Dualsteric GPCR targeting: a novel route to binding and signaling pathway selectivity.
Selective modulation of cell function by G protein-coupled receptor (GPCR) activation is highly desirable for basic research and therapy but difficult to achieve. We present a novel strategy toward this goal using muscarinic acetylcholine receptors as a model. The five subtypes bind their physiological transmitter in the highly conserved orthosteric site within the transmembrane domains of the receptors. Orthosteric muscarinic activators have no binding selectivity and poor signaling specificity. There is a less well conserved allosteric site at the extracellular entrance of the binding pocket. To gain subtype-selective receptor activation, we synthesized two hybrids fusing a highly potent oxotremorine-like orthosteric activator with M(2)-selective bis(ammonio)alkane-type allosteric fragments. Radioligand binding in wild-type and mutant receptors supplemented by receptor docking simulations proved M(2) selective and true allosteric/orthosteric binding. G protein activation measurements using orthosteric and allosteric blockers identified the orthosteric part of the hybrid to engender receptor activation. Hybrid-induced dynamic mass redistribution in CHO-hM(2) cells disclosed pathway-specific signaling. Selective receptor activation (M(2)>M(1)>M(3)) was verified in living tissue preparations. As allosteric sites are increasingly recognized on GPCRs, the dualsteric concept of GPCR targeting represents a new avenue toward potent agonists for selective receptor and signaling pathway activation.
Antony
Johannes
J
Pharmacology and Toxicology Section, Institute of Pharmacy, Rheinische Friedrich-Wilhelms-University, Bonn, Germany.
Kellershohn
Kerstin
K
Mohr-Andrä
Marion
M
Kebig
Anna
A
Prilla
Stefanie
S
Muth
Mathias
M
Heller
Eberhard
E
Disingrini
Teresa
T
Dallanoce
Clelia
C
Bertoni
Simona
S
Schrobang
Jasmin
J
Tränkle
Christian
C
Kostenis
Evi
E
Christopoulos
Arthur
A
Höltje
Hans-Dieter
HD
Barocelli
Elisabetta
E
De Amici
Marco
M
Holzgrabe
Ulrike
U
Mohr
Klaus
K
D000818:Animals; D002460:Cell Line; D019204:GTP-Binding Proteins / Q000819:agonists / Q000378:metabolism; D008024:Ligands; D008958:Models, Molecular; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011869:Radioligand Assay; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects
D008024:Ligands; D043562:Receptors, G-Protein-Coupled; D019204:GTP-Binding Proteins