università degli Studi di Milano, Milano, Italy (C.M., M.D.A., C.D.); Institute of Pharmacology and Toxicology, University of Würzburg, Würzburg, Germany (A.B.); Department of Pharmaceutical and Medicinal Chemistry, Institute of Pharmacy, University of Würzburg, Würzburg, Germany (J.K., M.M., U.H.); and Department of Pharmacology, University of North Carolina School of Medicine, Chapel Hill, North Carolina (T.K.).
Evi
E
Kostenis
Pharmacology and Toxicology Section, Institute of Pharmacy (A.D.M., J.H., C.T., K.M., R.S.), Research Training Group 1873 (A.D.M., E.K., K.M.), and Molecular-, Cellular-, and Pharmacobiology Section, Institute of Pharmaceutical Biology (E.K.), University of Bonn, Bonn, Germany; Dipartimento di Scienze Farmaceutiche, Sezione di Chimica Farmaceutica 'Pietro Pratesi,' Università degli Studi di Milano, Milano, Italy (C.M., M.D.A., C.D.); Institute of Pharmacology and Toxicology, University of Würzburg, Würzburg, Germany (A.B.); Department of Pharmaceutical and Medicinal Chemistry, Institute of Pharmacy, University of Würzburg, Würzburg, Germany (J.K., M.M., U.H.); and Department of Pharmacology, University of North Carolina School of Medicine, Chapel Hill, North Carolina (T.K.).
Klaus
K
Mohr
Pharmacology and Toxicology Section, Institute of Pharmacy (A.D.M., J.H., C.T., K.M., R.S.), Research Training Group 1873 (A.D.M., E.K., K.M.), and Molecular-, Cellular-, and Pharmacobiology Section, Institute of Pharmaceutical Biology (E.K.), University of Bonn, Bonn, Germany; Dipartimento di Scienze Farmaceutiche, Sezione di Chimica Farmaceutica 'Pietro Pratesi,' Università degli Studi di Milano, Milano, Italy (C.M., M.D.A., C.D.); Institute of Pharmacology and Toxicology, University of Würzburg, Würzburg, Germany (A.B.); Department of Pharmaceutical and Medicinal Chemistry, Institute of Pharmacy, University of Würzburg, Würzburg, Germany (J.K., M.M., U.H.); and Department of Pharmacology, University of North Carolina School of Medicine, Chapel Hill, North Carolina (T.K.) rschrage@uni-bonn.de.
Ramona
R
Schrage
D000494:Allosteric Regulation; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D006801:Humans; D008024:Ligands; D011485:Protein Binding; D015202:Protein Engineering; D043585:Receptor, Muscarinic M2 / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000819:agonists* / Q000378:metabolism; D014325:Tromethamine
D008024:Ligands; D043585:Receptor, Muscarinic M2; D043562:Receptors, G-Protein-Coupled; D014325:Tromethamine