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. 2023 Nov 10;6(12):1790–1800. doi: 10.1021/acsptsci.3c00214

Recommended Tool Compounds: Application of YM-254890 and FR900359 to Interrogate Gαq/11-Mediated Signaling Pathways

Jan Hendrik Voss 1,*
PMCID: PMC10714435  PMID: 38093837

Abstract

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The macrocyclic depsipeptides YM-254890 (YM) and FR900359 (FR) are natural products, which inhibit heterotrimeric Gαq/11 proteins with high potency and outstanding selectivity. Historically, pharmacological modulation of Gα proteins was only achieved by treatment with pertussis toxin and cholera toxin, whose application can be tedious and is restricted to the inhibition of Gαi/o proteins and activation of Gαs proteins, respectively. The breakthrough discovery and characterization of YM and FR rendered the closely related Gαq, Gα11, and Gα14 proteins amenable to pharmacological inhibition, and since then, both compounds have become widely used in molecular pharmacology and were also proven to be efficacious in animal models of disease. In the past years, both YM and FR were thoroughly characterized and have substantially contributed to an improved understanding of Gαq/11 signaling on a molecular and cellular level. Yet, the possibilities to interrogate Gαq/11 signaling in complex systems have only been exploited in a very limited number of studies, whose promising initial results warrant further application of YM and FR in basic and translational research. As both compounds have become commercially available as of late, this review focuses on their application in cell-based assays and in vivo systems, highlighting their qualities as tool compounds and providing instructions for their use.

Keywords: FR900359, G protein, tool compound, G protein-coupled receptor, review, YM-254890

G protein-coupled receptors (GPCRs) are the largest family of membrane proteins in the human genome, comprising approximately 800 members.1 GPCRs are the subject of many pharmacological studies, and drug development targeting GPCRs has been tremendously successful due to the modular nature of GPCRs in a complex signaling cascades, their well-accessible binding site, and their recognition of a wide range of chemotypes, e.g., small molecules, peptides, proteins, and lipids.2 The most immediate intracellular effector proteins of GPCRs are heterotrimeric guanine-nucleotide binding proteins (G proteins), which relay signaling from activated receptors to intracellular signaling pathways.3 G proteins are composed of a Gα, Gβ, and Gγ protein subunit, and are located on the inner leaflet of the plasma membrane. Here, the Gα protein is the key factor determining the downstream signaling pathway(s) initiated by an active-state GPCR.4 Notably, free Gβγ subunits can activate distinct effector proteins, yet the role of Gβγ effector specificity has only partially been uncovered.5,6 GPCR signaling converges on the level of G proteins. In total, the human genome encodes for only 16 Gα protein subunits, grouped by sequence similarity into the four subfamilies Gαs, Gαi/o, Gαq/11, and Gα12/13 (Figure 1), as well as 5 Gβ subunits, and 12 Gγ subunits.7

Figure 1.

Figure 1

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Pharmacological modulation of Gα protein subunits. Active state GPCRs promote guanine nucleotide exchange in Gα protein subunits, which then initiate downstream signaling cascades. Gαs proteins are ADP-ribosylated by cholera toxin (CTX), leading to a loss of catalytic activity in the Gαs protein, thereby keeping the Gαs protein in its active GTP-bound state. Gαi/o proteins (except for Gαz) are ADP-ribosylated in their C-termini, blocking interaction with the GPCR, which inactivates the G protein. The Gαq/11 family members Gαq, Gα11, and Gα14 are inactivated by the macrocyclic compounds YM and FR, which prevent nucleotide exchange from GDP to GTP upon receptor activation. To date, Gα12/13 proteins are not amenable to pharmacological modulation. YM: R1 = CH3, R2 = CH3; FR: R1 = C2H5, R2 = C(CH3)2.

A detailed investigation of GPCR-mediated signaling networks can be cumbersome, as most GPCRs do not exclusively activate a single Gα protein or Gα protein subfamily, but rather two or more Gα protein families, recruit β-arrestins, and display complex spatiotemporal signaling patterns.8−10 While several recently developed bioluminescence energy resonance transfer (BRET) biosensors facilitate cellular studies of individual GPCR-Gα interactions, this setup relies on the overexpression of artificial protein constructs in cultured cells.9,11,12 To study the downstream signaling of specific GPCRs in native cells and tissues or whole organisms, potent tool compounds selectively inhibiting individual Gα proteins or subfamilies are highly desirable. However, Gα proteins are much less amenable to pharmacological modulation than their activating receptors. While several compounds, mostly of peptidic nature, have been proposed to act as Gα protein modulators, most of them are not used routinely as tool compounds. This points to issues regarding potency, efficacy, cell-permeability, synthesizability, and subtype selectivity7

Historically, exclusively the bacterial toxins cholera toxin (CTX; activator of the Gαs protein) and pertussis toxin (PTX; inhibitor of the Gαi/o protein family with exception of Gαz) were used as pharmacological modulators of Gα proteins in basic research (Figure 1).13−15 In the early days of G protein research and GPCR pharmacology, CTX and PTX played a major role in identifying the contribution of Gαs or Gαi/o proteins to a given biochemical event, but their macromolecular structure, their toxicity, and their limited coverage of Gα proteins have drawbacks for their everyday use. The complex pathological mechanism of CTX, reviewed in ref (15), results in adenosine diphosphate (ADP)-ribosylation of the Gαs protein nucleotide binding site, which leads to a loss of its catalytic activity by keeping Gαs in a guanosine triphosphate (GTP)-bound state, subsequently uncoupling cAMP production from receptor activation. PTX inhibits Gαi/o proteins (with exception of the Gαz protein) by ADP-ribosylation of a C-terminal cysteine, thereby preventing the recognition of active-state GPCRs and subsequent receptor-mediated nucleotide exchange in ADP-ribosylated Gα proteins. Recently, a novel PTX-like protein, termed OZITX, was engineered to ADP-ribosylate a Gαi/o-family wide conserved C-terminal asparagine residue and is thus capable of targeting Gαz proteins.16

In the past decade, the macrocyclic depsipeptides YM-254890 (YM) and FR900359 (FR), also known by the name UBO-QIC, have become widely used tool compounds in molecular pharmacology as specific inhibitors of the Gαq/11 protein family (Figure 1).17 In a cellular context, activation of Gαq/11 proteins induces phospholipase C-β (PLC-β)-mediated production of the second messenger molecules diacylglycerol (DAG) and inositol trisphosphate (IP3) and subsequent liberation of Ca2+ from intracellular storages.18 Further signaling pathways triggered by Gαq/11 protein activation include (but are not limited to) several pro-mitogenic pathways, such as extracellular signal regulated kinase (ERK) phosphorylation, Akt phosphorylation, RhoA stimulation, and Hippo/YAP signaling, associating Gαq/11 protein activation with cancer malignancy.18,19 Accordingly, mutational activation of Gαq/11 proteins was found to be causative for uveal melanoma, the most prevalent type of eye cancer in humans.19−21

Both YM and FR act as guanine nucleotide dissociation inhibitors (GDI), i.e., they prevent the dissociation of guanosine diphosphate (GDP) from the inactive Gα protein triggered by interaction with an active GPCR, and therefore arrest the Gα protein in its inactive state, irresponsive to stimuli from active GPCRs.22,23 Out of the four members of the Gαq/11 protein family, YM and FR engage three of them (Gαq, Gα11, Gα14) with high affinity, sparing the evolutionarily more distant Gα15/16 proteins (Gα15 and Gα16 denote the human and mouse paralog, respectively).22,24 Following the recent commercialization of FR, this review, as a part of the review series on recommended tool compounds published in ACS Pharmacology and Translational Science,25 aims to deliver an in-depth summary focused on the pharmacology and application of YM and FR.

Chemical Structure and Biosynthesis

YM and FR are natural products of bacterial origin sharing a nearly identical, complex structure, which differs only in two residues (Figure 2A+B). Their macrocyclic backbone is composed out of seven building blocks (d-phenyllactic acid (d-Pla), N-methyl-dehydroalanine (N-Me-Dha), alanine (Ala), N-methyl-alanine (N-Me-Ala), hydroxyleucine (Hle), N–O-dimethyl-threonine (N,O-Me2-Thr), and N-acetyl-hydroxyleucine (N-Ac-Hle; N-acetyl-threonine (N-Ac-Thr) in YM, see Figure 2B). A further hydroxyleucine side chain, which is N-acetylated in case of YM (N-Ac-Hle) and N-propionylated (N-Pp-Hle) in case of FR, is attached to the hydroxyl group of the core hydroxyleucine residue.

Figure 2.

Figure 2

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Chemical structure and biosynthesis of YM and FR. (A) Common chemical structure of YM and FR (YM: R1 = CH3, R2 = CH3; FR: R1 = C2H5, R2 = C(CH3)2). Shared residues forming the macrocyclic backbone are highlighted in different colors. d-Pla, d-phenyllactic acid; N-Me-Dha, N-methyl-dehydroalanine; Ala, alanine; N-Me-Ala, N-methyl-alanine; Hle, hydroxyleucine; N,O-Me2-Thr, N,O-dimethyl-threonine. (B) Chemical structures of YM (left) and FR (right). Unique residues are highlighted in blue (for YM) and purple (for FR), respectively. N-Ac-Hle, N-acetyl-hydroxyleucine; N-Ac-Thr, N-acetyl-threonine; N-Pp-Hle, N-propionyl-hydroxyleucine. Procedures to isolate each natural product are listed below the respective chemical structure. (C) Schematic depiction of the FR biosynthetic pathway, as postulated by Hermes et al.29

YM was isolated from Chromobacterium sp. QS3666, a bacterial strain discovered in a soil sample from Tokyo, Japan.26 The biosynthetic process yielding YM is unknown, as this bacterial strain was never made publicly available and no related information about the biosynthetic gene cluster (BGC) producing YM can be retrieved from the original publication. FR had first been isolated from the leaves of the plant Ardisia crenata in 1988,27 but only later in 2015, it was pharmacologically characterized as a potent and specific Gαq/11 protein inhibitor.22 As FR potently inhibits insect Gαq proteins, it protects the plant from predatory insect larvae.28 Subsequently, a large effort has been put into the elucidation of FR biosynthesis, whose biosynthetic pathways will be outlined in brief here. For a more comprehensive review focusing on the discovery and biosynthesis of FR, see Hermes et al.29

FR is naturally produced by the bacterium Candidatus burkholderia crenata, which is located in leaf nodules of A. crenata.28,30 This obligate endosymbiont harbors a BGC on a plasmid encoding for eight genes, termed frsA-H. In the proposed synthesis pathway, a nonribosomal protein synthetase composed of the enzymes FrsD-G forms a seven-residue linear peptide chain which is cyclized by the FrsG transesterification domain. In a final step, the propionylated β-hydroxyleucine side chain is attached to the free hydroxyl group of the FR-core molecule by the FrsA transesterification domain (Figure 2C).29,31 A highly similar BGC was discovered in Chromobacterium vaccinii, which, in contrast to Cand. burkholderia crenata, is a cultivable producer of FR, yielding approximately 2.5 mg FR per liter of bacterial culture.31,32

The natural producers of both YM and FR do not exclusively synthesize the respective compounds, but also generate several related side products harboring minor chemical modifications.31−34 Several of these compounds, e.g., FR-1/2 or YM-254891/2, apparently retain the pharmacological properties of the parent compounds, yet already subtle structural deviations were shown to have a profound impact on binding kinetics due to the complex Gαq/11 inhibitor pharmacophore.33−35 More profound chemical differences, such as truncation of the branched N-Ac/Pp-Hle side chain, resulted in a large decrease in affinity.31,33 Intriguingly, natural derivatives of YM and FR contained similar modifications relative to the original compound, such modifications or truncation of the side chain, hinting at mechanistically similar biosynthetic pathways of YM and FR. Furthermore, another analogue of YM and FR, Sameuramide A, has been isolated from a marine tunicate,36 which is chemically identical to the FR derivative FR-3 (relative to FR, FR-3, and Sameuramide A contain a N-Pp-Hle instead of an N-Ac-Hle residue in its macrocyclic core). While—similar to YM—information about the BGC yielding Sameuramide A is not available, it is striking that nearly identical compounds with high affinity toward Gαq/11 proteins have apparently evolved on at least three occasions.

In addition to the isolation of the compounds from natural sources, the total synthesis of YM and FR was first reported in 2016.37 The synthesis approach allowed for a versatile exchange of several building blocks, which led to the generation of several novel YM analogues.37−40 In an accompanying structure–activity relationship, none of the generated molecules displayed an increased inhibitory potency in an inositol monophosphate (IP1) accumulation assay readout.37,41 Exploitation of this synthesis route in the future may generate diverse derivatives, which could display affinities toward other, currently undrugged Gα proteins, in particular the most closely related Gα15 protein.

Chemical and Pharmacokinetic Properties

As the chemical structures of YM and FR are very closely related, it would be intuitive for their chemical and pharmacokinetic properties to resemble each other closely. A direct comparison, however, detected in part substantial differences between both compounds.42 YM and FR display sufficient solubility in water for all biological applications displaying a similar solubility product of 189 μM for FR and 88 μM for YM, and a calculated log P value of 1.86 and 1.37, respectively.42 The solubility in ethanol and DMSO is high, and both compounds can be stored as a powder or in a 1 mM DMSO stock at 4 °C for longer periods of time without notable loss of activity. Chemical stability was found to be very high in simulated gastric fluid and mildly alkaline solution (pH 9), but impaired under strongly alkaline conditions (pH 11). Here, YM decomposed fully and rapidly, whereas only about 25% of FR degraded within a period of 4 h. FR-core, a biosynthesis intermediate lacking the N-Pp-Hle side chain, was identified as a decay product under alkaline conditions.42 Interestingly, both depsipeptides formed an isomer with equal mass under all conditions, which was identified in a later study as a biologically inactive degradation product that had undergone macrocyclic ring cleavage (between the backbone N,O-Me2-Thr and N-Ac-Hle residue of FR/N-Ac-r residue of YM) and dehydration (at the N-Ac-Hle residue of FR/N-Ac-r residue of YM).43

An in vitro assessment of YM and FR’s pharmacokinetic properties yielded mostly similar results for both compounds. Their bioavailability was predicted to be low in an intestinal absorption model (Caco2 permeation assay) with the basal-apical transportation rate being greater than the apical-basal transportation rate. The exceptionally high basal-apical transport rate suggests that both compounds are substrates of efflux transporters, such as P-glycoproteins. Given the direction of transport for these compounds, the study postulates a low oral bioavailability and low penetration into the central nervous system. In plasma and lung tissue, YM and FR were found to be relatively stable (<10% decay after 4 h). In contrast, both compounds were metabolized rather quickly in liver microsomes, displaying half-lives of 27 min for YM and a far shorter half-life of 8 min for FR, yet the metabolites and the metabolic pathways remain unknown. Both compounds did not inhibit the investigated drug-metabolizing cytochrome P450 (CYP) enzymes at therapeutically relevant concentrations. At excessive concentrations of 10 μM, YM and FR inhibited the CYP3A4 enzyme by slightly above 50%, but none of the other tested enzymes.42

Furthermore, in vivo distribution of both compounds was assessed in mouse, following 7 days of intratracheal application (5 μg YM and FR per animal and day). Organs of interest were homogenated and extracted with methanol, followed by a LC-MS/MS analysis protocol to quantify the levels of YM and FR in the respective organs.44 The highest levels of both compounds were detected in the kidney and in the lung. A high level of YM was also found in the liver. FR was not found here, which is likely due to its fast liver metabolization. Upon 21 days of intratracheal application (2.5 μg YM and FR, twice daily), near-exclusive drug accumulation in the lung was observed.42,45 In agreement with previously mentioned absorption model data classifying YM and FR as P-glycoprotein substrates, neither compound was detected in mouse brain tissue after intratracheal application.42 After a one-time oral application of 0.2 mg FR, the compound could be recovered from several organ extracts, implying that FR is at least to some degree orally bioavailable. After oral application, the highest concentrations of FR were recovered from the gut and remarkably also from the eye, with low compound levels detected in liver, kidney, lung, heart, and fatty tissue.44 As tissue distribution was addressed by using LC-MS/MS, metabolites, or other chemically modified derivatives of YM and FR were not quantified from homogenized organ extracts.

In summary, YM and FR do not fulfill the criteria for drug-like molecules in the classical sense defined by Lipinski’s rule of five due to their high molecular weight.46 Reflective of that, the oral bioavailability of both compounds is predicted to be low, which is the case for 61% of FDA-approved macrocyclic drugs.47 However, when applied by other routes, e.g., intratracheal or intraperitoneal application, YM and FR displayed sufficiently good stability in plasma and lung tissue, no excessive undesired tissue accumulation, and no CYP enzyme inhibition at relevant concentrations, making them generally suitable for in vivo application. A caveat for their in vivo use is the excessive and, especially for FR, quick metabolization yielding unidentified molecules of unknown biological activity.

Molecular Mechanism of Gαq Protein Inhibition by YM and FR

Both YM and FR were characterized as GDIs at Gαq, Gα11, and Gα14 proteins. The mechanism of action has been confirmed for both YM and FR by [3H]GDP-dissociation experiments with purified Gαq proteins in two independent sets of experiments.22,23 A crystal structure of YM in complex with the heterotrimeric, GDP-bound Gαqβ1γ2 protein complex (2.9 Å resolution) demonstrated that the compound binds to the linker I/switch I area of the Gαq protein. This area connects the protein’s Ras-like domain to its helical domain (see Figure 3A), and binding of YM and FR prevents domain separation, which is a prerequisite for nucleotide exchange—the rate-limiting step in Gα protein activation.23,48 In this crystal structure, direct interactions with the nearby Gβ subunit were not observed, however, the electron density of the ligand in this structure is in part ambivalent.49 In a recent preprint describing high-resolution crystal structures of YM and FR in complex with G11 (1.7 and 1.43 Å resolution, respectively), molecular interactions between the N-Ac/Pp-Hle side chain of Gαq/11 inhibitors and the Gβ residue R96 were observed. These structures and the supporting functional data suggest an additional mechanism of action for these compounds acting also as “molecular adhesives”, which stabilize a heterotrimeric G protein complex by linking the Gα and Gβγ subunits, thereby preventing molecular recognition of Gαq/11 effector proteins.71

Figure 3.

Figure 3

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Binding mode and mechanism of action of Gαq/11 inhibitors. (A) Surface representation of the heterotrimeric Gαqβ1γ2 protein in complex with YM (red) and GDP (yellow) (PDB 3AH8(23)), showing binding of YM to a site located at the linker I/switch I region between the Gαq Ras-like domain (RasD, blue) and its helical domain (HD, light blue) near the interface with the Gβ subunit (gray). (B) Binding of FR to the wild-type (wt) Gαq protein inhibits G protein-dependent signaling in readouts covering different signaling cascades, including mitogenic signaling pathways (exemplified by ERK and Akt phosphorylation, red) and canonical, PLC-β-mediated pathways (blue). (C) Binding of FR to constitutively GTP-bound, receptor-uncoupled GαqQ209L/P preferentially silences mitogenic signaling over PLC-β dependent signaling.

YM and FR bind Gαq proteins with high affinity and broadly inhibit Gαq/11 activity as shown in several signaling readouts (Figure 3B). Radioligand binding experiments with probes derived from YM and FR by catalytic hydrogenation of the exocyclic double bond reported pKD values of 7.96 and 8.45 for YM- and FR-derived radiotracers, respectively, in saturation binding assays using human platelet membrane preparations.24 Notably, binding of radiolabeled Gαq inhibitors is not allosterically modulated by salts, lipids, GPCR agonists, and nucleotides.24A direct comparison of binding parameters is provided in Table 1.

Table 1. Binding Constants and Biological Activity of YM and FR in Multiple Assay Systems.

binding constants YM FR reference
apparent affinity, saturation binding of derived radioligand, human platelet membranes, 37 °C (pKD) 7.96 8.45 Kuschak et al.24
apparent affinity, pseudohomologous competition binding between radioligand and unlabeled parent compound, human platelet membranes, 37 °C (pKi) 8.20 8.39 Kuschak et al.24
association half-life of derived radioligand, human platelet membranes, 37 °C (min) 3.6 6.7 Kuschak et al.24
dissociation half-life of derived radioligand, human platelet membranes, 37 °C (min) 3.8 92 Kuschak et al.24
dissociation half-life of parent compound, HEK293 cell membrane, 37 °C (min) 40 323 Voss et al.35
biological activity YM FR reference
inhibition of calcium mobilization, HEK293-Gαq cells (pIC50) 8.09 8.20 Voss et al.50
inhibition of carbachol-induced IP1 accumulation, CHO-M1R cells (pIC50) 7.03 7.49 Xiong et al.37
inhibition of ADP-induced platelet aggregation (IC50, μM) 0.26 n.d. Taniguchi et al.26,51
label-free dynamic mass redistribution, stimulated by an EC80 conc. of carbachol (pIC50) 6.3 6.3 Malfacini et al.52
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Kinetic experiments using these radiotracers unveiled a huge discrepancy in unbinding kinetics between the fast dissociating YM-derived radiotracer (dissociation half-life: 3.8 min) and the slow-dissociating FR-derived radiotracer (dissociation half-life: 92 min), consistent with previous reports claiming that FR-, but not YM-mediated inhibition of Gαq proteins, was washout-resistant.22,24 The molecular basis for pseudoirreversible binding of FR was pinpointed to be a stronger lipophilic interaction between the isopropyl group exclusively present in FR and the neighboring amino acids of the Gαq binding site.24,50 In a different study, establishing a structure–affinity and structure–kinetics relationship of unlabeled YM, FR, and their derivatives, an even longer dissociation half-life was determined for the parent, non-hydrogenated compound FR (323 min) using a competition association binding setup. Interestingly, the dissociation half-life of YM was also found to be far longer than that of its radiolabeled derivative (40 vs 3.8 min).35

Structure–activity relationships, structure–kinetics relationships, and mutagenesis experiments of the Gαq inhibitor binding site found that even minor modifications to the ligand–protein interaction all result in a major decrease of potency, affinity, and residence time, suggesting that major parts of YM and FR (and vice versa, of the Gαq inhibitor binding site) contribute to high-affinity ligand binding.35,37,50,53

Initial binding studies and functional characterization demonstrated a high degree of selectivity toward Gαq, Gα11, and Gα14 proteins (which share a fully conserved inhibitor binding site) over the closest paralogue, Gα15 (five residue exchanges in the binding site), and all other Gα protein families.22,24,52 Gα15 inhibition by FR is theoretically possible, but requires high inhibitor concentrations exceeding 1 μM, while YM could not inhibit the Gα15 protein at all in the tested concentration range.52 Two independent reports have claimed putative off-target action of YM and FR, i.e., not mediated by inhibition of Gαq/11 proteins; in one case it was hypothesized that the observed effects are caused by blockade of Gβγ activity following Gαi/o activation,54 in the other case by broad-spectrum inhibition of Gαq/11 and Gαs proteins and pathway-selective inhibition of Gαi/o-mediated signaling.55 Making use of an FR-resistant Gαq protein mutant, Patt et al. demonstrated that even at high concentrations, YM and FR did not display off-target effects in a plethora of readouts and any biological effect required the presence of wild-type Gαq proteins, inferring that the previously observed effects can be attributed to Gαq/11 inhibition of YM and FR.56

A further intriguing aspect of the mode-of-action of FR has come from its application to GTPase-deficient Gαq and Gα11 proteins bearing the Q209L/P mutations, which is causative for a notable fraction of uveal melanoma cases in humans.21 GαqQ209L/P proteins are constitutively bound to GTP and are therefore active irrespective of receptor input. On the basis of its classification as a GDI, it would be expected that FR cannot inhibit these mutant Gαq proteins. Unsuspectedly, FR (but so far not YM) was found to preferentially inhibit the mitogenic ERK and AKT signaling pathways in cell-based assays without attenuating canonical PLC-β-dependent signaling (measured by IP1 accumulation assays) in HCmel12 cells (Figure 3C).19 This finding could not be reproduced in several other GαqQ209L/P-expressing cell lines, leaving the inhibitory mechanism of FR on PLC-mediated signaling downstream of GαqQ209L incompletely understood at present.19 It may be possible that elevated IP1 levels in FR-unresponsive cells lines are mediated by high Gα16 activity.

Inhibition of mitogenic Gαq/11 signaling by application of FR to models expressing GαqQ209L/P mutants has been demonstrated in several instances. Treatment with FR resulted in reduced tumor growth in mouse xenografts,19,57 reduced glucose uptake in uveal melanoma cells in vitro and in vivo,58 and decreased colony formation of uveal melanoma cells in 3D cell culture.59 As these effects cannot be attributed to the GDI properties of FR, inhibitor binding was hypothesized to switch the nucleotide preference of GαqQ209L/P from GTP to GDP in an allosteric fashion.17 The additionally proposed “molecular adhesive” mechanism of action of YM and FR might provide an explanation for effector protein silencing downstream of constitutively active GαqQ209L/P.71

Application of YM and FR as Tool Compounds in Molecular Pharmacology

YM and FR possess several qualities of probe compounds:60 they are highly potent, showing a binding affinity in the low nanomolar to subnanomolar range,24 and a comparable inhibitory potency in calcium mobilization assays.50 In other assay readouts, such as dynamic mass redistribution assays or ERK phosphorylation assays, the potency has been found to be lower, but still in a satisfactory range (IC50 < 1 μM).52 Biophysical proof of target engagement has been demonstrated in several ways, most notably by X-ray crystallography of YM and FR in complex with the heterotrimeric, GDP-bound Gαq/11β1γ2 protein23 and by radioligand binding.24 Despite the unsaturated moiety in the dehydroalanine block of YM and FR that forms a potential Michael acceptor (commonly an α,β-unsaturated carbonyl group that can react with a nucleophile, forming a carbon–carbon bond), both compounds do not bind in a covalent fashion.22 However, no inactive analogue of those compounds is publicly available. Compounds such as YM-1 or FR-core, which share a major part of the structure and show greatly reduced affinity for the Gαq protein, were only synthesized or purified at laboratory scale. Conversely, no potent but structurally unrelated Gαq protein inhibitor is known to date.

Both molecules have been extensively characterized in cell-based assays in multiple readouts, including, but not limited to calcium mobilization, IP1 accumulation, platelet aggregation, serum response element reporter gene assays, and dynamic mass redistribution.38,50,51,56 Virtually any investigated readout based on Gαq/11 protein activity was found to be suitable to characterize the activity of YM and FR, and in turn, YM and FR can be used as tool compounds to block any of these pathways. Notably, the potency derived from different assay systems can vary to a large extent: calcium mobilization assays yielded IC50 values that corresponded fairly well to the affinity values determined in radioligand binding experiments,50 while IP1 accumulation assays yielded potencies that were approximately 1 order of magnitude lower,37 and label-free dynamic mass redistribution assays potencies were reduced by a further order of magnitude.52 Thus, the concentration of YM and FR required for full signal suppression is highly dependent on the employed assay system, and if possible, a concentration-response curve should be recorded prior to performing experiments in a yet uncharacterized readout to find the optimal working concentration for YM or FR. Several published potencies in different assay systems for YM and FR are listed in Table 1.

In a laboratory setting, YM and FR are well-usable compounds that show neither significant solubility nor stability issues, display no off-target effects even at high concentrations, bind quickly, and are not directly cytotoxic. These properties are clearly advantageous compared to PTX, which usually requires at least 4 h of incubation time or cotransfection of a constitutive PTX expression plasmid. For pilot experiments in cell-based readouts, a Gαq/11 inhibitor concentration of 100 nM and a preincubation time of 30 min prior to intervention or measurement is a good starting point. For most setups, this concentration will be sufficient as it is approximately 10 times higher than the apparent affinity of the drugs to the Gαq protein and will therefore inhibit around 90% of the Gαq/11 proteins. In cases requiring full inhibition of Gαq/11 proteins, an inhibitor concentration of 1 μM may be desirable. Higher inhibitor concentrations, such as 10 μM or even 1 mM, are typically not required, nevertheless off-target effects are not expected even for very high concentrations (except for a putative partial inhibition of the Gα15 protein,61 in the case it is expressed in the investigated system).52,56 Membrane permeation of YM and FR does not pose a problem, exemplified by YM- and FR radiotracer binding to intact platelets with similar association kinetics.24 A thirty-minute incubation prior to stimulation of a Gαq/11-mediated signal exceeds the association half-life of the compound by far and should therefore be sufficient for the inhibitor to bind at near-equilibrium conditions.62 Longer inhibitor exposure, e.g., in overnight experiments, is not expected to cause cytotoxic side effects as demonstrated by the application of FR in serum response element assays with a total FR incubation time of 7 h or more.19,56 Washing out YM is possible due to its shorter residence time at the Gαq protein (but can require several washing steps with in-between incubation periods), while FR binds in a pseudoirreversible and thus wash out-resistant manner.22,24

While no other similarly potent and selective compounds binding to other Gα protein families are available, inhibition of two other Gα proteins, Gαs and Gα15, by YM and FR was unlocked via reverse mutation of the respective Gα protein’s “inhibitor binding site” to the Gαq inhibitor binding site.52,63 This allows pharmacological inhibition of Gαs and Gα15-mediated signaling pathways in cellulo, but requires previous knockout of the respective native protein and will simultaneously lead to Gαq/11 protein inhibition (unless these proteins are knocked out as well and rescued by inhibitor-resistant Gαq variants). While suitable genome-edited human embryonic kidney 293 cell lines have been described,64 the translation of results to more native systems is difficult as pharmacological modulation of said Gα proteins is not possible outside of genome-edited, transfected cells.

YM and FR have been used as tool compounds in basic research and translational science on several occasions, with YM yielding 133 results and FR yielding 65 results in a PubMed keyword search (04.07.2023), with an increasing interest in YM after its commercialization, and in FR following its first comprehensive pharmacological characterization.17 Application of YM and FR as tool compounds in cellulo has significantly improved our current model of understanding G protein signaling. For instance, Pfeil et al. have characterized Gαq as a master switch for Gαiβγ-mediated calcium release, by demonstrating that pharmacological inhibition by FR or genetic ablation of Gαq proteins also abolished Gαiβγ-mediated calcium mobilization.65 Similarly, White et al. demonstrated that Gαq/11 inhibition reduced the duration of parathyroid hormone receptor-mediated cAMP production, providing insight in the spatiotemporal regulation of Gαs-driven signaling processes.66 Pharmacological inhibition of Gαq/11 signaling may also be advantageous in obesity, as Gαq/11 activation in adipose tissue leads to decreased energy expenditure and decreased uncoupling-protein 1 expression, and a decreased frequency of brown adipocytes.67 From a translational perspective, Onken et al. and Annala et al. independently demonstrated that FR can target constitutively active Gαq mutants driving uveal melanoma, thereby inhibiting oncogenic properties of constituvite Gαq/11 signaling, while having no effect on non-Gαq-mediated malignancies, opening up a possible future for Gαq/11 inhibition as a therapeutic mechanism to treat uveal melanoma.19,57

Employing YM and FR in Animal Studies

Besides using YM and FR in classical in cellulo or in vitro systems, inhibition of Gαq/11-mediated signaling in more complex systems and translational models is of high interest to disentangle the contribution of this signaling pathway in the context of various physiological and pathological conditions. However, the list of studies performed in vivo is rather short, despite their promising outcomes, and revolves around the investigation of asthma bronchiale, pain, thrombosis, and uveal melanoma.19,45,68 Importantly, Gαq proteins in rat and mouse are identical to the human Gαq protein with the exception of one amino acid in the helical domain (I91 V from human to mouse/rat). Consequentially, they share a virtually identical affinity for both YM and FR, confirmed by radioligand binding assays.24 Due to low predicted oral bioavailability, all animal studies performed with YM or FR to this date used intratracheal, intraperitoneal, or intrathecal application routes. The two most comprehensive animal studies focus on the bronchiodilating effect of FR in a model of asthma bronchiale45 and on antinociceptive effects exerted by YM.68

Matthey et al. showed that inhalation of 2.5 μg FR resulted in strong and long-lasting antiasthmatic effects of FR outperforming those of conventionally used antiasthmatic drugs, such as the β2-adrenoceptor agonist salmeterol, without inducing systemic side effects. Additionally, FR protected mice from allergen-induced airway hyperreactivity.45

Studying the effect of Gαq/11 signaling on pain transmission, Marwari et al. detected long-lasting antinociceptive effects by intrathecal application of YM, which intriguingly displayed a strong synergy when applied in combination with morphine by reducing the excitability of dorsal root ganglia.68

Due to the broad impact of Gαq signaling as a major signal transduction pathway downstream of GPCRs, systemic application of Gαq protein inhibitors was initially expected to be highly toxic at relevant concentrations. After the initial isolation of YM, Kawasaki et al. noticed that the compound displayed antithrombotic effects, but also decreased the heart pressure in anaesthetized rats and dogs at doses of 30 μg kg–1, administered as bolus injection.69 In agreement with this observation, Matthey et al. observed hypotensive effects in mice following the injection of 12.5 μg FR per animal into the jugular vein.45 At a dose of 2.5 μg FR per animal administered in the same fashion, no noticeable side effects were observed. A possible explanation for the lack of side effects observed by Matthey et al. might come from the preferred enrichment of the drug molecule in lung tissue detected in the same study by an LC/MS-MS protocol.45

Upon subcutaneous administration of 500 μg kg–1 YM, Marwari et al. observed reduced locomotion in mice. Local administration of YM by intrathecal injection, however, caused no side effects.68 This observation has not been reported by other studies so far. A further study investigated growth inhibition of uveal melanoma xenograft in mice, and administered 10 μg of FR by intraperitoneal injection, but did not report on the observation of any side effects.19

Thus, the limited information available from published studies does not allow a comprehensive assessment of side effects from systemic Gαq/11 inhibition. However, published reports seem to be conflicting, which may be caused by differences between species, strains, and administration routes. For instance, in one case, administration of 30 μg kg–1 YM (i.v. bolus injection) caused a significant drop in blood pressure in rats and dogs,69 while in another case, no blood pressure drop was reported despite using a more than 10-fold dose of YM (s.c. injection in mice).68 In the third case, a dose of 12.5 μg FR per mouse (i.v. bolus injection, corresponding to approximately 400 μg kg–1) was required to elicit a drop of blood pressure, which was not noticed at a lower dose of 2.5 μg per animal (approximately 80 μg kg–1).

Most importantly, the previously mentioned animal studies suggest that therapeutic application of FR and YM is indeed possible. Nevertheless, the route of administration and/or the dosage require careful consideration, depending on the investigated conditions, to eliminate toxic side effects while reaching suitable drug concentrations in the compartment of interest. Generally, high systemic exposure to the Gαq protein inhibitors should be limited if possible as shown by reports of inhibited locomotion and reduction of blood pressure observed in aforementioned studies. Compared to their widespread application in molecular pharmacology, the under-use of Gαq/11 protein inhibitors in vivo may stem in one part from the relatively high amount of expensive compound that needs to be purchased for such experiments, and in other parts from a concern regarding adverse drug effects from systemic Gαq/11 inhibition. Due to the higher metabolization rate of FR, it may be advisible to use YM to inhibit Gαq/11 proteins in vivo. A possible future development of a locally targeted YM/FR-therapy, either by chemical modification or a sophisticated delivery system, will greatly facilitate the investigation of the Gαq/11 signaling contribution to further pathologies in vivo by decreasing the risk of systemic side effects at elevated drug concentrations, and may even constitute a much-needed treatment for uveal melanoma patients.

Summary and Recommendations

The natural products YM and FR are potent and selective inhibitors of Gαq, Gα11, and Gα14 proteins and are currently the only potent and commercially available nonprotein inhibitors of heterotrimeric G proteins. They both fulfill most of the criteria defined for probe compounds, making them ideally suited for the investigation of Gαq/11-mediated processes. In contrast to protocols for PTX and CTX, which involve either overnight incubation, cell permeabilization, or transfection of an expression vector, the application of YM and FR is straightforward, and thus they have become well-accepted and frequently used tool compounds in molecular pharmacology. In combination with genetic ablation of Gαq/11 proteins or with Gα protein-specific biosensors, YM and FR add to a powerful toolbox to unambiguously illuminate processes such as GPCR-Gα coupling or the role of Gαq/11 proteins in signal transduction processes,9,65,67 and their potential can be extended to engineered inhibitor-sensitive Gαs and Gα15/16 proteins.52,63 Both compounds can be recommended without any further restrictions for most in vitro applications. For experiments which include washing steps, the usage of FR may be preferred due to its pseudoirreversible target binding. In contrast, application of YM over FR may be preferable for in vivo experiments due to its slower metabolization rate.

A recent and noteworthy publication reported two peptide-based, state-selective modulators of the Gαs protein, unlocking pharmacological modulation of the Gαs protein and further proving the druggability of Gα proteins by cyclic peptides. However, the published compounds have not been employed as tool compounds outside of the original study so far and are not commercially available.70 While new chemotypes unlocking the inhibition of other wild-type Gα proteins or targeted YM/FR-delivery systems to specific tissues are highly desirable for basic and translational research, the promising and sometimes also surprising outcome of experiments with YM and FR encourages the further investigation of Gαq/11 proteins in complex systems and pathways. This is not limited to cellular signal transduction readouts or animal studies, but also extends to in vitro systems consisting of primary cells or tissues, organoids, or organ-on-a-chip models, which are less amenable to genetic manipulation of any sort compared to cancer cell cultures. Especially in the investigation of complex systems, the Gαq/11 signaling pathway may still hold a few surprises that can be uncovered by the application of YM and FR to further shape the understanding of G protein-mediated signaling processes.

Acknowledgments

All figures were created with the help of biorender.com. This work was funded by the German Research Foundation (project number 520506488). I thank Prof. Dr. Christa E. Müller (University of Bonn), Dr. Lukas Grätz (Karolinska Institutet), Julia Kinsolving (Karolinska Institutet), and Prof. Dr. Gunnar Schulte (Karolinska Institutet) for helpful discussions regarding this article.

Glossary

Abbreviations

Ac

acetyl

ADP

adenosine diphosphate

BGC

biosynthetic gene cluster

BRET

bioluminescence energy resonance transfer

CTX

cholera toxin

CYP

cytochrome P 450

DAG

diacylglycerol

DAG

diacylglycerol

Dha

dehydroalanine

ERK

extracellular signal-regulated kinase

FR

FR900359

G protein

Heterotrimeric guanine nucleotide-binding protein

GDI

guanine nucleotide dissociation inhibitor

GDP

guanosine diphosphate

GPCR

G protein-coupled receptor

GTP

guanosine triphosphate

Hle

hydroxyleucine

IP1/3

inositolmono/trisphosphate

LC-MS/MS

liquid chromatography - tandem mass spectrometry

Pla

phenyllactic acid

PLC-β

phospholipase C-β

Pp

propionyl

PTX

pertussis toxin

YM

YM-254890

The author declares no competing financial interest.

References

  1. Fredriksson R.; Lagerström M. C.; Lundin L.-G.; Schiöth H. B. The G-protein-coupled receptors in the human genome form five main families. Phylogenetic analysis, paralogon groups, and fingerprints. Mol. Pharmacol. 2003, 63, 1256–1272. 10.1124/mol.63.6.1256. [DOI] [PubMed] [Google Scholar]
  2. Hauser A. S.; Attwood M. M.; Rask-Andersen M.; Schiöth H. B.; Gloriam D. E. Trends in GPCR drug discovery: new agents, targets and indications. Nat. Rev. Drug Discovery 2017, 16, 829–842. 10.1038/nrd.2017.178. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Oldham W. M.; Hamm H. E. Heterotrimeric G protein activation by G protein-coupled receptors. Nat. Rev. Mol. Cell Biol. 2008, 9, 60–71. 10.1038/nrm2299. [DOI] [PubMed] [Google Scholar]
  4. Milligan G.; Kostenis E. Heterotrimeric G-proteins. A short history. Br. J. Pharmacol. 2006, 147, S46–55. 10.1038/sj.bjp.0706405. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Dupré D. J.; Robitaille M.; Rebois R. V.; Hébert T. E. The role of Gβγ subunits in the organization, assembly, and function of GPCR signaling complexes. Annu. Rev. Pharmacol. Toxicol. 2009, 49, 31–56. 10.1146/annurev-pharmtox-061008-103038. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Masuho I.; Skamangas N. K.; Muntean B. S.; Martemyanov K. A. Diversity of the Gβγ complexes defines spatial and temporal bias of GPCR signaling. Cell Syst. 2021, 12, 324–337. e5 10.1016/j.cels.2021.02.001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Voss J. H.; Müller C. E. Heterotrimeric G protein α-subunits - structures, peptide-derived inhibitors, and mechanisms. Curr. Med. Chem. 2022, 29, 6359–6378. 10.2174/0929867329666220308112424. [DOI] [PubMed] [Google Scholar]
  8. Inoue A.; Raimondi F.; Kadji F. M. N.; Singh G.; Kishi T.; Uwamizu A.; Ono Y.; Shinjo Y.; Ishida S.; Arang N.; Kawakami K.; Gutkind J. S.; Aoki J.; Russell R. B. Illuminating G protein-coupling selectivity of GPCRs. Cell 2019, 177, 1933–1947. e25 10.1016/j.cell.2019.04.044. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Avet C.; Mancini A.; Breton B.; Le Gouill C.; Hauser A. S.; Normand C.; Kobayashi H.; Gross F.; Hogue M.; Lukasheva V.; St-Onge S.; Carrier M.; Héroux M.; Morissette S.; Fauman E. B.; Fortin J.-P.; Schann S.; Leroy X.; Gloriam D. E.; Bouvier M.. Effector membrane translocation biosensors reveal G protein and βarrestin coupling profiles of 100 therapeutically relevant GPCRs. eLife 2022, 11 10.7554/eLife.74101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hauser A. S.; Avet C.; Normand C.; Mancini A.; Inoue A.; Bouvier M.; Gloriam D. E.. Common coupling map advances GPCR-G protein selectivity. eLife 2022, 11 10.7554/eLife.74107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Schihada H.; Shekhani R.; Schulte G. Quantitative assessment of constitutive G protein-coupled receptor activity with BRET-based G protein biosensors. Sci. Signal. 2021, 14, eabf1653 10.1126/scisignal.abf1653. [DOI] [PubMed] [Google Scholar]
  12. Olsen R. H. J.; DiBerto J. F.; English J. G.; Glaudin A. M.; Krumm B. E.; Slocum S. T.; Che T.; Gavin A. C.; McCorvy J. D.; Roth B. L.; Strachan R. T. TRUPATH, an open-source biosensor platform for interrogating the GPCR transducerome. Nat. Chem. Biol. 2020, 16, 841–849. 10.1038/s41589-020-0535-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Moss J.; Stanley S. J.; Burns D. L.; Hsia J. A.; Yost D. A.; Myers G. A.; Hewlett E. L. Activation by thiol of the latent NAD glycohydrolase and ADP-ribosyltransferase activities of Bordetella pertussis toxin (islet-activating protein). J. Biol. Chem. 1983, 258, 11879–11882. 10.1016/S0021-9258(17)44314-6. [DOI] [PubMed] [Google Scholar]
  14. Katada T.; Tamura M.; Ui M. The A protomer of islet-activating protein, pertussis toxin, as an active peptide catalyzing ADP-ribosylation of a membrane protein. Arch. Biochem. Biophys. 1983, 224, 290–298. 10.1016/0003-9861(83)90212-6. [DOI] [PubMed] [Google Scholar]
  15. Sanchez J.; Holmgren J. Cholera toxin - a foe & a friend. Ind. J. Med. Res. 2011, 133, 153–163. [PMC free article] [PubMed] [Google Scholar]
  16. Keen A. C.; Pedersen M. H.; Lemel L.; Scott D. J.; Canals M.; Littler D. R.; Beddoe T.; Ono Y.; Shi L.; Inoue A.; Javitch J. A.; Lane J. R. OZITX, a pertussis toxin-like protein for occluding inhibitory G protein signalling including Gαz. Commun. Biol. 2022, 5, 256. 10.1038/s42003-022-03191-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kostenis E.; Pfeil E. M.; Annala S. Heterotrimeric Gq proteins as therapeutic targets?. J. Biol. Chem. 2020, 295, 5206. 10.1074/jbc.REV119.007061. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Sánchez-Fernández G.; Cabezudo S.; García-Hoz C.; Benincá C.; Aragay A. M.; Mayor F.; Ribas C. Gαq signalling. The new and the old. Cell. Signal. 2014, 26, 833–848. 10.1016/j.cellsig.2014.01.010. [DOI] [PubMed] [Google Scholar]
  19. Annala S.; Feng X.; Shridhar N.; Eryilmaz F.; Patt J.; Yang J.; Pfeil E. M.; Cervantes-Villagrana R. D.; Inoue A.; Häberlein F.; Slodczyk T.; Reher R.; Kehraus S.; Monteleone S.; Schrage R.; Heycke N.; Rick U.; Engel S.; Pfeifer A.; Kolb P.; König G.; Bünemann M.; Tüting T.; Vázquez-Prado J.; Gutkind J. S.; Gaffal E.; Kostenis E. Direct targeting of Gαq and Gα11 oncoproteins in cancer cells. Sci. Signal. 2019, 12, eaau5948 10.1126/scisignal.aau5948. [DOI] [PubMed] [Google Scholar]
  20. Onken M. D.; Makepeace C. M.; Kaltenbronn K. M.; Kanai S. M.; Todd T. D.; Wang S.; Broekelmann T. J.; Rao P. K.; Cooper J. A.; Blumer K. J. Targeting nucleotide exchange to inhibit constitutively active G protein α subunits in cancer cells. Sci. Signal. 2018, 11, eeao6852 10.1126/scisignal.aao6852. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. van Raamsdonk C. D.; Griewank K. G.; Crosby M. B.; Garrido M. C.; Vemula S.; Wiesner T.; Obenauf A. C.; Wackernagel W.; Green G.; Bouvier N.; Sozen M. M.; Baimukanova G.; Roy R.; Heguy A.; Dolgalev I.; Khanin R.; Busam K.; Speicher M. R.; O’Brien J.; Bastian B. C. Mutations in GNA11 in uveal melanoma. N. Engl. J. Med. 2010, 363, 2191–2199. 10.1056/NEJMoa1000584. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Schrage R.; Schmitz A.-L.; Gaffal E.; Annala S.; Kehraus S.; Wenzel D.; Büllesbach K. M.; Bald T.; Inoue A.; Shinjo Y.; Galandrin S.; Shridhar N.; Hesse M.; Grundmann M.; Merten N.; Charpentier T. H.; Martz M.; Butcher A. J.; Slodczyk T.; Armando S.; Effern M.; Namkung Y.; Jenkins L.; Horn V.; Stößel A.; Dargatz H.; Tietze D.; Imhof D.; Galés C.; Drewke C.; Müller C. E.; Hölzel M.; Milligan G.; Tobin A. B.; Gomeza J.; Dohlman H. G.; Sondek J.; Harden T. K.; Bouvier M.; Laporte S. A.; Aoki J.; Fleischmann B. K.; Mohr K.; König G. M.; Tüting T.; Kostenis E. The experimental power of FR900359 to study Gq-regulated biological processes. Nat. Commun. 2015, 6, 10156. 10.1038/ncomms10156. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Nishimura A.; Kitano K.; Takasaki J.; Taniguchi M.; Mizuno N.; Tago K.; Hakoshima T.; Itoh H. Structural basis for the specific inhibition of heterotrimeric Gq protein by a small molecule. Proc. Natl. Acad. Sci. U. S. A. 2010, 107, 13666–13671. 10.1073/pnas.1003553107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kuschak M.; Namasivayam V.; Rafehi M.; Voss J. H.; Garg J.; Schlegel J. G.; Abdelrahman A.; Kehraus S.; Reher R.; Küppers J.; Sylvester K.; Hinz S.; Matthey M.; Wenzel D.; Fleischmann B. K.; Pfeifer A.; Inoue A.; Gütschow M.; König G. M.; Müller C. E. Cell-permeable high-affinity tracers for Gq proteins provide structural insights, reveal distinct binding kinetics and identify small molecule inhibitors. Br. J. Pharmacol. 2020, 177, 1898–1916. 10.1111/bph.14960. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Call for manuscripts for ACS Pharmacology & Translational Science review series on recommended tool compounds. ACS Pharmacol. Transl. Sci. 2022, 5, 516–517 10.1021/acsptsci.2c00135. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Taniguchi M.; Nagai K.; Arao N.; Kawasaki T.; Saito T.; Moritani Y.; Takasaki J.; Hayashi K.; Fujita S.; Suzuki K.; Tsukamoto S. YM-254890, a novel platelet aggregation inhibitor produced by Chromobacterium sp. QS3666. J. Antibiot. 2003, 56, 358–363. 10.7164/antibiotics.56.358. [DOI] [PubMed] [Google Scholar]
  27. Fujioka M.; Koda S.; Morimoto Y.; Biemann K. Structure of FR900359, a cyclic depsipeptide from Ardisia crenata sims. J. Org. Chem. 1988, 53, 2820–2825. 10.1021/jo00247a030. [DOI] [Google Scholar]
  28. Crüsemann M.; Reher R.; Schamari I.; Brachmann A. O.; Ohbayashi T.; Kuschak M.; Malfacini D.; Seidinger A.; Pinto-Carbó M.; Richarz R.; Reuter T.; Kehraus S.; Hallab A.; Attwood M.; Schiöth H. B.; Mergaert P.; Kikuchi Y.; Schäberle T. F.; Kostenis E.; Wenzel D.; Müller C. E.; Piel J.; Carlier A.; Eberl L.; König G. M. Heterologous expression, bosynthetic studies, and ecological function of the selective Gq-signaling inhibitor FR900359. Angew. Chem. Int. Ed. Engl. 2018, 57, 836–840. 10.1002/anie.201707996. [DOI] [PubMed] [Google Scholar]
  29. Hermes C.; König G. M.; Crüsemann M. The chromodepsins - chemistry, biology and biosynthesis of a selective Gq inhibitor natural product family. Nat. Prod. Rep. 2021, 38, 2276. 10.1039/D1NP00005E. [DOI] [PubMed] [Google Scholar]
  30. Carlier A.; Fehr L.; Pinto-Carbó M.; Schäberle T.; Reher R.; Dessein S.; König G.; Eberl L. The genome analysis of Candidatus Burkholderia crenata reveals that secondary metabolism may be a key function of the Ardisia crenata leaf nodule symbiosis. Environ. Microbiol. 2016, 18, 2507–2522. 10.1111/1462-2920.13184. [DOI] [PubMed] [Google Scholar]
  31. Hermes C.; Richarz R.; Wirtz D. A.; Patt J.; Hanke W.; Kehraus S.; Voß J. H.; Küppers J.; Ohbayashi T.; Namasivayam V.; Alenfelder J.; Inoue A.; Mergaert P.; Gütschow M.; Müller C. E.; Kostenis E.; König G. M.; Crüsemann M. Thioesterase-mediated side chain transesterification generates potent Gq signaling inhibitor FR900359. Nat. Commun. 2021, 12, 144. 10.1038/s41467-020-20418-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Hanke W.; Patt J.; Alenfelder J.; Voss J. H.; Zdouc M. M.; Kehraus S.; Kim J. B.; Grujičić G. V.; Namasivayam V.; Reher R.; Müller C. E.; Kostenis E.; Crüsemann M.; König G. M. Feature-based molecular networking for the targeted identification of Gq-inhibiting FR900359 derivatives. J. Nat. Prod. 2021, 84, 1941–1953. 10.1021/acs.jnatprod.1c00194. [DOI] [PubMed] [Google Scholar]
  33. Taniguchi M.; Suzumura K.-I.; Nagai K.; Kawasaki T.; Takasaki J.; Sekiguchi M.; Moritani Y.; Saito T.; Hayashi K.; Fujita S.; Tsukamoto S.; Suzuki K. YM-254890 analogues, novel cyclic depsipeptides with Gαq/11-inhibitory activity from Chromobacterium sp. QS3666. Bioorg. Med. Chem. 2004, 12, 3125–3133. 10.1016/j.bmc.2004.04.006. [DOI] [PubMed] [Google Scholar]
  34. Reher R.; Kuschak M.; Heycke N.; Annala S.; Kehraus S.; Dai H.-F.; Müller C. E.; Kostenis E.; König G. M.; Crüsemann M. Applying molecular networking for the detection of natural sources and analogues of the selective Gq protein inhibitor FR900359. J. Nat. Prod. 2018, 81, 1628–1635. 10.1021/acs.jnatprod.8b00222. [DOI] [PubMed] [Google Scholar]
  35. Voss J. H.; Crüsemann M.; Bartling C. R.; Kehraus S.; Inoue A.; König G. M.; Strømgaard K.; Müller C. E. Structure-affinity and structure-residence time relationships of macrocyclic Gαq protein inhibitors. iScience 2023, 26, 106492. 10.1016/j.isci.2023.106492. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Machida K.; Arai D.; Katsumata R.; Otsuka S.; Yamashita J. K.; Ye T.; Tang S.; Fusetani N.; Nakao Y. Sameuramide A, a new cyclic depsipeptide isolated from an ascidian of the family Didemnidae. Bioorg. Med. Chem. 2018, 26, 3852–3857. 10.1016/j.bmc.2018.06.042. [DOI] [PubMed] [Google Scholar]
  37. Xiong X.-F.; Zhang H.; Underwood C. R.; Harpsøe K.; Gardella T. J.; Wöldike M. F.; Mannstadt M.; Gloriam D. E.; Bräuner-Osborne H.; Strømgaard K. Total synthesis and structure-activity relationship studies of a series of selective G protein inhibitors. Nat. Chem. 2016, 8, 1035–1041. 10.1038/nchem.2577. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Xiong X.-F.; Zhang H.; Boesgaard M. W.; Underwood C. R.; Bräuner-Osborne H.; Strømgaard K. Structure-activity relationship studies of the natural product Gq/11 protein inhibitor YM-254890. ChemMedChem. 2019, 14, 865. 10.1002/cmdc.201900018. [DOI] [PubMed] [Google Scholar]
  39. Zhang H.; Nielsen A. L.; Boesgaard M. W.; Harpsøe K.; Daly N. L.; Xiong X.-F.; Underwood C. R.; Haugaard-Kedström L. M.; Bräuner-Osborne H.; Gloriam D. E.; Strømgaard K. Structure-activity relationship and conformational studies of the natural product cyclic depsipeptides YM-254890 and FR900359. Eur. J. Med. Chem. 2018, 156, 847–860. 10.1016/j.ejmech.2018.07.023. [DOI] [PubMed] [Google Scholar]
  40. Zhang H.; Xiong X.-F.; Boesgaard M. W.; Underwood C. R.; Bräuner-Osborne H.; Strømgaard K. Structure-activity relationship studies of the cyclic depsipeptide natural product YM-254890, targeting the Gq protein. ChemMedChem. 2017, 12, 830–834. 10.1002/cmdc.201700155. [DOI] [PubMed] [Google Scholar]
  41. Zhang H.; Nielsen A. L.; Strømgaard K. Recent achievements in developing selective Gq inhibitors. Med. Res. Rev. 2020, 40, 135–157. 10.1002/med.21598. [DOI] [PubMed] [Google Scholar]
  42. Schlegel J. G.; Tahoun M.; Seidinger A.; Voss J. H.; Kuschak M.; Kehraus S.; Schneider M.; Matthey M.; Fleischmann B. K.; König G. M.; Wenzel D.; Müller C. E. Macrocyclic Gq protein inhibitors FR900359 and/or YM-254890 – fit for translation?. ACS Pharmacol. Transl. Sci. 2021, 4, 888–897. 10.1021/acsptsci.1c00021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Pistorius D.; Buntin K.; Weber E.; Richard E.; Bouquet C.; Wollbrett S.; Regenass H.; Peón V.; Böhm M.; Kessler R.; Gempeler T.; Haberkorn A.; Wimmer L.; Lanshoeft C.; Davis J.; Hainzl D.; D’Alessio J. A.; Manchado E.; Petersen F. Promoter-driven overexpression in Chromobacterium vaccinii facilitates access to FR900359 and yields novel low abundance analogs. Chemistry 2022, 28, e202103888 10.1002/chem.202103888. [DOI] [PubMed] [Google Scholar]
  44. Kuschak M.; Schlegel J. G.; Schneider M.; Kehraus S.; Voss J. H.; Seidinger A.; Matthey M.; Wenzel D.; Fleischmann B. K.; König G. M.; Müller C. E. Sensitive LC-MS/MS method for the quantification of macrocyclic Gαq protein inhibitors in biological samples. Front. Chem. 2020, 8, 833. 10.3389/fchem.2020.00833. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Matthey M.; Roberts R.; Seidinger A.; Simon A.; Schröder R.; Kuschak M.; Annala S.; König G. M.; Müller C. E.; Hall I. P.; Kostenis E.; Fleischmann B. K.; Wenzel D.. Targeted inhibition of Gq signaling induces airway relaxation in mouse models of asthma. Sci. Transl. Med. 2017, 9 10.1126/scitranslmed.aag2288. [DOI] [PubMed] [Google Scholar]
  46. Lipinski C. A.; Lombardo F.; Dominy B. W.; Feeney P. J. Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. Adv. Drug Delivery Rev. 2001, 46, 3–26. 10.1016/S0169-409X(00)00129-0. [DOI] [PubMed] [Google Scholar]
  47. Garcia Jimenez D.; Poongavanam V.; Kihlberg J. Macrocycles in drug discovery—learning from the past for the future. J. Med. Chem. 2023, 66, 5377–5396. 10.1021/acs.jmedchem.3c00134. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Dror R. O.; Mildorf T. J.; Hilger D.; Manglik A.; Borhani D. W.; Arlow D. H.; Philippsen A.; Villanueva N.; Yang Z.; Lerch M. T.; Hubbell W. L.; Kobilka B. K.; Sunahara R. K.; Shaw D. E. SIGNAL TRANSDUCTION. Structural basis for nucleotide exchange in heterotrimeric G proteins. Science 2015, 348, 1361–1365. 10.1126/science.aaa5264. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Tietze D.; Kaufmann D.; Tietze A. A.; Voll A.; Reher R.; König G.; Hausch F. Structural and dynamical basis of G protein inhibition by YM-254890 and FR900359: an inhibitor in action. J. Chem. Inf. Model. 2019, 59, 4361–4373. 10.1021/acs.jcim.9b00433. [DOI] [PubMed] [Google Scholar]
  50. Muehle J.; Rodrigues M. J.; Alenfelder J.; Jürgenliemke L.; Guixà-González R.; Bacchin A.; Andres F.; Hennig M.; Schihada H.; Crüsemann M.; König G. M.; Kostenis E.; Schertler G.; Deupi X. Cyclic peptide inhibitors stabilize Gq/11 heteromers. bioRxiv 2023, 10.1101/2023.10.24.563737. [DOI] [Google Scholar]
  51. Voss J. H.; Nagel J.; Rafehi M.; Guixà-González R.; Malfacini D.; Patt J.; Kehraus S.; Inoue A.; König G. M.; Kostenis E.; Deupi X.; Namasivayam V.; Müller C. E. Unraveling binding mechanism and kinetics of macrocyclic Gαq protein inhibitors. Pharmacol. Res. 2021, 173, 105880. 10.1016/j.phrs.2021.105880. [DOI] [PubMed] [Google Scholar]
  52. Takasaki J.; Saito T.; Taniguchi M.; Kawasaki T.; Moritani Y.; Hayashi K.; Kobori M. A novel Gαq/11-selective inhibitor. J. Biol. Chem. 2004, 279, 47438–47445. 10.1074/jbc.M408846200. [DOI] [PubMed] [Google Scholar]
  53. Malfacini D.; Patt J.; Annala S.; Harpsøe K.; Eryilmaz F.; Reher R.; Crüsemann M.; Hanke W.; Zhang H.; Tietze D.; Gloriam D. E.; Bräuner-Osborne H.; Strømgaard K.; König G. M.; Inoue A.; Gomeza J.; Kostenis E. Rational design of a heterotrimeric G protein α subunit with artificial inhibitor sensitivity. J. Biol. Chem. 2019, 294, 5747–5758. 10.1074/jbc.RA118.007250. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Reher R.; Kühl T.; Annala S.; Benkel T.; Kaufmann D.; Nubbemeyer B.; Odhiambo J. P.; Heimer P.; Bäuml C. A.; Kehraus S.; Crüsemann M.; Kostenis E.; Tietze D.; König G. M.; Imhof D. Deciphering specificity determinants for FR900359-derived Gq α inhibitors based on computational and structure-activity studies. ChemMedChem. 2018, 13, 1634–1643. 10.1002/cmdc.201800304. [DOI] [PubMed] [Google Scholar]
  55. Gao Z.-G.; Jacobson K. A. On the selectivity of the Gαq inhibitor UBO-QIC. A comparison with the Gαi inhibitor pertussis toxin. Biochem. Pharmacol. 2016, 107, 59–66. 10.1016/j.bcp.2016.03.003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Peng Q.; Alqahtani S.; Nasrullah M. Z. A.; Shen J. Functional evidence for biased inhibition of G protein signaling by YM-254890 in human coronary artery endothelial cells. Eur. J. Pharmacol. 2021, 891, 173706. 10.1016/j.ejphar.2020.173706. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Patt J.; Alenfelder J.; Pfeil E. M.; Voss J. H.; Merten N.; Eryilmaz F.; Heycke N.; Rick U.; Inoue A.; Kehraus S.; Deupi X.; Müller C. E.; König G. M.; Crüsemann M.; Kostenis E. An experimental strategy to probe Gq contribution to signal transduction in living cells. J. Biol. Chem. 2021, 296, 100472. 10.1016/j.jbc.2021.100472. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Onken M. D.; Makepeace C. M.; Kaltenbronn K. M.; Choi J.; Hernandez-Aya L.; Weilbaecher K. N.; Piggott K. D.; Rao P. K.; Yuede C. M.; Dixon A. J.; Osei-Owusu P.; Cooper J. A.; Blumer K. J. Targeting primary and metastatic uveal melanoma with a G protein inhibitor. J. Biol. Chem. 2021, 296, 100403. 10.1016/j.jbc.2021.100403. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Onken M. D.; Noda S. E.; Kaltenbronn K. M.; Frankfater C.; Makepeace C. M.; Fettig N.; Piggott K. D.; Custer P. L.; Ippolito J. E.; Blumer K. J. Oncogenic Gq/11 signaling acutely drives and chronically sustains metabolic reprogramming in uveal melanoma. J. Biol. Chem. 2022, 298, 101495. 10.1016/j.jbc.2021.101495. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Lapadula D.; Farias E.; Randolph C. E.; Purwin T.; McGrath D.; Charpentier T.; Zhang L.; Wu S.; Terai M.; Sato T.; Tall G. G.; Zhou N.; Wedegaertner P.; Aplin A. E.; Aguirre-Ghiso J.; Benovic J. L. Effects of oncogenic Gαq and Gα11 inhibition by FR900359 in uveal melanoma. Mol. Cancer. Res. 2019, 17, 963. 10.1158/1541-7786.MCR-18-0574. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Müller S.; Ackloo S.; Arrowsmith C. H.; Bauser M.; Baryza J. L.; Blagg J.; Böttcher J.; Bountra C.; Brown P. J.; Bunnage M. E.; Carter A. J.; Damerell D.; Dötsch V.; Drewry D. H.; Edwards A. M.; Edwards J.; Elkins J. M.; Fischer C.; Frye S. V.; Gollner A.; Grimshaw C. E.; IJzerman A.; Hanke T.; Hartung I. V.; Hitchcock S.; Howe T.; Hughes T. V.; Laufer S.; Li V. M.; Liras S.; Marsden B. D.; Matsui H.; Mathias J.; O’Hagan R. C.; Owen D. R.; Pande V.; Rauh D.; Rosenberg S. H.; Roth B. L.; Schneider N. S.; Scholten C., Singh Saikatendu K.; Simeonov A.; Takizawa M.; Tse C.; Thompson P. R.; Treiber D. K.; Viana A. Y.; Wells C. I.; Willson T. M.; Zuercher W. J.; Knapp S.; Mueller-Fahrnow A.. Donated chemical probes for open science. eLife 2018, 7 10.7554/eLife.34311. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Kukkonen J. P. G-protein inhibition profile of the reported Gq/11 inhibitor UBO-QIC. Biochem. Biophys. Res. Commun. 2016, 469, 101–107. 10.1016/j.bbrc.2015.11.078. [DOI] [PubMed] [Google Scholar]
  63. Hulme E. C.; Trevethick M. A. Ligand binding assays at equilibrium. Validation and interpretation. Br. J. Pharmacol. 2010, 161, 1219–1237. 10.1111/j.1476-5381.2009.00604.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. Boesgaard M. W.; Harpsøe K.; Malmberg M.; Underwood C. R.; Inoue A.; Mathiesen J. M.; König G. M.; Kostenis E.; Gloriam D. E.; Bräuner-Osborne H. Delineation of molecular determinants for FR900359 inhibition of Gq/11 unlocks inhibition of Gαs. J. Biol. Chem. 2020, 295, 13850. 10.1074/jbc.RA120.013002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. Hisano Y.; Kono M.; Cartier A.; Engelbrecht E.; Kano K.; Kawakami K.; Xiong Y.; Piao W.; Galvani S.; Yanagida K.; Kuo A.; Ono Y.; Ishida S.; Aoki J.; Proia R. L.; Bromberg J. S.; Inoue A.; Hla T. Lysolipid receptor cross-talk regulates lymphatic endothelial junctions in lymph nodes. J. Exp. Med. 2019, 216, 1582–1598. 10.1084/jem.20181895. [DOI] [PMC free article] [PubMed] [Google Scholar]
  66. Pfeil E. M.; Brands J.; Merten N.; Vögtle T.; Vescovo M.; Rick U.; Albrecht I.-M.; Heycke N.; Kawakami K.; Ono Y.; Ngako Kadji F. M.; Hiratsuka S.; Aoki J.; Häberlein F.; Matthey M.; Garg J.; Hennen S.; Jobin M.-L.; Seier K.; Calebiro D.; Pfeifer A.; Heinemann A.; Wenzel D.; König G. M.; Nieswandt B.; Fleischmann B. K.; Inoue A.; Simon K.; Kostenis E. Heterotrimeric G protein subunit Gαq is a master switch for Gβγ-mediated calcium mobilization by Gi-coupled GPCRs. Mol. Cell 2020, 80, 940–954. 10.1016/j.molcel.2020.10.027. [DOI] [PubMed] [Google Scholar]
  67. White A. D.; Jean-Alphonse F. G.; Fang F.; Peña K. A.; Liu S.; König G. M.; Inoue A.; Aslanoglou D.; Gellman S. H.; Kostenis E.; Xiao K.; Vilardaga J.-P. Gq/11-dependent regulation of endosomal cAMP generation by parathyroid hormone class B GPCR. Proc. Natl. Acad. Sci. U. S. A. 2020, 117, 7455–7460. 10.1073/pnas.1918158117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  68. Klepac K.; Kilić A.; Gnad T.; Brown L. M.; Herrmann B.; Wilderman A.; Balkow A.; Glöde A.; Simon K.; Lidell M. E.; Betz M. J.; Enerbäck S.; Wess J.; Freichel M.; Blüher M.; König G.; Kostenis E.; Insel P. A.; Pfeifer A. The Gq signalling pathway inhibits brown and beige adipose tissue. Nat. Commun. 2016, 7, 10895. 10.1038/ncomms10895. [DOI] [PMC free article] [PubMed] [Google Scholar]
  69. Marwari S.; Kowalski C.; Martemyanov K. A. Exploring pharmacological inhibition of Gq/11 as an analgesic strategy. Br. J. Pharmacol. 2022, 179, 5196–5208. 10.1111/bph.15935. [DOI] [PMC free article] [PubMed] [Google Scholar]
  70. Kawasaki T.; Taniguchi M.; Moritani Y.; Uemura T.; Shigenaga T.; Takamatsu H.; Hayashi K.; Takasaki J.; Saito T.; Nagai K. Pharmacological properties of YM-254890, a specific Gαq/11 inhibitor, on thrombosis and neointima formation in mice. Thromb. Haemost. 2005, 94, 184–192. 10.1160/TH04-09-0635. [DOI] [PubMed] [Google Scholar]
  71. Dai S. A.; Hu Q.; Gao R.; Blythe E. E.; Touhara K. K.; Peacock H.; Zhang Z.; von Zastrow M.; Suga H.; Shokat K. M. State-selective modulation of heterotrimeric Gαs signaling with macrocyclic peptides. Cell 2022, 185, 3950–3965. 10.1016/j.cell.2022.09.019. [DOI] [PMC free article] [PubMed] [Google Scholar]

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