For the prototypical M2 receptor, it has been previously demonstrated that dualsteric partial agonists can stabilize both active and inactive receptor states, but it remains unclear whether orthosteric partial agonists have a similar mechanism. Here, we apply dynamic pharmacophore models to unveil binding mode ensembles for classical M2 partial agonists. We report correlations between the spatial distribution of lipophilic contacts and ligand efficacy and demonstrate the applicability of dynamic pharmacophore models to analyze subtle binding mode changes. Partial agonism at the receptor level can translate into clinically desired effects while reducing adverse side effects. Thus, an understanding of the underlying structural mechanism is essential for tailored drug design.

Pilocarpine (1), a secondary metabolite of several Pilocarpus species, is a therapeutically used partial agonist of muscarinic acetylcholine receptors (mAChRs). The available pharmacological data and structure-activity relationships do not provide comparable data for all five receptor subtypes. In this study, pilocarpine (1), its epimer isopilocarpine (2), racemic analogues pilosinine (3) and desmethyl pilosinine (4), and the respective hybrid ligands with a naphmethonium fragment (5-C6 to 8-C6) were synthesized and analyzed in mini-G nano-BRET assays at the five mAChRs. In line with earlier studies, pilocarpine was the most active compound among the orthosteric ligands 1-4. Computational docking of pilocarpine and isopilocarpine to the active M2 receptor suggests that the trans-configuration of isopilocarpine leads to a loss of the hydrogen bond from the lactone carbonyl to N6.52, explaining the lower activity of isopilocarpine. Hybrid formation of pilocarpine (1) and isopilocarpine (2) led to an inverted activity rank, with the trans-configured isopilocarpine hybrid (6-C6) being more active. The hydrogen bond of interest is formed by the isopilocarpine hybrid (6-C6) but not by the pilocarpine hybrid (5-C6). Hybridization thus leads to a modified binding mode of the orthosteric moiety, as the binding mode of the hybrid is dominated by the high-affinity allosteric moiety.

In the last few years, fluorescence resonance energy transfer (FRET) receptor sensors have contributed to the understanding of GPCR ligand binding and functional activation. FRET sensors based on muscarinic acetylcholine receptors (mAChRs) have been employed to study dual-steric ligands, allowing for the detection of different kinetics and distinguishing between partial, full, and super agonism. Herein, we report the synthesis of the two series of bitopic ligands, 12-Cn and 13-Cn, and their pharmacological investigation at the M-1, M-2, M-4, and M-5 FRET-based receptor sensors. The hybrids were prepared by merging the pharmacophoric moieties of the M-1/M-4-preferring orthosteric agonist Xanomeline 10 and the M-1-selective positive allosteric modulator 77-LH-28-1 (1-[3-(4-butyl-1-piperidinyl)propyl]-3,4-dihydro-2(1H)-quinolinone) 11. The two pharmacophores were connected through alkylene chains of different lengths (C3, C5, C7, and C9). Analyzing the FRET responses, the tertiary amine compounds 12-C5, 12-C7, and 12-C9 evidenced a selective activation of M-1 mAChRs, while the methyl tetrahydropyridinium salts 13-C5, 13-C7, and 13-C9 showed a degree of selectivity for M-1 and M-4 mAChRs. Moreover, whereas hybrids 12-Cn showed an almost linear response at the M-1 subtype, hybrids 13-Cn evidenced a bell-shaped activation response. This different activation pattern suggests that the positive charge anchoring the compound 13-Cn to the orthosteric site ensues a degree of receptor activation depending on the linker length, which induces a graded conformational interference with the binding pocket closure. These bitopic derivatives represent novel pharmacological tools for a better understanding of ligand-receptor interactions at a molecular level.

Allosteric coupling describes a reciprocal process whereby G-protein-coupled receptors (GPCRs) relay ligand-induced conformational changes from the extracellular binding pocket to the intracellular signaling surface. Therefore, GPCR activation is sensitive to both the type of extracellular ligand and intracellular signaling protein. We hypothesized that ligand-specific allosteric coupling may result in preferential (i.e., biased) engagement of downstream effectors. However, the structural basis underlying ligand-dependent control of this essential allosteric mechanism is poorly understood. Here, we show that two sets of extended muscarinic acetylcholine receptor M1 agonists, which only differ in linker length, progressively constrain receptor signaling. We demonstrate that stepwise shortening of their chemical linker gradually hampers binding pocket closure, resulting in divergent coupling to distinct G-protein families. Our data provide an experimental strategy for the design of ligands with selective G-protein recognition and reveal a potentially general mechanism of ligand-specific allosteric coupling.

The muscarinic M1 acetylcholine receptor is an important drug target for the treatment of various neurological disorders. Designing M1 receptor-selective drugs has proven challenging, mainly due to the high conservation of the acetylcholine binding site among muscarinic receptor subtypes. Therefore, less conserved and topographically distinct allosteric binding sites have been explored to increase M1 receptor selectivity. In this line, bitopic ligands, which target orthosteric and allosteric binding sites simultaneously, may provide a promising strategy. Here, we explore the allosteric, M1-selective BQCAd scaffold derived from BQCA as a starting point for the design, synthesis, and pharmacological evaluation of a series of novel bitopic ligands in which the orthosteric moieties and linker lengths are systematically varied. Since β-arrestin recruitment seems to be favorable to therapeutic implication, all the compounds were investigated by G protein and β-arrestin assays. Some bitopic ligands are partial to full agonists for G protein activation, some activate β-arrestin recruitment, and the degree of β-arrestin recruitment varies according to the respective modification. The allosteric BQCAd scaffold controls the positioning of the orthosteric ammonium group of all ligands, suggesting that this interaction is essential for stimulating G protein activation. However, β-arrestin recruitment is not affected. The novel set of bitopic ligands may constitute a toolbox to study the requirements of β-arrestin recruitment during ligand design for therapeutic usage.

Deterioration or inborn malformations of the cardiac conduction system (CCS) interfere with proper impulse propagation in the heart and may lead to sudden cardiac death or heart failure. Patients afflicted with arrhythmia depend on antiarrhythmic medication or invasive therapy, such as pacemaker implantation. An ideal way to treat these patients would be CCS tissue restoration. This, however, requires precise knowledge regarding the molecular mechanisms underlying CCS development. Here, we aimed to identify regulators of CCS development. We performed a compound screen in zebrafish embryos and identified tolterodine, a muscarinic receptor antagonist, as a modifier of CCS development. Tolterodine provoked a lower heart rate, pericardiac edema, and arrhythmia. Blockade of muscarinic M3, but not M2, receptors induced transcriptional changes leading to amplification of sinoatrial cells and loss of atrioventricular identity. Transcriptome data from an engineered human heart muscle model provided additional evidence for the contribution of muscarinic M3 receptors during cardiac progenitor specification and differentiation. Taken together, we found that muscarinic M3 receptors control the CCS already before the heart becomes innervated. Our data indicate that muscarinic receptors maintain a delicate balance between the developing sinoatrial node and the atrioventricular canal, which is probably required to prevent the development of arrhythmia.

For probing biomolecular interactions or conformational changes in a live-cell setting the distance depending Fluorescence resonance energy transfer (FRET) is often the method of choice. The G-protein-coupled receptor (GPCR) superfamily mediates cellular responses and communication across cellular membranes, and is the largest known class of molecular targets with proven therapeutic value. On the one hand, real-time monitoring of membrane receptors and their interactions is vital to understand how they act in concert. On the other hand, the design of GPCR FRET probes is crucial to ensure unhampered functionality and binding kinetics of the molecular complex. Thus, such FRET probes usually require labeling compromises concerning donor-acceptor positions, distances and orientations, which limit the detectability of emitter signals in general, and the dynamic range of the FRET changes in particular. Here, we present an approach to optimize the energy transfer without changing the design of the FRET probe. We show that gold coated glass cover slips allow reinforcing the otherwise forbidden donor-acceptor energy transfer by virtual optimization of the dipole orientation. First, we confirm the resulting enhanced FRET on our nano-coatings for the inactive M1 muscarinic acetylcholine receptors (mAChRs) labeled with a CFP-FlAsH pair in classical bleaching experiments. Second, we demonstrate the advantage of this enhanced FRET technique for ligand binding studies in live cells by the increased dynamic FRET response between the inactive and active state of the M1 mAChR receptor. We believe that our techniques has particular potential for pharmaceutical drug screening to boost non-ideal FRET probes, and thus amplify signal responses without interfering with the well-characterized molecular assay.

The G-protein-coupled receptor (GPCR) superfamily mediates cellular responses and communication across cellular membranes and is the largest known class of molecular targets with proven therapeutic value. For probing conformational changes of GPCRs and others in a live cell setting, fluorescence resonance energy transfer (FRET) is usually the method of choice. FRET probes often require careful labeling procedures, elaborate characterization, and assay optimization to provide both physiologically relevant probes with unaltered pharmacology and a sufficient dynamic range of the FRET changes. Here, we present an approach to optimize the energy transfer without changing the design of the FRET probe. We show that gold-coated glass coverslips reinforce the otherwise forbidden donor acceptor energy transfer by virtual optimization of the dipole orientation. First, we confirm the resulting enhanced FRET efficiency on our nanocoatings for the inactive M1 muscarinic acetylcholine receptor (mAChR) labeled with a FRET pair of cyan fluorescent protein and fluorescein arsenical hairpin binder in classical bleaching experiments. Second, we show the advantage of this enhanced FRET technique for ligand binding studies in live cells, by the increased dynamic FRET response between the inactive and active states of the muscarinic acetylcholine receptor M1 subtype. Our method is not limited to GPCRs and thus has general potential for surface-bound FRET approaches. We believe our technique is particularly suited for pharmaceutical drug screening to boost FRET probes, in which it is highly desired to amplify signal responses without interfering with the well-characterized assay.

Within the past decade, a large increase in structural knowledge from crystallographic studies has significantly fostered our understanding of the structural biology of G protein-coupled receptors (GPCRs). However, information on dynamic events upon receptor activation or deactivation is not yet readily accessed by these structural approaches. GPCR-based fluorescence resonance energy transfer or bioluminescence resonance energy transfer sensors or sensors for interacting proteins (e.g., G proteins or arrestins) can in part cover this gap. The principal design of such sensors was reported 15 years ago. Since then, sensors for almost 20 different GPCRs have been designed. If used with necessary controls and cautious interpretation, such sensors can contribute significantly to our understanding of the basic mechanisms of GPCR function and beyond. In this review, we will discuss the recent developments in this area of GPCR dynamics.

The investigation of the mode and time course of the activation of G-protein-coupled receptors (GPCRs), in particular muscarinic acetylcholine (mACh or M) receptors, is still in its infancy despite the tremendous therapeutic relevance of M receptors and GPCRs in general. We herein made use of a dualsteric ligand that can concomitantly interact with the orthosteric, that is, the neurotransmitter, binding site and an allosteric one. We synthetically incorporated a photoswitchable (photochromic) azobenzene moiety. We characterized the photophysical properties of this ligand called BQCAAI and investigated its applicability as a pharmacological tool compound with a set of FRET techniques at the M1 receptor. BQCAAI proved to be an unprecedented molecular tool; it is the first photoswitchable dualsteric ligand, and its activity can be regulated by light. We also applied BQCCAI to investigate the time course of several receptor activation processes.

Aiming to design partial agonists as well as allosteric modulators for the M1 muscarinic acetylcholine (M1AChR) receptor, two different series of bipharmacophoric ligands and their structural analogues were designed and synthesized. The hybrids were composed of the benzyl quinolone carboxylic acid (BQCA)-derived subtype selective allosteric modulator 3 and the orthosteric building block 4-((4,5-dihydroisoxazol-3-yl)oxy)-N,N-dimethylbut-2-yn-1-amine (base of iperoxo) 1 or the endogenous ligand 2-(dimethylamino)ethyl acetate (base of acetylcholine) 2, respectively. The two pharmacophores were linked via alkylene chains of different lengths (C4, C6, C8, and C10). Furthermore, the corresponding structural analogues of 1 and 2 and of modified BQCA 3 with varying alkyl chain length between C2 and C10 were investigated. Fluorescence resonance energy transfer (FRET) measurements in a living single cell system were investigated in order to understand how these compounds interact with a G protein-coupled receptor (GPCR) on a molecular level and how the single moieties contribute to ligand receptor interaction. The characterization of the modified orthosteric ligands indicated that a linker attached to an orthoster rapidly attenuates the receptor response. Linker length elongation increases the receptor response of bitopic ligands, until reaching a maximum, followed by a gradual decrease. The optimal linker length was found to be six methylene groups at the M1AChR. A new conformational change is described that is not of inverse agonistic origin for long linker bitopic ligands and was further investigated by exceptional fragment-based screening approaches.

AbstractÜber die Art und den zeitlichen Verlauf der Aktivierung von G‐Protein‐gekoppelten Rezeptoren (GPCRs), insbesondere der muskarinischen Acetylcholinrezeptoren (mACh‐ oder M‐Rezeptoren), ist trotz der enormen therapeutischen Relevanz von M‐Rezeptoren und GPCRs wenig bekannt. In der vorliegenden Arbeit haben wir deshalb einen dualsteren Liganden eingesetzt, der sowohl mit der orthosteren Bindungsstelle des Rezeptors, d. h. derjenigen des Neurotransmitters, als auch mit einer allosteren Bindungsstelle interagiert, wobei synthetisch eine photoschaltbare (photochrome) Azobenzoleinheit eingebaut wurde. Wir haben die photophysikalischen Eigenschaften des BQCAAI benannten Liganden charakterisiert und seine Einsetzbarkeit als pharmakologisches Werkzeug mit einem Satz von FRET‐Techniken am M1‐Rezeptor untersucht. BQCAAI stellt den ersten photoschaltbaren dualsteren Liganden dar, und seine Aktivität kann durch Licht reguliert werden. Wir haben BQCAAI eingesetzt, um den zeitlichen Ablauf verschiedener Prozesse der Rezeptoraktivierung zu untersuchen.