Small molecules targeting heterotrimeric G proteins

European Journal of Pharmacology 826 (2018) 169–178

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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar

Molecular and cellular pharmacology

Small molecules targeting heterotrimeric G proteins

T

Mohammed Akli Ayoub Biology Department, College of Science, United Arab Emirates University, PO Box 15551, Al Ain, United Arab Emirates

A R T I C LE I N FO

A B S T R A C T

Keywords: GPCR G protein Pharmacology Signaling Drug Small molecule

G protein-coupled receptors (GPCRs) represent the largest family of cell surface receptors regulating many human and animal physiological functions. Their implication in human pathophysiology is obvious with almost 30–40% medical drugs commercialized today directly targeting GPCRs as molecular entities. However, upon ligand binding GPCRs signal inside the cell through many key signaling, adaptor and regulatory proteins, including various classes of heterotrimeric G proteins. Therefore, G proteins are considered interesting targets for the development of pharmacological tools that are able to modulate their interaction with the receptors, as well as their activation/deactivation processes. In this review, old attempts and recent advances in the development of small molecules that directly target G proteins will be described with an emphasis on their utilization as pharmacological tools to dissect the mechanisms of activation of GPCR-G protein complexes. These molecules constitute a further asset for research in the “hot” areas of GPCR biology, areas such as multiple G protein coupling/signaling, GPCR-G protein preassembly, and GPCR functional selectivity or bias. Moreover, this review gives a particular focus on studies in vitro and in vivo supporting the potential applications of such small molecules in various GPCR/G protein-related diseases.

1. Introduction GPCRs represent the largest cell surface and membrane receptor family mediating cellular responses of a large variety of activating extracellular signals (Bockaert and Pin, 1998; Katritch et al., 2013; Lefkowitz, 2013; Pierce et al., 2002). This illustrates the central role of GPCRs in many physiological functions, making them one of the largest families of drug targets (Eglen and Reisine, 2011; Hauser et al., 2017; Lappano and Maggiolini, 2011; Lundstrom, 2009; Spiegel and Weinstein, 2004). Indeed, the attractiveness of GPCRs as drug targets for the pharmaceutical industry led to the development of many selective small molecules that are proposed to directly modulate the function of GPCRs. Moreover, considerable advances in our understanding of the molecular pharmacology of GPCRs and their signaling and regulation have significantly contributed to the explosion of drug discovery programs (Kobilka, 2011; Limbird, 2004). The structural insights obtained from the recently reported crystallographic GPCR structures, as well as the advances in the field of chemical and computational biology, will certainly contribute to further advances in GPCR drug discovery (Hauser et al., 2017; Kobilka, 2011; Rasmussen et al., 2011a; Rosenbaum et al., 2009). Moreover, recently antibodies and nanobodies targeting GPCRs constitute a promising way towards the development of new generations of more selective drugs to fine tune GPCR function (Ayoub et al., 2017; Hutchings et al., 2010; Rasmussen et al., 2011a, 2011b). All these advances have contributed to the

development of new classes of drugs with better pharmacological properties in terms of selectivity, efficacy and safety. Since heterotrimeric G proteins play the pivotal role in GPCR activation and signaling, they have been obvious targets for researchers wanting to modulate GPCR function (Bockaert, 1991; Bonacci et al., 2006; Cabrera-Vera et al., 2003; Johnston et al., 2008; Smrcka, 2013). Indeed, GPCRs signal mostly through their intimate physical and functional coupling with various classes of guanosine di/tri-phosphate (GDP/GTP) nucleotide-binding proteins (G proteins) including the major ones, Gs, Gi/o, Gq/11, and G12/13 proteins (Bockaert, 1991; Bockaert et al., 1987; Cabrera-Vera et al., 2003; Gilman, 1987; Oldham and Hamm, 2008). At the molecular level, GPCR-G protein coupling occurs in a well ordered cycle where ligand binding promotes receptor activation followed by receptor-G protein interaction resulting in GDP to GTP exchange on the Gα subunit (Fig. 1). This is followed by subunit dissociation and/or conformational changes within the Gα/Gβγ trimer and each subunit triggers the activation of multiple intracellular pathways (such as cyclic adenosine 3′,5′-monophosphate (cAMP), inositol trisphosphate (IP3), Ca2+, diacylglycerol (DAG), mitogen-activated protein kinase (MAP kinase), Rho/Rac), which control important physiological responses (Fig. 1) (Bockaert, 1991; Cabrera-Vera et al., 2003; Ghanouni et al., 2001; Oldham and Hamm, 2008). For regulation of G protein activation and its termination, this is mediated by the GTPase activity of G proteins, as well as regulators of G protein signaling (RGSs) that stimulate GTP hydrolysis on the Gα subunit (Fig. 1) (De Vries et al.,

E-mail address: [email protected]. https://doi.org/10.1016/j.ejphar.2018.03.003 Received 11 January 2018; Received in revised form 27 February 2018; Accepted 2 March 2018 Available online 06 March 2018 0014-2999/ © 2018 Elsevier B.V. All rights reserved.

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Fig. 1. The classical GPCR-G protein activation/deactivation cycle. In the absence of activation, the Gα-GDP and Gβγ subunits of the heterotrimeric G proteins are associated with each other without necessarily physically interacting with the receptor. Upon a ligand binding to its GPCR, conformational changes occur within the receptor leading to an intimate interaction and coupling with the heterotrimeric G protein. This results in G protein activation characterized by GDP/GTP exchange followed by the dissociation between Gα-GTP complex from the Gβγ subunits. Each activated subunit is then able to interact with and modulate the function of its specific intracellular effectors leading to canonical Gα- and Gβγdependent, but also Gα/Gβγ-independent signaling pathways (e.g. β-arrestin, Src). As a regulation mechanism, the GTPase activity of Gα hydrolyzes GTP to GDP and along with the involvement of selective RGSs, this inactivates the Gα subunit. Finally, the Gα-GDP and Gβγ subunits re-associate to be ready for another GPCR-G protein activation cycle.

pharmacological effects on G protein activation in various in vitro and in vivo models will be described (Table 1).

2000). A further mechanism of GPCR regulation involves the phosphorylation of GPCRs by specific G protein coupled-receptor kinases (GRKs) followed by their interaction with β-arrestins causing GPCR desensitization and internalization (Luttrell and Lefkowitz, 2002; Moore et al., 2007; Reiter and Lefkowitz, 2006). Of course, this classical view of GPCR-G protein activation and desensitization appears very simplistic since it is now evident that GPCRs are able to engage various G protein-independent signaling pathways (Fig. 1) (Lefkowitz and Shenoy, 2005; Luttrell and Lefkowitz, 2002; Reiter and Lefkowitz, 2006). Indeed, upon their internalization GPCRs promote intracellular signaling pathways including β-arrestin- and Src-dependent responses and even intracellular G protein-dependent responses (Lefkowitz and Shenoy, 2005; Luttrell and Lefkowitz, 2002; Reiter and Lefkowitz, 2006). Overall, this highlights the complexity of GPCR pharmacology and signaling systems, where G proteins still play the central role in GPCR function.

3. Small molecules targeting Gα subunits 3.1. From toxins to small molecules From our knowledge of the key role of G proteins in GPCR signaling and function, it is obvious that molecules that interfere or directly interact with G proteins and modulate their activation would be of great interest. This was amazingly illustrated by the famous harmful bacterial toxins, cholerae toxin (CTX), Bordetella pertussis toxin (PTX), heat-labile enterotoxin of Escherichia coli, and Pasteurella multocida toxin (PMT), which differentially act on the Gα subunit of heterotrimeric G proteins and thereby cause severe infections (Aktories, 2011; Mangmool and Kurose, 2011; Orth and Aktories, 2010). Their common mode of action is based on the chemical modification of specific and critical residues on the Gα subunits involved either in the GTP hydrolysis reaction (CTX and PMT) or in the physical interaction of the G proteins with GPCRs (PTX) (Table 2). In addition to their infectious potential, these toxins have been extensively used to study GPCR/G protein-mediated signaling, especially to dissect the involvement of each of the different classes of G proteins in GPCR biology and physiology. Furthermore, these toxins strongly inspired scientists to develop other molecules that can bind and interfere with Gα protein activation and its physical and functional interaction with either GPCRs or Gβ/Gγ subunits. In fact, over the years G proteins have constituted interesting targets for the development of molecules that can bind the different G protein subunits and modulate their activation/deactivation cycle. This is mainly based on our knowledge of the molecular and structural events involved in both GPCR-Gα protein and Gα-Gβγ physical interactions and their activation upon ligand binding and receptor activation. Consequently, many peptides and small molecules capable of binding to Gα subunits in a more or less selective manner have been

2. G proteins as key targets As a key element in GPCR function, G proteins constitute interesting molecular targets. This has led to the development of many pharmacological tools used for better understanding of GPCR function and more importantly for their potential use as therapeutic targets to treat GPCR-linked disorders. Indeed, several approaches for pharmacological targeting of G proteins have been used, and many selective and nonselective peptides and small molecules that target either Gα or Gβγ subunits have been developed. These tools have been used in mechanistic-based studies to understand GPCR-G protein signaling, as well as for their potential application in treating GPCR-linked diseases (Table 1). In the current topical context of GPCR-biased signaling, G protein-selective molecules present a major asset to better dissect GPCR signaling and its implication in pathophysiology (Fig. 2). In this review, a particular emphasis will be placed on small molecules targeting G proteins and their different modes of action (Fig. 2). In addition, their 170

Gαs?

Cell-dependent selectivity

Gαq

BIM-46174

BIM-46187

YM-254890

171

Disruption of Gβγ-GRK2 association Physical dissociation of Gα and Gβγ

Gβγ

No GDP/GTP exchange

Disruption of Gα-Gβγ association

Gβγ

M119 M158 M201 S12155

Inhibition of GDP release and GDP/GTP exchange

Gαi < Gαq

Inhibition of GDP release and GDP/GTP exchange Inhibition of Gα-Gβγ physical dissociation and conformationchanges

Inhibition of GDP release and GDP/GTP exchange

Inhibition of GDP/GTP exchange (empty Gα) Inhibition of GPCR-Gα physical interaction Inhibition of Gα-Gβγ physical dissociation

Inhibition of GDP release and GDP/GTP exchange Inhibition of Gαs-AC interaction Disruption of Gα-Gβγ association Reversible inhibition of GDP/GTP exchange

Mode of action

Quinazoline derivatives

Gαq

Gαs

Suramin and analogues

YM-280193 WU-07047 FR900359

G protein Subunit selectivity

Small Molecule

Competitive ELISA Surface plasmon resonance

Docking, screening, and saturationtransfer difference nuclear magnetic resonance Competitive ELISA

All-atom molecular dynamic simulation

Crystallo-graphy

Far-ultraviolet CD

N/A

Recombinant Gαs

Binding evidence

Inhibition of GDP/GTP exchange Reversion of Gαi/o-inhibited cAMP response Inhibition of GPCR and Gβγmediated PLCβ2/3 and PI3γkinase activation Stimulation of Gβγ/PLCmediated Ca2+ production Activation of Gβγ-induced ERK and Akt pathways

Inhibition of GPCR-induced Ca2+/IP1 production Inhibition of GPCR-induced ERK1/2 activation Inhibition of Rho/Rho kinase C activity

Inhibition of GPCR-induced Ca2+ production

Inhibition of GPCR-induced cAMP production Inhibition of GPCR-induced cAMP/Ca2+/IP1 production

Inhibition of GPCR-induced cAMP production

Inhibition of GPCR-induced cAMP production

Signaling effects

GPCR-mediated chemotaxis and inflammation in HL60 neutrophil cell line Chemotaxis in HL60 cell line and primary neutrophils

Label-free cellular-based assay on various GPCRs Differentiation of human and murine brown adipocytes Gαq-mediated signaling in murine and human airway smooth muscle cells Broncho-constrictor responses and relaxation in airway tissues ex vivo Real-time GTPγS binding assay GPCR-promoted cAMP production

Anti-proliferative effects in cancer melanoma cells BRET between G protein subunits

GTPγS binding assay

Purified GPCR and Gα proteins BRET/FRET between GPCRs-Gα and Gβγ subunits GPCR-promoted Ca2+ production in platelets and cell lines

GPCR-mediated signaling and proliferation in various cancer cell lines GTPγS binding assay GPCR-mediated signaling and proliferation in cancer cell lines GTPγS binding assay

Purified Gα, Gβγ, and AC GTPγS binding assay

in vitro Model

Table 1 Different small molecules targeting heterotrimeric G proteins and their related signaling and functions (For references refer to the corresponding sections in the main text).

Morphine-induced anti-nociception in PLCβ3-/- mice

Brown and beige murine adipose tissue Broncho-relaxation in mice Airway hyper-responsiveness in the mouse model of asthma

Anti-thrombotic and thrombolytic effects in a rat model of arterial thrombosis Platelet functions and thrombus formation under high-shear stress Vasoconstriction in mouse arteries

Anti-hyperalgesic effects in rat pain models

Anti-cancer effects in a human tumor xenograft model

GPCR- and G protein-mediated signaling in rat sympathetic neurons

in vivo Model

M.A. Ayoub

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Fig. 2. The different modes of action of Gα subunit inhibitors. The small molecule binds to the Gα subunit and inhibits GDP/GTP exchange by disrupting either GDP release, GTP binding, receptor-Gα protein interaction or even the dissociation or the molecular rearrangements between Gα-Gβγ subunits.

inhibit G proteins (Fig. 3). Indeed, it was developed by Bayer Co in 1916 as a drug to treat African sleeping disease (African trypanosomiasis) and river blindness (Joshi et al., 2005; Reincke et al., 1994; Schulz-Key et al., 1985). Later, suramin was also tested in the case of recurrent high-grade gliomas (Grossman et al., 2001; Takano et al., 1994). The evidence of suramin acting directly on G proteins came in 1996 from Freissmuth and colleagues who tested suramin and its analogs on purified G proteins (Freissmuth et al., 1996). They showed that suramin bound specifically to the Gαs subunit and efficiently inhibited GDP release with an IC50 around 250 nM. They also showed that suramin inhibited GDP/GTP exchange on Gαi/o class (Freissmuth et al., 1996). Later, suramin analogs, NF449 and NF503, were developed with selective antagonistic action on Gαs relative to other Gα proteins (Hohenegger et al., 1998). Suramin and its analogs were shown to inhibit GPCR-promoted GDP release and nucleotide exchange on Gα

developed. These molecules interfere with Gα activation and interaction with either GPCRs or Gβγ subunits (Fig. 2) (Table 1). Depending on their selectivity and mode of action, the molecules/peptides targeting G proteins may have a double application in the field of GPCR biology and physiology. Indeed, such molecules can be used to decipher the molecular basis of G protein activation/deactivation processes as well as GPCR function and regulation. In addition, they may also be used as potential therapeutics in human diseases where a link with a dysfunction and/or a dysregulation of GPCR/G protein-mediated signaling has been demonstrated.

3.2. Suramin and its analogs Suramin is an organic polysulfonated naphthylamine-benzamide derivative and constitutes one of the oldest molecules that target and

Table 2 Different adenosine di-phosphate (ADP)-ribosylating toxins and their effects on G proteins activation and signaling. Toxins

Bacterium

G protein targets/mode of action

Signaling effects

Cholera toxin

Vibrio cholerae

ADP-ribosylation of a critical arginine residue in the GTPase activity domain of Gαs subfamily (Gαs and Gαolf).

GTPase activity of the stimulatory Gαs is inhibited.

Pertussis toxin

Bordetella pertussis

ADP-ribosylation of a key cysteine residue in the C-terminus of Gαi subfamily (Gαi, Gαo, and Gαt).

Heat-labile enterotoxin

Escherichia coli

ADP-ribosylation of a critical arginine residue in the GTPase activity domain of Gαs subfamily (Gαs and Gαolf).

Pasteurella toxin

Pasteurella multocida

Deamidation of a conserved glutamine residue in switch 1 of various Gα (Gαi/o, Gαq, and Gα13) and its conversion to glutamic acid.

Gαs protein is permanently activated (irreversible Gαs-GTP form). Permanent activation of adenylyl cyclase and production of cAMP. The physical and functional coupling between the receptor and Gαi protein is inhibited (permanent Gαi-GDP form). Inhibition of Gαi-mediated signaling (no inhibition of adenylyl cyclase and cAMP production). GTPase activity of the stimulatory Gαs is inhibited. Gαs protein is permanently activated (irreversible Gαs-GTP form). Permanent activation of adenylyl cyclase and production of cAMP. GTPase activity of the Gα proteins is inhibited (irreversible Gα-GTP form). Permanent activation of Gα proteins-mediated signaling.

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Fig. 3. Structures of the different small molecules targeting Gα subunits.

(Prévost et al., 2006). Interestingly, BIM-46174 also showed anti-cancer properties including inhibition of cell proliferation, survival, and invasion using in vitro, ex vivo and in vivo systems (Prévost et al., 2006). Although the exact molecular mechanism involved in BIM-46174's action was not known, the inhibition of CTX- as well as GPCR-mediated cAMP accumulation by BIM-46174 was fully reversible with 80–100% of cAMP recovered upon a washout step (Prévost et al., 2006). This suggests a reversible inhibition of nucleotide exchange on Gαs subunit by BIM-46174. A few years later, a disulfide bridged dimer of BIM-46174 was developed and designed as BIM-46187 (Fig. 3). The first study on BIM46187 reported its potent anti-hyperalgesic activity in vivo by targeting GPCRs that play a central role in pain (Favre-Guilmard et al., 2008). Using a combination of various assays based on bioluminescence and fluorescence resonance energy transfer (BRET and FRET) technologies, BIM-46187 was further characterized by Ayoub et al. using transient expression of various GPCRs and G proteins in COS7 cells as well as a purified and reconstituted GPCR/G protein system (Ayoub et al., 2009). Interestingly, BIM-46187 was widely effective at inhibiting GPCR/G protein downstream signaling pathways in COS7 cells with a concentration-dependent inhibition (IC50 = 1–3 μM) of cAMP and IP1 production and serum response element (SRE) gene expression mediated by various GPCRs (vasopressin V2 receptor, β2-adrenergic receptor, 5-HT2c serotonin receptor, protease-activated receptor 1, lysophosphatidic acid receptor, and gamma-aminobutyric acid (GABAB) receptor) (Ayoub et al., 2009). This suggested a pan inhibitory effect of BIM-46187 on the heterotrimeric Gs, Gi/o, Gq, and G12/13 classes with no effect on small G proteins such as Rac (Ayoub et al., 2009). From the mechanistic point of view, the real-time BRET assays performed in live cells and FRET assays on purified proteins allowed the investigation of two aspects of G subunit functioning, its physical association with both the receptor and Gβγ subunits as well as its activation and/or conformational changes. This was mostly studied using protease-activated

subunits (Freissmuth et al., 1996; Hohenegger et al., 1998) (Table 1). This was demonstrated on the adrenergic receptor-Gαs pair where the transition of the receptor to the high affinity agonist binding state was blocked by suramin. The large structure of the suramin molecule indicates that suramin binds and masks a large surface of the Gα subunit, including the one which is involved in the interaction of Gαs with its effector, adenylyl cyclase. In fact, Freissmuth et al. showed a competitive or exclusive binding on the Gαs subunit between suramin and the purified adenylyl cyclase, which significantly prevented the inhibitory effect of suramin on GDP/GTP exchange (Freissmuth et al., 1996). Suramin has also been shown to physically inhibit receptor-G protein coupling by disrupting the association between the purified Gα and Gβγ subunits (Chung and Kermode, 2005). However, many other intracellular effects of suramin have been reported independent of its direct action on G proteins (Williams et al., 2017).

3.3. Imidazopyrazines, BIM-46174 and BIM-46187 In the 2000's two imidazopyrazine derivatives, BIM-46174 and BIM46187, were developed by IPSEN-Institut Henri Beaufour (France) (Fig. 3) (Ayoub et al., 2009; Favre-Guilmard et al., 2008; Prévost et al., 2006). The original compound, BIM-46174 (7-[2-amino-1-oxo-3-thiopropyl]-8-cyclohexylmethyl-2-phenyl-5,6,7,8-tetrahydro-imidazo [1,2a]pyrazine), was identified in a cell-based screening assay as a specific inhibitor of Gαs protein and thereby the cAMP pathway, but not GPCRs or adenylyl cyclase (Prévost et al., 2006). For this study, the MCF7 breast cancer cell line and inhibition of Gαs-induced cAMP accumulation were used. BIM-46174 was found to selectively inhibit the cAMP increase induced by CTX via the activation of Gαs, but not the cAMP response promoted by forskolin activating the membrane adenylyl cyclase (Prévost et al., 2006). Moreover, further characterization clearly showed the inhibitory effect of BIM-46174 on Gαs-mediated cAMP accumulation upon the activation of various Gαs-coupled GPCRs 173

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more efficient than the monomeric form for inhibiting Gαq signaling in CHO and HEK293 cell lines (Schmitz et al., 2014). Interestingly, it has been suggested that the extracellular monomeric BIM might be entirely converted to the dimeric form under specific conditions. Molecular dynamic simulations suggest three potential binding sites of BIM-46187 on Gαq that are consistent with BIM-46187 compromising intradomain movements within Gα (Schmitz et al., 2014). This includes the conformational changes required for GTP binding in the switch region as well as the large displacement of the helical domain away from the Raslike domain important for GDP/GTP exchange. Therefore, it has been proposed that BIM-46187 stabilizes Gα proteins in an intermediate conformation with an empty nucleotide binding pocket by allowing GDP to be released but preventing GTP entry (Schmitz et al., 2014). This conclusion is consistent with the data reported by Ayoub et al. on the effect of BIM-46187 on the purified leukotriene LTB4 receptor/Gαi2 protein complex showing that BIM-46187 did not block GDP binding but inhibited GTP binding (Ayoub et al., 2009).

receptor 1 for BRET and leukotriene B4 receptor for the purified/reconstituted system (Ayoub et al., 2009). Indeed, the study by Ayoub et al. clearly demonstrated that BIM-46187 inhibited both the pre-assembly between protease-activated receptor 1 and its cognate G proteins (Gαi1, Gαo, and Gα12) and thrombin-induced association/activation of protease-activated receptor 1-G protein complexes (IC50 = 2–7 µM) (Ayoub et al., 2009). In addition, BIM-46187 significantly impaired FRET signals measured as a direct interaction between leukotriene B4 receptor and Gαi2 protein (Ayoub et al., 2009). Far-ultraviolet circular dichroism (CD) experiments further supported that BIM46187 directly binds to Gαi2 subunit (but not Gβ1γ2) with an affinity around 330 nM (Ayoub et al., 2009). This conclusion was supported by a recent study on the binding of BIM-46187 on Gαq analyzed by molecular dynamic simulations (Schmitz et al., 2014). In regards to nucleotide binding on the Gα subunit, BIM-46187 did not inhibit the binding of GDP on the Gαi2 subunit, indicating that it binds to GDPbound G protein (Ayoub et al., 2009). By contrast, the GTPγS incorporation assay on purified and reconstituted leukotriene B4 receptor/Gαi2 protein complex indicated that BIM-46187 inhibited GDP/ GTP exchange on the Gαi2 subunit in a non-competitive way (IC50 = 360 nM) (Ayoub et al., 2009). All together, these observations suggest that BIM-46187 binds to GDP-bound Gα subunit on a region, which perturbs its physical interaction with the receptor but not with the Gβγ dimer. This binding seems to functionally disconnect the Gα subunit from the activated receptor resulting in an inhibition of GTP binding. However, from these data it is not clear whether or not BIM-46187 blocks GDP release from the Gα subunit. Finally, BRET and FRET, along with size exclusion chromatography experiments indicated that BIM46187 did not prevent the association between Gαi/o and Gβ1γ2 subunits but dramatically decreased the ligand-induced conformational changes within the heterotrimeric Gα/Gβγ proteins activated by various co-expressed GPCRs (Ayoub et al., 2009). These effects of BIM-46187 were specific to G proteins since no inhibition was observed on agonist-promoted β-arrestin recruitment as shown by BRET in intact cells and FRET using purified receptor and βarrestin (Ayoub et al., 2009). Therefore, based on its pan inhibitory effect on Gα proteins, many studies reported the utilization of BIM46187 as the pharmacological tool of choice to better dissect the multiplicity and complexity of GPCR pharmacology and signaling. In these studies, BIM-46187 was used to inhibit G protein-dependent signaling pathways in order to assess and quantify the contribution of other GPCR-mediated signaling pathways (e.g. β-arrestin, Src) that are G protein-independent (Baba et al., 2013; Labasque et al., 2010; Perkovska et al., 2017). More recently, a study by Schmitz et al. reported wide profiling of BIM-46187 on various GPCR/G protein pairs co-expressed in different cell backgrounds including Chinese hamster ovary (CHO), human embryonic kidney 293 (HEK293), and COS7 cells (Schmitz et al., 2014). As a result, the efficiency of BIM-46187 on different Gα proteins varied from one cell type to another, with an evident preference for Gαq in most of the conditions (Schmitz et al., 2014). Also, the differing levels of expression and density of the different classes of Gα proteins in the various cell lines would contribute to this discrepancy (Schmitz et al., 2014). From the structural and the chemical standpoints, BIM-46187 is the dimer of BIM-46174 and the disulfide bridge does not seem to affect its permeability through the plasma membrane of the cells (Ayoub et al., 2009; Prévost et al., 2006; Schmitz et al., 2014). Also, this chemical crosslinking should breakdown once in the cytoplasm of the cells under high reducing conditions. In addition, the data from purified/reconstituted systems where BIM-46187 was supposed to stay intact are consistent with the cell-based data (Ayoub et al., 2009; Prévost et al., 2006; Schmitz et al., 2014). Together, these observations suggest that BIM-46174 constitutes the active compound with the presence of the disulfide bridge in BIM-46187 that may improve the kinetics of action or the stoichiometry compared with the monomer version. Schmitz et al. reported that the dimeric form of the compound (BIM-46187) was

3.4. Depsipeptides, YM254890 and FR900359, as selective Gαq inhibitors As agents acting on G proteins, the two cyclic depsipeptides, YM254890 and FR900359, constitute fascinating molecules since they were isolated from Chromobacterium and plant Ardisia crenata, respectively (Fig. 3) (Fujioka et al., 1988; Schrage et al., 2015; Takasaki et al., 2004). Their common feature is the specific inhibition of Gαq-mediated signaling shown in many elegant in vitro and in vivo studies. Indeed, YM254890 was the first studied compound with evidence for its antithrombotic and thrombolytic activities in vivo (Kawasaki et al., 2003). YM254890 has also been shown to inhibit purinergic P2Y1 receptorpromoted intracellular calcium increase and platelet aggregation, with an IC50 ranging from 0.1 to 1 μM (Taniguchi et al., 2003; Uemura et al., 2006). In addition, YM-254890 reduces the serum response factor (SRF)-mediated gene transcription in cells expressing the oncogenic Gαq protein mutant (R183C), suggesting a potential application of YM254890 in cancer (Takasaki et al., 2004). As the mechanism of action, YM254890 binds to Gαq and inhibits nucleotide exchange on Gαq by preventing GDP release (Nishimura et al., 2010; Takasaki et al., 2004). In contrast to BIM-46187, which has been shown to interfere with GPCR-G protein association, radioligand binding experiments on purinergic P2Y1 receptor excluded any effect of YM-254890 on the physical interaction between the receptor and Gαq, as well as on their functional coupling (Takasaki et al., 2004). More interestingly, YM254890 has been successfully crystallized with the Gαq subunit, providing the first structural information about a G protein-small molecule complex (Nishimura et al., 2010). From the X-ray crystal structure analysis of the complex between Gαq/βγ and YM-254890, it has been proposed that the binding surface of YM-254890 on Gαq overlaps between the Ras-like GTPase and the helical domains of the Gαq protein (Fig. 4) (Nishimura et al., 2010). This includes binding to the α1 helix and β2 strands of the GTPase domain and the αA helix of the helical domain as well as the hydrophobic cleft between two key interdomain linkers, linker 1 and linker 2 (or Switch 1), connecting these two critical domain of Gαq protein (Fig. 4) (Nishimura et al., 2010). Consequently, because of the importance of the molecular rearrangements of both the Ras-like GTPase and helical domains of the Gα subunit for GDP dissociation, such a binding of YM-254890 stabilizes the inactive GDP-bound form via direct interactions with switch I impairing the linker flexibility and thereby inhibiting GDP release (Nishimura et al., 2010). The selectivity of YM-254890 for the Gαq family may be explained by the conserved residues among members of the Gαq family (Gαq, 11, 14, 15, and 16) that are engaged in the contact, notably within the sequence VPTTGIIEYP in switch 1 (Nishimura et al., 2010). Those residues are significantly different in the other Gα subunit classes. Thus, YM-254890 is considered as a unique “interfacial inhibitor” of G proteins with its interaction at a domain-domain interface of Gαq protein and by binding to the hinge region connecting its two 174

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FR900359. More recently, a study by Matthey et al. showed that local application of FR900359 to the airways of mice in vivo prevented airway constriction without acute effects on blood pressure and heart rate. Moreover, FR900359 induced airway relaxation in mouse, pig, and human airway tissues ex vivo, suggesting that pharmacological inhibition of Gαq proteins by FR900359 may be a therapeutic alternative to treat asthma (Matthey et al., 2017). 3.5. Quinazoline derivatives as Gαi-selective inhibitors As mentioned earlier, PTX is the inhibitor of choice that has been widely used to block Gαi/o proteins. Recently, Appleton et al. reported several small molecules that displayed a partial but a selective inhibitory action on Gαi (Appleton et al., 2014) (Table 1). The study reported peptide analysis on the G protein regulatory (GPR) motif of RGS12/14 and AGS3 (activator of G protein signaling 3) that acts as a GDI for Gαi (Kimple et al., 2001; Peterson et al., 2000). This corresponds to a peptide of 20–40 amino acids that preferentially binds Gαi/ o-GDP preventing the spontaneous release of GDP, so GDP/GTP exchange. From this analysis, multiple small molecules have been identified using computational docking and screening on Gαi1-GDP-Mg2+, Gαi1-GTP-Mg2+, and Gαq-GDP-Mg2+ (Appleton et al., 2014). Among 280,000 compounds, fifteen were selected and validated for their effect on both Gαi1 and Gαq subunits. These compounds showed partial inhibition (25–40%) of nucleotide exchange with differential selectivity patterns between Gαi1 and Gαq. Indeed, two compounds (0990/ 0990CL and 4630) showed significant selectivity for Gαi1 over Gαq and three (8005, 8770, and 4799) were more selective for Gαq, whereas compounds 2967, 6715, and 1026 inhibited nucleotide exchange on both Gαi1 and Gαq (Appleton et al., 2014). Moreover, some compounds were also validated in cAMP assay in cells expressing the α2-adrenergic receptor, showing a partial restoration (∼ 30%) of cAMP reduction promoted by Gαi/o activation. Nuclear magnetic resonance (NMR) experiment demonstrated the interaction of 0990CL with the purified Gαi1 in its GDP-bound form. Finally, the structure-activity relationship analysis confirmed the importance of quinazoline scaffold in such a GDI action (Appleton et al., 2014).

Fig. 4. Crystal structure of the heterotrimeric Gαi/q-Gβγ proteins in complex with YM254890. Adapted from (Nishimura et al., 2010).

key domains, Ras-like GTPase and the helical domains. However, despite its unique and interesting feature as a selective Gαq inhibitor, YM254890 was unavailable for many years until recently it has been commercialized by a Japanese company (Wako Pure Chemical Industries, Ltd, Osaka, Japan). In the meantime, and based on the structural knowledge of the Gαq/YM-254890 complex, two analogs, YM280193 and WU-07047, have been developed (Kaur et al., 2015; Rensing et al., 2015). These two analogs showed less potency and efficiency to inhibit Gαq activation (Kaur et al., 2015; Rensing et al., 2015). Therefore, one challenge remains to synthesize other YM254890 derivatives or compounds that could target not only Gαq but also other Gα proteins. The second cyclic depsipeptide is FR900359, which has been proposed as an alternative to YM254890 (Fig. 3) (Fujioka et al., 1988; Klepac et al., 2016; Schrage et al., 2015). This compound was first isolated in 1988 from the leaves of the ornamental plant Ardisia crenata, which constitutes a big asset from the scientific point of view compared to YM-254890 regarding its extraction and utilization. However, the mode of action of FR900359 and its putative effects or selectivity for a specific Gα class were unknown until recently. Indeed, in 2015 Schrage et al. published the first paper reporting the molecular basis of FR900359 acting as a potent and a selective Gαq inhibitor in vitro and ex vivo (Schrage et al., 2015). This elegant study combined various in vitro assays to dissect the mechanisms of action of FR900359. In addition, the study also provided evidence for the potential application of FR900359 in cancer using a melanoma cell line (Schrage et al., 2015). FR900359 has been shown to block melanoma cell growth by arresting cells in G1 phase. Regarding its mode of action, unlike BIM-46187 but like YM254890, FR900359 seems to function as a guanine nucleotide dissociation inhibitor (GDI) by preventing GDP dissociation (Schrage et al., 2015). Docking and molecular dynamics simulation suggested that binding of FR900359 to Gαq reduces local flexibility in switch region I and II, as revealed by all-atom molecular dynamic simulations, and this effect results in the inhibition of GDP release (Schrage et al., 2015). Furthermore, the specificity of FR900359 towards Gαq/11 was shown by BRET experiments, measuring in real-time and live cells, ligand-induced conformational changes between Gβ1γ2 and various Gα subunits activated by various GPCRs (Schrage et al., 2015). In addition to the seminal work by Schrage et al. recent in vitro, in vivo and ex vivo studies have used FR900359 to reveal the involvement of the Gαq-mediated signaling pathway in various pathophysiologies including obesity and asthma (Klepac et al., 2016; Matthey et al., 2017). In fact, FR900359 has been reported to restore the differentiation and the activity of human and murine brown/beige adipose tissue by inhibiting the Gαq-mediated signaling pathway engaged by various GPCRs (Klepac et al., 2016). This reveals interesting potential opportunities in the treatment of obesity using a G protein inhibitor like

4. Small molecules targeting Gβγ subunits The understanding of the importance of Gβγ subunits in GPCR and G protein signaling came from many genetic studies showing that silencing the expression of Gβγ subunits significantly disrupted G protein-mediated signaling (Hwang et al., 2005; Khan et al., 2013; Krumins and Gilman, 2006; Smrcka, 2008). Moreover, it is well documented that selective pharmacological inhibition of Gβγ subunits results into the inhibition of various downstream signaling pathways mediated by GPCRs (Bonacci et al., 2006; Lehmann et al., 2008; Smrcka, 2013; Smrcka et al., 2008; Surve et al., 2014). Regarding their role in the G protein heterotrimer function, Gβγ subunits are required for the interaction of the heterotrimeric G protein with GPCRs (Bockaert, 1991; Cabrera-Vera et al., 2003; Khan et al., 2013; Smrcka, 2008). In addition to GDP/GTP exchange, which occurs on the Gα subunit of the heterotrimeric G protein upon GPCR activation, the molecular rearrangements between the Gα and Gβγ subunits and their physical dissociation into free Gα-GTP and Gβγ subunits constitute critical events in G proteinmediated signaling. This dissociation allows each part of the G protein heterotrimer to interact with and modulate multiple intracellular signaling effectors (Fig. 1) (Bockaert, 1991; Cabrera-Vera et al., 2003; Khan et al., 2013; Smrcka, 2008). For Gβγ subunits, the well-known effectors include adenylyl cyclase, phosphoinositide 3-kinase γ (PI3γkinase), phospholipase C (PLC β2/β3), N-type Ca2+ channels, and inwardly rectifying K+ channels (Khan et al., 2013; Oldham and Hamm, 2008; Smrcka, 2008). These signaling effectors mediate key cell responses such as neutrophil chemotaxis and vascular cell proliferation (Khan et al., 2013; Lehmann et al., 2008; Oldham and Hamm, 2008; 175

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Smrcka and colleagues, arguing that binding of small molecules at a protein-protein interaction interface (or “hot spot”) on Gβγ would successfully disrupt interactions with their signaling partners (Johnston et al., 2008; Scott et al., 2001; Smrcka, 2013; Smrcka et al., 2008). Indeed, a critical peptide, SIGK (SIGKAFKILGYPDYD), has been identified as a “hot spot” on the Gβ1 subunit, which largely corresponds to the binding surface of the switch II region of Gα (Davis et al., 2005; Johnston et al., 2008; Scott et al., 2001; Smrcka et al., 2008). This was further supported by the observations that incubation of SIGK with the preformed G protein heterotrimer caused its dissociation (Ghosh et al., 2003; Goubaeva et al., 2003; Smrcka, 2008). Moreover, SIGK has been shown to selectively inhibit Gβγ-dependent regulation of specific effectors, suggesting the importance of this surface in Gβγ-effector interactions as well (Johnston et al., 2008; Scott et al., 2001; Smrcka, 2008; Smrcka et al., 2008). 4.2. M119 class molecules Smrcka and colleagues used virtual docking using the crystal structure of Gβ1γ2 bound to SIGK (Davis et al., 2005) as well as a phage-display approach coupled with enzyme-linked immunosorbent assay (ELISA) on many potential Gβγ binders to investigate their ability to inhibit binding of a “hot spot”-binding SIGK peptide to Gβγ (Bonacci et al., 2006). Compounds that inhibited 50% or more the binding of SIGK-phage to Gβγ were then selected and their apparent affinity for Gβγ was determined with IC50 values ranging from 100 nM to 60 μM (Bonacci et al., 2006). Among these molecules, M119 was one of the most potent compounds with high apparent affinity for Gβ1γ2 (IC50 = 200 nM) (Fig. 5). M119 has been shown to display selective inhibitory effects in vitro on Gαi1-Gβγ interactions and membrane translocation of GRK2 and its association with Gβγ (Bonacci et al., 2006). In addition, M119 inhibited, in a concentration-dependent manner, Gβγ-mediated effector activation including PLCβ2/3 and PI3γkinase activation and their related cell response including neutrophil chemotaxis and inflammation (Bonacci et al., 2006; Lehmann et al., 2008). Finally, M119 significantly decreased N-formylmethionineleucyl-phenylalanine (fMLP)-mediated chemotactic signaling pathways in HL-60 leukocytes and morphine/opioid receptor–dependent analgesia in mice (Bonacci et al., 2006; Lehmann et al., 2008). By contrast, M119 did not block fMLP-induced activation of Gαi1-dependent extracellular signal-regulated kinases (ERK1/2) activation in HL-60 cells (Bonacci et al., 2006). Additionally, M119 had no effect on carbachol-dependent Ca2+ release in HEK293 cells stably expressing the Gαq-linked M3-muscarinic receptor (Bonacci et al., 2006). These observations nicely illustrate the specificity of M119 on Gβγ-dependent responses in vitro and in vivo, and further support the concept that Gβγ can be selectively targeted without disrupting the activity of Gα within the heterotrimeric G protein. Some other related compounds, such as M119B, M158C, and M201, have been shown to present differential inhibitory profiles on Gβγ function compared to M119 (Fig. 5) (Bonacci et al., 2006). Indeed, while both M119 and M158C reduced fMLP-mediated calcium increase via the activation of PLCβ2 in HL-60, the compound M201 had no effect. Moreover, M119 blocked the association between Gβ1γ2 and PLCβ2 whereas M201 had a positive effect by increasing this interaction. However, M201 was able to inhibit GRK2 translocation more strongly than M119 (Bonacci et al., 2006). Finally, both M119 and M158C, but not M201, significantly inhibited Gβγ-mediated PI3γ-kinase activation (Bonacci et al., 2006). Thus, the whole study indicates that while all these compounds bind Gβγ, they cause different modulations to the interaction of Gβγ with its effectors, thereby resulting in different alterations in cells. More recently, Smrcka and colleagues also reported another interesting Gβγ-selective compound, S12155, with biased effects (selective modulation of one signaling pathway among others) on Gβγ-mediated signaling (Fig. 5) (Surve et al., 2014). In fact, while S12155 did not

Fig. 5. Structures of the different small molecules targeting Gβγ subunits.

Smrcka, 2008). Therefore, Gβγ subunits represent interesting targets for therapy in G protein-related diseases as well as the mechanistic-based research on GPCR pharmacology and signaling. From the therapeutic standpoint, much evidence supports the beneficial effects of targeting Gβγ subunits in inflammation, heart failure, hypertension, cancer, and pain (Smrcka, 2008; Smrcka et al., 2008). Whereas in fundamental research, selective targeting of Gβγ will undoubtedly have a strong benefit to better decipher the molecular basis of GPCR signaling multiplicity. This should have useful applications with respect to GPCRbiased signaling, with the aim of investigating the importance of Gαindependent versus Gα-dependent and/or Gβγ-dependent signaling in GPCR function (Fig. 1).

4.1. From peptides to small molecules In this decade, many attempts have been carried out to specifically target Gβγ subunits and this was mainly for understanding their role in GPCR and G protein signaling and physiology. As a result, many peptides, and more interestingly, small molecules have been developed as Gβγ binders and modulators (Fig. 5) (Table 1). The most famous pharmacological tool on Gβγ is the GRK2ct (βARKct) consisting of a cterminal 194 amino-acid peptide of GRK2 that contains the Gβγ binding domain and known to interfere with GRK2 activity through disrupting its Gβγ-mediated membrane translocation (Daaka et al., 1997; Hollins et al., 2009; Koch et al., 1994). The structure of GRK2-Gβγ complex has been solved showing that the c-terminus of GRK2 actually interacts with Gβγ subunits at the interface that overlaps with that involved in their association with Gα subunit (Lodowski et al., 2003). Consequently, GRK2ct interferes with Gβγ activity without affecting Gα activation and its downstream signaling (Inglese et al., 1994; Koch et al., 1994, 1994). This constituted the molecular basis for the development of multiple selective Gβγ binders that inhibit Gβγ-mediated signaling without necessarily disrupting other GPCR/G protein-dependent signaling pathways. Indeed, interesting peptides and small molecules capable of interacting with Gβγ subunits have been developed with inhibitory actions on specific effectors downstream of Gβγ. The strategy used for development of selective-Gβγ binders and inhibitors was based on disrupting the physical and functional interaction between Gβγ subunits and their signaling effectors. This was based on a “hot spot” model for Gβγ-effector recognition developed by 176

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inhibit Gβγ-mediated PLC and PI3γ-kinase activation, it nicely activated ERK1/2 and Akt (Surve et al., 2014). Also, S12155 binds to Gβγ subunits and promotes their dissociation from Gαi1-GDP even in the absence of activation of a GPCR and any nucleotide exchange on the Gα subunit (Surve et al., 2014). This process has been shown to be sufficient to promote cell migration in vitro (Surve et al., 2014). The mechanism of this action is not known but one would argue that the binding of S122155 to Gβγ is enough to cause physical dissociation within the G protein heterotrimer, but such a binding is not efficient in term of Gβγ inhibition. Of course, this compound could be useful to elucidate the contribution of Gβγ in GPCR-mediated signaling.

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5. Conclusion The development of G protein-selective small molecules along with GPCR-selective drugs constitute important advances in the GPCR field. This has led to selectively targeting both sides of the coin with the aim of better understanding the molecular basis of the GPCR-G protein axis and its implication in pathophysiology. Indeed, the whole set of small molecules targeting G proteins described in this review present dual interest: firstly, for fundamental research on GPCR pharmacology and signaling, and secondly, for their potential application in GPCR-related disorders. The different studies using these molecules have improved our understanding of the physical and functional GPCR-G protein interaction. The recent advances on the multiplicity and the diversity of GPCR signaling and their regulation constitute a solid rationale for targeting G proteins as a key molecular component in the GPCR system. The concept of GPCR bias is an excellent example of the importance of such molecules targeting G proteins in order to better understand GPCR function in physiology. Our understanding of their mode of action and their impact on the activation/deactivation of the different G protein subunits should open new era for the development of new classes of molecules with better selectivity and/or efficiency. Acknowledgments Special thanks to Dr. Elizabeth Johnstone (Harry Perkins Institute of Medical Research, Perth, Australia) for her critical reading and corrections of the manuscript. Funding This work was supported by the United Arab Emirates University through its 2018–2020 Research Start-up competition Grant no. G00002595. Conflict of interest The author declares no conflict of interest. References Aktories, K., 2011. Bacterial protein toxins that modify host regulatory GTPases. Nat. Rev. Microbiol. 9, 487–498. Appleton, K.M., Bigham, K.J., Lindsey, C.C., Hazard, S., Lirjoni, J., Parnham, S., Hennig, M., Peterson, Y.K., 2014. Development of inhibitors of heterotrimeric Gαi subunits. Bioorg. Med. Chem. 22, 3423–3434. Ayoub, M.A., Damian, M., Gespach, C., Ferrandis, E., Lavergne, O., De Wever, O., Banères, J.-L., Pin, J.-P., Prévost, G.P., 2009. Inhibition of heterotrimeric G protein signaling by a small molecule acting on Galpha subunit. J. Biol. Chem. 284, 29136–29145. Ayoub, M.A., Crépieux, P., Koglin, M., Parmentier, M., Pin, J.-P., Poupon, A., Reiter, E., Smit, M., Steyaert, J., Watier, H., et al., 2017. Antibodies targeting G protein-coupled receptors: recent advances and therapeutic challenges. MAbs 9, 735–741. Baba, K., Benleulmi-Chaachoua, A., Journé, A.-S., Kamal, M., Guillaume, J.-L., Dussaud, S., Gbahou, F., Yettou, K., Liu, C., Contreras-Alcantara, S., et al., 2013. Heteromeric MT1/MT2 melatonin receptors modulate photoreceptor function. Sci. Signal. 6, ra89. Bockaert, J., 1991. G proteins and G-protein-coupled receptors: structure, function and interactions. Curr. Opin. Neurobiol. 1, 32–42. Bockaert, J., Pin, J.P., 1998. Use of a G-protein-coupled receptor to communicate: An evolutionary succes. C. R. Acad. Sci. III 321, 529–551.

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