Activation Mechanism of the β2-Adrenergic Receptor

Design of ligands that provide receptor selectivity has emerged as a new paradigm for drug discovery of G protein-coupled receptors, and may, for certain families of receptors, only be achieved via identification of chemically diverse allosteric modulators. Here, the extracellular vestibule of the M 2 muscarinic acetylcholine receptor (mAChR) is targeted for structure-based design of allosteric modulators. Accelerated molecular dynamics (aMD) simulations were performed to construct structural ensembles that account for the receptor flexibility. Compounds obtained from the National Cancer Institute (NCI) were docked to the receptor ensembles. Retrospective docking of known ligands showed that combining aMD simulations with Glide induced fit docking (IFD) provided much-improved enrichment factors, compared with the Glide virtual screening workflow. Glide IFD was thus applied in receptor ensemble docking, and 38 top-ranked NCI compounds were selected for experimental testing. In [ 3 H]Nmethylscopolamine radioligand dissociation assays, approximately half of the 38 lead compounds altered the radioligand dissociation rate, a hallmark of allosteric behavior. In further competition binding experiments, we identified 12 compounds with affinity of ≤30 μM. With final functional experiments on six selected compounds, we confirmed four of them as new negative allosteric modulators (NAMs) and one as positive allosteric modulator of agonist-mediated response at the M 2 mAChR. Two of the NAMs showed subtype selectivity without significant effect at the M 1 and M 3 mAChRs. This study demonstrates an unprecedented successful structure-based approach to identify chemically diverse and selective GPCR allosteric modulators with outstanding potential for further structure-activity relationship studies.GPCR | allosteric modulators | ensemble docking | affinity | cooperativity

Section: Significancementioning

confidence: 99%

Section: Significancementioning

confidence: 99%

Accelerated structure-based design of chemically diverse allosteric modulators of a muscarinic G protein-coupled receptor

Miao

Goldfeld

Moo

et al. 2016

“…The structure, dynamics, and function of GPCRs result from underlying free energy landscapes (4). However, quantitative characterization of the GPCR activation and ligand-dependent free energy profiles has proved challenging (4)(5)(6)(7)(8)(9)(10)(11)(12).…”

mentioning

confidence: 99%

“…The M 2 receptor has been crystallized in both an inactive state bound by the inverse agonist 3-quinuclidinylbenzilate (QNB) (13) and an active state bound by the full agonist iperoxo (IXO) and a G-protein mimetic nanobody (14). The receptor activation is characterized by rearrangements of the transmembrane (TM) helices 5,6, and 7, particularly closing of the ligand-binding pocket, outward tilting of the TM6 cytoplasmic end, and close interaction of Tyr206 5.58 and Tyr440 7.53 in the G-proteincoupling site (14). The residue superscripts denote the Ballesteros-Weinstein (BW) numbering of GPCRs (15).…”

mentioning

confidence: 99%

Graded activation and free energy landscapes of a muscarinic G-protein–coupled receptor

Miao

McCammon

2016

143

170

G-protein-coupled receptors (GPCRs) recognize ligands of widely different efficacies, from inverse to partial and full agonists, which transduce cellular signals at differentiated levels. However, the mechanism of such graded activation remains unclear. Using the Gaussian accelerated molecular dynamics (GaMD) method that enables both unconstrained enhanced sampling and free energy calculation, we have performed extensive GaMD simulations (∼19 μs in total) to investigate structural dynamics of the M 2 muscarinic GPCR that is bound by the full agonist iperoxo (IXO), the partial agonist arecoline (ARC), and the inverse agonist 3-quinuclidinyl-benzilate (QNB), in the presence or absence of the G-protein mimetic nanobody. In the receptornanobody complex, IXO binding leads to higher fluctuations in the protein-coupling interface than ARC, especially in the receptor transmembrane helix 5 (TM5), TM6, and TM7 intracellular domains that are essential elements for GPCR activation, but less flexibility in the receptor extracellular region due to stronger binding compared with ARC. Two different binding poses are revealed for ARC in the orthosteric pocket. Removal of the nanobody leads to GPCR deactivation that is characterized by inward movement of the TM6 intracellular end. Distinct low-energy intermediate conformational states are identified for the IXO-and ARC-bound M 2 receptor. Both dissociation and binding of an orthosteric ligand are observed in a single all-atom GPCR simulation in the case of partial agonist ARC binding to the M 2 receptor. This study demonstrates the applicability of GaMD for exploring free energy landscapes of large biomolecules and the simulations provide important insights into the GPCR functional mechanism. cellular signaling | ligand recognition | protein-protein interactions | allostery | drug discovery G -protein-coupled receptors (GPCRs) are primary targets of about one-third of currently marketed drugs. They recognize ligands of widely different efficacies, from inverse to partial and full agonists, which transduce cellular signals at differentiated levels. Increasing experimental and computational evidence suggests that GPCRs exist in an ensemble of different conformations that interconvert dynamically during activation and ligand recognition (1-3). The structure, dynamics, and function of GPCRs result from underlying free energy landscapes (4). However, quantitative characterization of the GPCR activation and ligand-dependent free energy profiles has proved challenging (4-12).The M 2 muscarinic GPCR is widely distributed in mammalian tissues. It plays a key role in regulating the human heart rate and heart contraction forces. The M 2 receptor has been crystallized in both an inactive state bound by the inverse agonist 3-quinuclidinylbenzilate (QNB) (13) and an active state bound by the full agonist iperoxo (IXO) and a G-protein mimetic nanobody (14). The receptor activation is characterized by rearrangements of the transmembrane (TM) helices 5, 6, and 7, particularly closing of the ligan...

“…The structure-function relationship of several class A members (e.g., rhodopsin and β2-adrenergic receptor) has been investigated in great details via various approaches including site-directed mutagenesis, X-ray crystallography (7,8), and molecular modeling (9)(10)(11). Although no crystal structure is available for any OR, sitedirected mutagenesis and/or computational modeling have shed light on structure-function relationship for a few ORs (12-18).…”

mentioning

confidence: 99%

Responsiveness of G protein-coupled odorant receptors is partially attributed to the activation mechanism

March²,

et al. 2015

Mammals detect and discriminate numerous odors via a large family of G protein-coupled odorant receptors (ORs). However, little is known about the molecular and structural basis underlying OR response properties. Using site-directed mutagenesis and computational modeling, we studied ORs sharing high sequence homology but with different response properties. When tested in heterologous cells by diverse odorants, MOR256-3 responded broadly to many odorants, whereas MOR256-8 responded weakly to a few odorants. Out of 36 mutant MOR256-3 ORs, the majority altered the responses to different odorants in a similar manner and the overall response of an OR was positively correlated with its basal activity, an indication of ligand-independent receptor activation. Strikingly, a single mutation in MOR256-8 was sufficient to confer both high basal activity and broad responsiveness to this receptor. These results suggest that broad responsiveness of an OR is at least partially attributed to its activation likelihood.