Covalent agonists for studying G protein-coupled receptor activation

Abstract

Structural studies on G protein-coupled receptors (GPCRs) provide important insights into the architecture and function of these important drug targets. However, the crystallization of GPCRs in active states is particularly challenging, requiring the formation of stable and conformationally homogeneous ligand-receptor complexes. Native hormones, neurotransmitters, and synthetic agonists that bind with low affinity are ineffective at stabilizing an active state for crystallogenesis. To promote structural studies on the pharmacologically highly relevant class of aminergic GPCRs, we here present the development of covalently binding molecular tools activating G s -, G i -, and G q -coupled receptors. The covalent agonists are derived from the monoamine neurotransmitters noradrenaline, dopamine, serotonin, and histamine, and they were accessed using a general and versatile synthetic strategy. We demonstrate that the tool compounds presented herein display an efficient covalent binding mode and that the respective covalent ligand-receptor complexes activate G proteins comparable to the natural neurotransmitters. A crystal structure of the β 2 -adrenoreceptor in complex with a covalent noradrenaline analog and a conformationally selective antibody (nanobody) verified that these agonists can be used to facilitate crystallogenesis. (1), and the resulting biochemical instability of the solubilized protein (2, 3). Protein crystallography, the most powerful tool for the study of GPCR structure, requires the formation of stable and conformationally homogeneous ligand-receptor complexes (4). High-affinity agonists with dissociation constants in the low to subnanomolar range and low off-rates facilitate stabilization of the protein throughout the process of expression, purification, and crystallogenesis (2); however, endogenous neurotransmitters usually show poor binding affinity. Low binding affinity with rapid association and dissociation rates leads to conformational heterogeneity that prevents the formation of diffraction-quality crystals. The rapid dissociation rate of agonists also makes it difficult to generate active-state stabilizing proteins, such as the camelid antibodies (nanobodies) that have been used to obtain active-state structures of the β 2 -adrenergic receptor (β 2 AR) (5) and M2 muscarinic receptor (6).To prevent ligand dissociation, irreversible ligation of electrophilic moieties like halomethylketones, isothiocyanates, Michael acceptors, or aziridinium groups of small-molecule ligands with a suitably positioned nucleophilic residue in the receptor has been used (7-16). However, irreversible ligands often suffer from incomplete cross-linking (15) and reduced receptor activation when covalent binding leads to loss of agonist efficacy (10, 16). Furthermore, their highly electrophilic nature and the abundance of nucleophilic groups in biological systems may lead to a low coupling selectivity (7, 8).Disulfide-based cross-linking approaches (17, 18) offer the advantage that the covalent binding of disulfide-...

Structure-based discovery of opioid analgesics with reduced side effects

“…Radioligand depletion assays to test the irreversible binding of compound PZM29 were performed as described previously 46 . Human embryonic kidney 293 (HEK 293) cells were transiently transfected with μOR or the cysteine mutant μOR:N127C using the Mirus TransIT-293 transfection reagent (MoBiTec, Goettingen, Germany), grown for 48 h, harvested, and homogenates were prepared as described 47 .…”

GPCRs through the keyhole: the role of protein flexibility in ligand binding to β-adrenoceptors

“…(Bokoch et al, 2010;Cherezov et al, 2007;Christopher et al, 2013;Hanson et al, 2008;J. Huang, Chen, Zhang, & Huang, 2013;Miller-Gallacher et al, 2014;Moukhametzianov et al, 2011;Rasmussen et al, 2007;2011a;2011b;Ring et al, 2014;Rosenbaum et al, 2011;Wacker et al, 2010;Warne, Edwards, Leslie, & Tate, 2012;Warne et al, 2011;Weichert et al, 2014;Zou, Weis, & Kobilka, 2012) The holo/liganded structures reveal a consistent mode of interaction with the receptors whether the receptor is in its inactive (R) or active (R*) state ( Figure 1). The orthosteric binding pockets of β1-and β2-ARs are almost identical.…”

“…(Cherezov et al, 2007;Christopher et al, 2013;Hanson et al, 2008;J. Huang et al, 2013;Miller-Gallacher et al, 2014;Moukhametzianov et al, 2011;Rasmussen et al, 2007;2011a;2011b;Ring et al, 2014;Rosenbaum et al, 2011;Wacker et al, 2010;Warne et al, 2008;2011;2012;Weichert et al, 2014;Zou et al, 2012) The ethanolamine nitrogen atom is protonated at physiological pH and the carbinol stereochemistry of the higher affinity isomer is as shown in Table 1. An extensive hydrogen-bonding network exists between the core and several further important residues (represented in Figure 1 as yellow arrows indicating the direction of H-bond donation).…”

“…Biogenic amines such as adrenaline possess a catechol head group (1,2-dihydroxyphen-4-yl) attached directly to the core ethanolamine, which forms H-bonds with Ser 5.43 and Ser 5.46 on H5, respectively. (Rasmussen et al, 2011a;2011b;Ring et al, 2013;Rosenbaum et al 2011;Warne et al, 2011;Weichert et al, 2014 to fully facilitate movement of H5; resulting in an antagonist, inverse agonist, or partial agonist ligand. (Bokoch et al, 2010;Cherezov et al, 2007;Christopher et al, 2013;Hanson et al, 2008;J.…”

Elements toward novel therapeutic targeting of the adrenergic system

“…Herein, we briefly illustrate some concepts on how to go from the description of the physiological properties and the pathophysiological roles that have been described by recent publications to a variety of pharmacologic implications and therapeutic applications via applying those new concepts toward finding out new treatments. Importantly, data related to the physiological and pathological implication of the adrenergic receptors supported by structural analytic approaches (Leioatts et al, 2014;Nguyen et al, 2014;Weichert et al, 2014;Zhu et al, 2014) will lead to the definition of new compounds (candidates) for the drug screening. Indeed, the adrenergic receptors structures has similarities with the other GPCRs (Shukla et al, 2014) which make extrapolating some data from some other GPCR, such as pharmacogenetics (Thompson et al, 2014) and signal transmission (Zalewska et al, 2014), a source of details about the structures of the receptors.…”