Klaus Mohr

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 M2‐selective bis(ammonio)alkane‐type allosteric fragments. Radioligand binding in wild‐type and mutant receptors supplemented by receptor docking simulations proved M2 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‐hM2 cells disclosed pathway‐specific signaling. Selective receptor activation (M2>M1>M3) 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, J., Kellershohn, K., Mohr‐Andrä, M., Kebig, A., Prilla, S., Muth, M., Heller, E., Disingrini, T., Dallanoce, C., Bertoni, S., Schrobang, J., Tränkle, C., Kostenis, E., Christopoulos, A., Höltje, H.‐D., Barocelli, E., De Amici, M., Holzgrabe, U., Mohr, K. Dualsteric GPCR targeting: a novel route to binding and signaling pathway selectivity. FASEB J. 23, 442–450 (2009)

Applying label-free dynamic mass redistribution technology to frame signaling of G protein-coupled receptors noninvasively in living cells

Label-free dynamic mass redistribution (DMR) is a cutting-edge assay technology that enables real-time detection of integrated cellular responses in living cells. It relies on detection of refractive index alterations on biosensor-coated microplates that originate from stimulus-induced changes in the total biomass proximal to the sensor surface. Here we describe a detailed protocol to apply DMR technology to frame functional behavior of G protein–coupled receptors that are traditionally examined with end point assays on the basis of detection of individual second messengers, such as cAMP, Ca2+ or inositol phosphates. The method can be readily adapted across diverse cellular backgrounds (adherent or suspension), including primary human cells. Real-time recordings can be performed in 384-well microtiter plates and be completed in 2 h, or they can be extended to several hours depending on the biological question to be addressed. The entire procedure, including cell harvesting and DMR detection, takes 1–2 d.

Rational design of dualsteric GPCR ligands: quests and promise

Dualsteric ligands represent a novel mode of targeting G protein‐coupled receptors (GPCRs). These compounds attach simultaneously to both, the orthosteric transmitter binding site and an additional allosteric binding area of a receptor protein. This approach allows the exploitation of favourable characteristics of the orthosteric and the allosteric site by a single ligand molecule. The orthosteric interaction provides high affinity binding and activation of receptors. The allosteric interaction yields receptor subtype‐selectivity and, in addition, may modulate both, efficacy and intracellular signalling pathway activation. Insight into the spatial arrangement of the orthosteric and the allosteric site is far advanced in the muscarinic acetylcholine receptor, and the design of dualsteric muscarinic agonists has now been accomplished. Using the muscarinic receptor as a paradigm, this review summarizes the way from suggestive evidence for an orthosteric/allosteric overlap binding to the rational design and experimental validation of dualsteric ligands. As allosteric interactions are increasingly described for GPCRs and as insight into the spatial geometry of ligand/GPCR‐complexes is growing impressively, the rational design of dualsteric drugs is a promising new approach to achieve fine‐tuned GPCR‐modulation.

Two-point kinetic experiments to quantify allosteric effects on radioligand dissociation

Allosteric modulation of a receptor may be proved experimentally by demonstrating an altered radioligand dissociation in the presence of the allosteric modulator. Two-point kinetic experiments provide a screening-type approach to determine the delay of radioligand dissociation caused by allosteric modulation. In this article, Evi Kostenis and Klaus Mohr describe a pitfall in the data analysis that may lead to a suboptimum determination of the allosteric potency in the case of monophasic dissociations, and suggest how this problem may be resolved.

The experimental power of FR900359 to study Gq-regulated biological processes

Despite the discovery of heterotrimeric αβγ G proteins ∼25 years ago, their selective perturbation by cell-permeable inhibitors remains a fundamental challenge. Here we report that the plant-derived depsipeptide FR900359 (FR) is ideally suited to this task. Using a multifaceted approach we systematically characterize FR as a selective inhibitor of Gq/11/14 over all other mammalian Gα isoforms and elaborate its molecular mechanism of action. We also use FR to investigate whether inhibition of Gq proteins is an effective post-receptor strategy to target oncogenic signalling, using melanoma as a model system. FR suppresses many of the hallmark features that are central to the malignancy of melanoma cells, thereby providing new opportunities for therapeutic intervention. Just as pertussis toxin is used extensively to probe and inhibit the signalling of Gi/o proteins, we anticipate that FR will at least be its equivalent for investigating the biological relevance of Gq.

Allosteric ligands for G protein-coupled receptors: A novel strategy with attractive therapeutic opportunities

Allosteric receptor ligands bind to a recognition site that is distinct from the binding site of the endogenous messenger molecule. As a consequence, allosteric agents may attach to receptors that are already transmitter‐bound. Ternary complex formation opens an avenue to qualitatively new drug actions at G protein‐coupled receptors (GPCRs), in particular receptor subtype selective potentiation of endogenous transmitter action. Consequently, suitable exploitation of allosteric recognition sites as alternative molecular targets could pave the way to a drug discovery paradigm different from those aimed at mimicking or blocking the effects of endogenous (orthosteric) receptor activators. The number of allosteric ligands reported to modulate GPCR function is steadily increasing and some have already reached routine clinical use. This review aims at introducing into this fascinating field of drug discovery and at providing an overview about the achievements that have already been made. Various case examples will be discussed in the framework of GPCR classification (family A, B, and C receptors). In addition, the behavior at muscarinic receptors of hybrid derivatives incorporating both an allosteric and an orthosteric fragment in a common molecular skeleton will be illustrated.

Decoding Signaling and Function of the Orphan G Protein–Coupled Receptor GPR17 with a Small-Molecule Agonist

Activation of GPR17 prevents oligodendrocyte maturation and reveals that inhibiting GPR17 may be a therapeutic strategy to treat multiple sclerosis. Overcoming a Myelination Maturity Block Demyelinating diseases, such as multiple sclerosis (MS), are characterized by the failure of oligodendrocytes to mature and produce myelin, the protective sheaths surrounding axons. The role of the orphan G protein (heterotrimeric guanine nucleotide–binding protein)–coupled receptor GPR17 in this process is debated. Hennen et al. identified a GPR17-selective small-molecule agonist and showed that application of this agonist induced G protein–mediated signaling that prevented maturation of cultured oligodendrocytes. The findings establish an inhibitory role for GPR17 in the cellular maturation process that enables remyelination of injured axons and suggest that GPR17 may be pharmacologically targeted to treat MS. Replacement of the lost myelin sheath is a therapeutic goal for treating demyelinating diseases of the central nervous system (CNS), such as multiple sclerosis (MS). The G protein (heterotrimeric guanine nucleotide–binding protein)–coupled receptor (GPCR) GPR17, which is phylogenetically closely related to receptors of the “purinergic cluster,” has emerged as a modulator of CNS myelination. However, whether GPR17-mediated signaling positively or negatively regulates this critical process is unresolved. We identified a small-molecule agonist, MDL29,951, that selectively activated GPR17 even in a complex environment of endogenous purinergic receptors in primary oligodendrocytes. MDL29,951-stimulated GPR17 engaged the entire set of intracellular adaptor proteins for GPCRs: G proteins of the Gαi, Gαs, and Gαq subfamily, as well as β-arrestins. This was visualized as alterations in the concentrations of cyclic adenosine monophosphate and inositol phosphate, increased Ca2+ flux, phosphorylation of extracellular signal–regulated kinases 1 and 2 (ERK1/2), as well as multifeatured cell activation recorded with label-free dynamic mass redistribution and impedance biosensors. MDL29,951 inhibited the maturation of primary oligodendrocytes from heterozygous but not GPR17 knockout mice in culture, as well as in cerebellar slices from 4-day-old wild-type mice. Because GPCRs are attractive targets for therapeutic intervention, inhibiting GPR17 emerges as therapeutic strategy to relieve the oligodendrocyte maturation block and promote myelin repair in MS.

Allosteric modulators of ligand binding to muscarinic acetylcholine receptors

Abstract Allosteric modulators of muscarinic receptors act at a site apart from the common ligand binding site of the receptor protein. These compounds affect both of the molecular events that determine the level of equilibrium binding, namely ligand association and dissociation. As a consequence, ligand binding can be elevated, reduced or remain unchanged, depending on the receptor subtype, the type of ligand and the type of allosteric modulator. Thus, allosterically acting drugs represent a novel approach for affecting muscarinic receptors. Notably, they may act as subtype-selective enhancers of acetylcholine binding. This might be exploited therapeutically, to improve cognition or to promote antinociception, for example.

A biased ligand for OXE-R uncouples Gα and Gβγ signaling within a heterotrimer

Differential targeting of heterotrimeric G protein versus β-arrestin signaling are emerging concepts in G protein-coupled receptor (GPCR) research and drug discovery, and biased engagement by GPCR ligands of either β-arrestin or G protein pathways has been disclosed. Herein we report on a new mechanism of ligand bias to titrate the signaling specificity of a cell-surface GPCR. Using a combination of biomolecular and virtual screening, we identified the small-molecule modulator Gue1654, which inhibits Gβγ but not Gα signaling triggered upon activation of Gα(i)-βγ by the chemoattractant receptor OXE-R in both recombinant and human primary cells. Gue1654 does not interfere nonspecifically with signaling directly at or downstream of Gβγ. This hitherto unappreciated mechanism of ligand bias at a GPCR highlights both a new paradigm for functional selectivity and a potentially new strategy to develop pathway-specific therapeutics.

Allosteric Interactions with Muscarinic Acetylcholine Receptors: Complex Role of the Conserved Tryptophan M2422Trp in a Critical Cluster of Amino Acids for Baseline Affinity, Subtype Selectivity, and Cooperativity

In general, the M2 subtype of muscarinic acetylcholine receptors has the highest sensitivity for allosteric modulators and the M5 subtype the lowest. The M2/M5 selectivity of some structurally diverse allosteric agents is known to be completely explained by M2177Tyr and M2423Thr in receptors whose orthosteric site is occupied by the conventional ligand N-methylscopolamine (NMS). This study explored the role of the conserved M2422Trp and the adjacent M2423Thr in the binding of alkane-bisammonio type modulators, gallamine, and diallylcaracurine V. Experiments were performed with human M2 or M5 receptors or mutants thereof. It was found that M2422Trp and M2423Thr independently influenced allosteric agent binding. The presence of M2423Thr may enhance the affinity of binding, depending on the allosteric agent, either directly or indirectly (by avoiding sterical hindrance through its M5 counterpart 478His). Replacement of M2422Trp and of the corresponding M5477Trp by alanine revealed a pronounced contribution of these epitopes to subtype independent baseline affinity in NMS-bound and NMS-free receptors for all agents except diallylcaracurine V. In a few instances, this tryptophan also influenced cooperativity and subtype selectivity. Docking simulations using a three-dimensional M2 receptor model revealed that the aromatic rings of M2177Tyr and M2422Trp, in a concerted action, might fix one of the aromatic moieties of alkane-bisammonio compounds between them. Thus, M2422Trp and the spatially adjacent M2177Tyr, as well as M2423Thr, form a cluster of amino acids within the allosteric binding cleft that is pivotal for both M2/M5 subtype selectivity and baseline affinity of allosteric agents.