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Heterotrimeric Gq proteins as therapeutic targets?

Open AccessPublished:March 02, 2020DOI:https://doi.org/10.1074/jbc.REV119.007061
      Heterotrimeric G proteins are the core upstream elements that transduce and amplify the cellular signals from G protein–coupled receptors (GPCRs) to intracellular effectors. GPCRs are the largest family of membrane proteins encoded in the human genome and are the targets of about one-third of prescription medicines. However, to date, no single therapeutic agent exerts its effects via perturbing heterotrimeric G protein function, despite a plethora of evidence linking G protein malfunction to human disease. Several recent studies have brought to light that the Gq family–specific inhibitor FR900359 (FR) is unexpectedly efficacious in silencing the signaling of Gq oncoproteins, mutant Gq variants that mostly exist in the active state. These data not only raise the hope that researchers working in drug discovery may be able to potentially strike Gq oncoproteins from the list of undruggable targets, but also raise questions as to how FR achieves its therapeutic effect. Here, we place emphasis on these recent studies and explain why they expand our pharmacological armamentarium for targeting Gq protein oncogenes as well as broaden our mechanistic understanding of Gq protein oncogene function. We also highlight how this novel insight impacts the significance and utility of using G(q) proteins as targets in drug discovery efforts.

      Introduction

      GTP/GDP exchange and the intrinsic activity of GTP-binding proteins constitute widespread regulatory mechanisms in cells. These are utilized by heterotrimeric αβγ G proteins, downstream effectors of G protein–coupled receptors (GPCRs),
      The abbreviations used are: GPCR
      G protein–coupled receptor
      FR
      FR900359
      YM
      YM254890
      GEF
      guanine nucleotide exchange factor
      GAP
      GTPase-activating protein
      UM
      uveal melanoma
      GDI
      guanine nucleotide dissociation inhibitor.
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      ). Therefore, development of G protein–targeting pharmacological agents that are active in intact cells, on the level of an isolated organ and ideally also in the living organism, would offer unique opportunities to explore the biological consequences that arise from more broad inhibition of signaling components.
      G proteins are grouped into four major families (Gq, Gi, Gs, and G12) based on α subunit homology and function (
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      G proteins: transducers of receptor-generated signals.
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      G protein pathways.
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      Receptor-mediated activation of heterotrimeric G-proteins: current structural insights.
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      Heterotrimeric G protein activation by G-protein-coupled receptors.
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      Conformational flexibility and structural dynamics in GPCR-mediated G protein activation: a perspective.
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      Heterotrimeric G-proteins: a short history.
      ). Missense mutations to codons within almost all of these (Gq, Gi, and Gs) result in diverse pathological conditions, yet all but Gq are lacking effective pharmacological inhibitors (i.e. remain untapped from a drug development perspective) (
      • Gilman A.G.
      G proteins: transducers of receptor-generated signals.
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      G protein pathways.
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      Heterotrimeric G protein activation by G-protein-coupled receptors.
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      Heterotrimeric G-proteins: a short history.
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      ). However, cell-permeable small-molecule inhibitors specifically targeting the Gαi/o branch have yet to be identified. Therefore, this review will focus primarily on the more recent discoveries obtained with the Gq family–specific inhibitors FR900359 (FR) and YM254890 (YM) (Fig. 1) and will highlight the conceptual advances originating therefrom for basic biological research and drug discovery. Specifically, we will single out a subset of Gq protein activities, namely aberrant signaling in cancer, to advance the ideas on drug–G protein interaction for therapeutic advantage. Because much of today's progress in this field traces back to a resurgence of interest in Gq protein inhibitors, a brief historical perspective will also be included.
      Figure thumbnail gr1
      Figure 1Chemical structures of Gq inhibitors FR and YM. Colored areas highlight the components of the amino acid building blocks that differ between FR and YM, accounting for the higher hydrophobicity of FR as well as for the distinct pharmacological features of the two inhibitors (
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      G protein signaling

      The delicate balance between on and off states

      To maintain organismal homeostasis, mammalian cells require an exquisite balance between G protein activation and deactivation. They achieve this by tight control over GDP/GTP exchange and GTP hydrolysis rates. Ligand-activated GPCRs act as guanine nucleotide exchange factors (GEFs) to stimulate GDP/GTP exchange on the G protein α subunit (Fig. 2). Upon GTP binding, Gα changes its conformation, and this is followed by separation of the heterotrimer (the extent of physical separation may vary however (
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      • Gilman A.G.
      G proteins: transducers of receptor-generated signals.
      ,
      • Neves S.R.
      • Ram P.T.
      • Iyengar R.
      G protein pathways.
      ,
      • Johnston C.A.
      • Siderovski D.P.
      Receptor-mediated activation of heterotrimeric G-proteins: current structural insights.
      ,
      • Oldham W.M.
      • Hamm H.E.
      Heterotrimeric G protein activation by G-protein-coupled receptors.
      ,
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      • Meiler J.
      • Hamm H.E.
      Conformational flexibility and structural dynamics in GPCR-mediated G protein activation: a perspective.
      ,
      • Milligan G.
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      Heterotrimeric G-proteins: a short history.
      ). GTP hydrolysis by the inherent GTPase activity, which is often supported by GTPase-activating proteins (GAPs), then terminates G signaling and allows GαGDP to associate with Gβγ to return the G protein to the inactive state (Fig. 2) (
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      G proteins: transducers of receptor-generated signals.
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      ). This activation-inactivation cycle suffices to explain why guanine nucleotide dissociation inhibitors (GDIs), such as FR and YM, are efficient terminators of G protein signaling; they block the rate-limiting step of the cycle, which is GDP release (Fig. 2) (
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      The experimental power of FR900359 to study Gq-regulated biological processes.
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      Structural basis for the specific inhibition of heterotrimeric Gq protein by a small molecule.
      ). It also rationalizes why G protein activity may be elevated in cancer cells because (i) GPCRs and/or their activating ligands are present in excess, (ii) cancer cells may harbor constitutively active receptor variants, (iii) cancer cells may have activating mutations within the Gα protein itself (
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      Illuminating the Onco-GPCRome: novel G protein-coupled receptor-driven oncocrine networks and targets for cancer immunotherapy.
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      Onco-GPCR signaling and dysregulated expression of microRNAs in human cancer.
      ,
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      • Stawiski E.W.
      • Handel T.M.
      • Seshagiri S.
      • Gutkind J.S.
      The emerging mutational landscape of G-proteins and G-protein coupled receptors in cancer.
      ), or (iv) may be deficient in expression of GAPs as well as carry mutated versions of these effective terminators of G protein–dependent signaling (
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      ). Unlike the conventional GPCR-targeted therapies that intervene with categories (i) and (ii), the therapeutic concept discussed in this review is also, and perhaps especially, effective for category (iii). GAPs, category (iv), are not within the scope of this review and interested readers may refer to several excellent reviews on this topic elsewhere (
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      ).
      Figure thumbnail gr2
      Figure 2Schematic of the guanine nucleotide cycle and Gα signaling states. Heterotrimeric G protein signaling commences when ligand-activated GPCRs act as GEFs, causing the release of bound GDP and its replacement by GTP via a short-lived intermediate “empty pocket” state. Exchange of the bound nucleotide results in ternary complex disassembly, separation of Gα from Gβγ, and initiation of downstream signaling. Intrinsic GTP hydrolysis, which is accelerated by GAPs, then resets GαGDP to form the inactive heterotrimer. FR and YM block G protein signaling by preventing GDP release. They freeze the heterotrimer in an inactive conformation by intercalating between the interdomain cleft at a site distinct from the nucleotide-binding pocket, thereby preventing domain separation (
      • Schrage R.
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      • Annala S.
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      • Bald T.
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      • Shinjo Y.
      • Galandrin S.
      • Shridhar N.
      • Hesse M.
      • Grundmann M.
      • Merten N.
      • et al.
      The experimental power of FR900359 to study Gq-regulated biological processes.
      ,
      • 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.
      ).

      When the balance is tipped toward the on state

      It has been known for many years that activating point mutations in Gα proteins are important causative factors in several human cancers (
      • O'Hayre M.
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      ). Of the four families of heterotrimeric G proteins, gain-of-function mutations were found in GNAS (Gαs) (
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      • Leyme A.
      • Marivin A.
      • Casler J.
      • Nguyen L.T.
      • Garcia-Marcos M.
      Different biochemical properties explain why two equivalent Gα subunit mutants cause unrelated diseases.
      ), and GNAQ/GNA11 (Gαq/Gα11) (
      • Lamba S.
      • Felicioni L.
      • Buttitta F.
      • Bleeker F.E.
      • Malatesta S.
      • Corbo V.
      • Scarpa A.
      • Rodolfo M.
      • Knowles M.
      • Frattini M.
      • Marchetti A.
      • Bardelli A.
      Mutational profile of GNAQQ209 in human tumors.
      ,
      • Van Raamsdonk C.D.
      • Bezrookove V.
      • Green G.
      • Bauer J.
      • Gaugler L.
      • O'Brien J.M.
      • Simpson E.M.
      • Barsh G.S.
      • Bastian B.C.
      Frequent somatic mutations of GNAQ in uveal melanoma and blue naevi.
      ,
      • 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.
      • et al.
      Mutations in GNA11 in uveal melanoma.
      ,
      • Möller I.
      • Murali R.
      • Müller H.
      • Wiesner T.
      • Jackett L.A.
      • Scholz S.L.
      • Cosgarea I.
      • van de Nes J.A.
      • Sucker A.
      • Hillen U.
      • Schilling B.
      • Paschen A.
      • Kutzner H.
      • Rütten A.
      • Böckers M.
      • Scolyer R.A.
      • Schadendorf D.
      • Griewank K.G.
      Activating cysteinyl leukotriene receptor 2 (CYSLTR2) mutations in blue nevi.
      ) gene loci. Whereas GNAS and GNAI mutations occur in subsets of human endocrinopathies (
      • Spiegel A.M.
      • Weinstein L.S.
      Inherited diseases involving G proteins and G protein-coupled receptors.
      ,
      • Lyons J.
      • Landis C.A.
      • Harsh G.
      • Vallar L.
      • Grünewald K.
      • Feichtinger H.
      • Duh Q.Y.
      • Clark O.H.
      • Kawasaki E.
      • Bourne H.R.
      Two G protein oncogenes in human endocrine tumors.
      ,
      • Weinstein L.S.
      • Shenker A.
      • Gejman P.V.
      • Merino M.J.
      • Friedman E.
      • Spiegel A.M.
      Activating mutations of the stimulatory G protein in the McCune-Albright syndrome.
      ,
      • Shenker A.
      • Weinstein L.S.
      • Sweet D.E.
      • Spiegel A.M.
      An activating Gsα mutation is present in fibrous dysplasia of bone in the McCune-Albright syndrome.
      ), the first activating somatic GNAO1 mutation was found in breast cancer (
      • Kan Z.
      • Jaiswal B.S.
      • Stinson J.
      • Janakiraman V.
      • Bhatt D.
      • Stern H.M.
      • Yue P.
      • Haverty P.M.
      • Bourgon R.
      • Zheng J.
      • Moorhead M.
      • Chaudhuri S.
      • Tomsho L.P.
      • Peters B.A.
      • Pujara K.
      • et al.
      Diverse somatic mutation patterns and pathway alterations in human cancers.
      ). Within the GNAQ and GNA11 genes, two particular codons are frequently mutated: arginine 183 and glutamine 209. Mutations at these two positions cause diminished GTPase function and so are linked to gain-of-signaling phenotypes (
      • Campbell A.P.
      • Smrcka A.V.
      Targeting G protein-coupled receptor signalling by blocking G proteins.
      ,
      • Takasaki J.
      • Saito T.
      • Taniguchi M.
      • Kawasaki T.
      • Moritani Y.
      • Hayashi K.
      • Kobori M.
      A novel Gαq/11-selective inhibitor.
      ,
      • 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.
      • et al.
      The experimental power of FR900359 to study Gq-regulated biological processes.
      ,
      • O'Hayre M.
      • Degese M.S.
      • Gutkind J.S.
      Novel insights into G protein and G protein-coupled receptor signaling in cancer.
      ,
      • O'Hayre M.
      • Vázquez-Prado J.
      • Kufareva I.
      • Stawiski E.W.
      • Handel T.M.
      • Seshagiri S.
      • Gutkind J.S.
      The emerging mutational landscape of G-proteins and G-protein coupled receptors in cancer.
      ,
      • Maziarz M.
      • Leyme A.
      • Marivin A.
      • Luebbers A.
      • Patel P.P.
      • Chen Z.
      • Sprang S.R.
      • Garcia-Marcos M.
      Atypical activation of the G protein Gαq by the oncogenic mutation Q209P.
      ). Interestingly, both are also considered oncogenic driver mutations in ocular (uveal) melanoma (UM), an aggressive malignancy of the adult eye (
      • Sisley K.
      • Doherty R.
      • Cross N.A.
      What hope for the future? GNAQ and uveal melanoma.
      ,
      • Bastian B.C.
      The molecular pathology of melanoma: an integrated taxonomy of melanocytic neoplasia.
      ,
      • Luke J.J.
      • Triozzi P.L.
      • McKenna K.C.
      • Van Meir E.G.
      • Gershenwald J.E.
      • Bastian B.C.
      • Gutkind J.S.
      • Bowcock A.M.
      • Streicher H.Z.
      • Patel P.M.
      • Sato T.
      • Sossman J.A.
      • Sznol M.
      • Welch J.
      • Thurin M.
      • et al.
      Biology of advanced uveal melanoma and next steps for clinical therapeutics.
      ,
      • Singh A.D.
      • Turell M.E.
      • Topham A.K.
      Uveal melanoma: trends in incidence, treatment, and survival.
      ,
      • Carvajal R.D.
      • Schwartz G.K.
      • Tezel T.
      • Marr B.
      • Francis J.H.
      • Nathan P.D.
      Metastatic disease from uveal melanoma: treatment options and future prospects.
      ). Aside from mutationally activated Gα subunits, an additional recurrent hotspot mutation in UM was recently identified in the CYSLTR2 gene, which codes for the G protein–coupled cysteinyl-leukotriene receptor type 2: CysLTR2L129Q (
      • Moore A.R.
      • Ceraudo E.
      • Sher J.J.
      • Guan Y.
      • Shoushtari A.N.
      • Chang M.T.
      • Zhang J.Q.
      • Walczak E.G.
      • Kazmi M.A.
      • Taylor B.S.
      • Huber T.
      • Chi P.
      • Sakmar T.P.
      • Chen Y.
      Recurrent activating mutations of G-protein-coupled receptor CYSLTR2 in uveal melanoma.
      ). A hallmark feature of this mutant receptor is an overactive Gq signaling cascade coupled with impaired arrestin-mediated down-regulation, abolished responsiveness to its cognate endogenous ligands, and insensitivity to CysLTR2 antagonist/inverse agonist ligands (
      • Ceraudo E.
      • Horioka M.
      • Mattheisen J.M.
      • Hitchman T.D.
      • Moore A.R.
      • Kazmi M.A.
      • Chi P.
      • Chen Y.
      • Sakmar T.P.
      • Huber T.
      Uveal melanoma oncogene CYSLTR2 encodes a constitutively active GPCR highly biased toward Gq signaling.
      ). It follows that inhibitors of Gq function such as FR or YM should have therapeutic potential to suppress the aberrant activity of this signaling module originating on either the receptor or the G protein level. In other words, targeting a convergence point in signal transduction with a single agent might bring therapeutic benefit irrespective of the precise nature of the upstream activating oncoprotein.

      Pharmacological inhibitors of G protein function: Focus on FR900359

      Discovery of a cyclic depsipeptide with the code name FR900359 from a methanol extract of the evergreen plant Ardisia crenata dates back to 1988 (Fig. 3) (
      • Fujioka M.
      • Koda S.
      • Morimoto Y.
      • Biemann K.
      Structure of FR900359, a cyclic depsipeptide from Ardisia crenata sims.
      ). Along with the elucidation of its chemical structure, a preliminary description of biological effects was provided: FR inhibits platelet aggregation, decreases blood pressure, and is cytotoxic to cultured rat fibroblasts and myelocytic leukemia cells (data not shown in Ref.
      • Fujioka M.
      • Koda S.
      • Morimoto Y.
      • Biemann K.
      Structure of FR900359, a cyclic depsipeptide from Ardisia crenata sims.
      ). Whereas all of the observed biological effects may be explained entirely by specific inhibition of Gq family proteins, it was not until 2010 that FR was rediscovered as “compound 362-63-08” in a plant extract library screen searching for inhibitors of the gut hormone cholecystokinin type 1 (CCK1, formerly CCK-A) receptor (Fig. 3) (
      • Nesterov A.
      • Hong M.
      • Hertel C.
      • Jiao P.
      • Brownell L.
      • Cannon E.
      Screening a plant extract library for inhibitors of cholecystokinin receptor CCK1 pathways.
      ). The structural similarity of compound 362-63-08 with YM together with its in vitro selectivity profile led the authors to conclude that the screening hit 362-63-08 does not target the receptor itself but rather hinders CCK1 receptor signaling by specific inhibition of its signal transducing Gq/11 proteins (
      • Nesterov A.
      • Hong M.
      • Hertel C.
      • Jiao P.
      • Brownell L.
      • Cannon E.
      Screening a plant extract library for inhibitors of cholecystokinin receptor CCK1 pathways.
      ). Selective inhibition by FR of Gq, G11, and G14 over all other mammalian G proteins, its molecular mechanism of GDI action, and the potential to probe the Gq contribution to complex biological processes in physiology and disease were not addressed until 2015, when a comprehensive study provided in vitro and ex vivo characterization at a level of detail sufficient to reinvigorate the field of Gq protein inhibitors (
      • 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.
      • et al.
      The experimental power of FR900359 to study Gq-regulated biological processes.
      ) (Fig. 3). Indeed, this very study impacted G protein inhibitor research in manifold beneficial ways: it (i) created scientific community awareness for the existence of a most valuable signal transduction inhibitor, (ii) triggered independent confirmatory studies to re-examine FR's selectivity profile (
      • Gao Z.-G.
      • Jacobson K.A.
      On the selectivity of the Gαq inhibitor UBO-QIC: a comparison with the Gαi inhibitor pertussis toxin.
      ,
      • Inamdar V.
      • Patel A.
      • Manne B.K.
      • Dangelmaier C.
      • Kunapuli S.P.
      Characterization of UBO-QIC as a Gαq inhibitor in platelets.
      ,
      • Kukkonen J.P.
      G-protein inhibition profile of the reported Gq/11 inhibitor UBO-QIC.
      ), (iii) helped fuel the competitive efforts to identify the best-suited synthetic methodology for preparing the complex molecule by chemical synthesis (
      • 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.
      ,
      • Rensing D.T.
      • Uppal S.
      • Blumer K.J.
      • Moeller K.D.
      Toward the selective inhibition of G proteins: total synthesis of a simplified YM-254890 analog.
      ,
      • Kaur H.
      • Harris P.W.R.
      • Little P.J.
      • Brimble M.A.
      Total synthesis of the cyclic depsipeptide YM-280193, a platelet aggregation inhibitor.
      ,
      • 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 Gqα inhibitors based on computational and structure-activity studies.
      ), (iv) sparked broad interest for the application of FR and YM to explore the biological consequences that arise from specific Gq inhibition (
      • Campbell A.P.
      • Smrcka A.V.
      Targeting G protein-coupled receptor signalling by blocking G proteins.
      ,
      • Karpinsky-Semper D.
      • Volmar C.-H.
      • Brothers S.P.
      • Slepak V.Z.
      Differential effects of the Gβ5-RGS7 complex on muscarinic M3 receptor-induced Ca2+ influx and release.
      ,
      • Wauson E.M.
      • Guerra M.L.
      • Dyachok J.
      • McGlynn K.
      • Giles J.
      • Ross E.M.
      • Cobb M.H.
      Differential regulation of ERK1/2 and mTORC1 through T1R1/T1R3 in MIN6 cells.
      ,
      • Carr 3rd, R.
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      • Zhang J.
      • Lam H.
      • An S.S.
      • Tall G.G.
      • Panettieri Jr., R.A.
      • Benovic J.L.
      Interdicting Gq activation in airway disease by receptor-dependent and receptor-independent mechanisms.
      ,
      • Kim S.H.
      • MacIntyre D.A.
      • Hanyaloglu A.C.
      • Blanks A.M.
      • Thornton S.
      • Bennett P.R.
      • Terzidou V.
      The oxytocin receptor antagonist, Atosiban, activates pro-inflammatory pathways in human amnion via Gαi signalling.
      ,
      • Liao Y.
      • Lu B.
      • Ma Q.
      • Wu G.
      • Lai X.
      • Zang J.
      • Shi Y.
      • Liu D.
      • Han F.
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      Human neuropeptide S receptor is activated via a Gαq protein-biased signaling cascade by a human neuropeptide S analog lacking the C-terminal 10 residues.
      ,
      • Bolognini D.
      • Moss C.E.
      • Nilsson K.
      • Petersson A.U.
      • Donnelly I.
      • Sergeev E.
      • König G.M.
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      • Tobin A.B.
      • Milligan G.
      A novel allosteric activator of free fatty acid 2 receptor displays unique Gi-functional bias.
      ,
      • Badolia R.
      • Inamdar V.
      • Manne B.K.
      • Dangelmaier C.
      • Eble J.A.
      • Kunapuli S.P.
      Gq pathway regulates proximal C-type lectin-like receptor-2 (CLEC-2) signaling in platelets.
      ,
      • 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.
      ,
      • 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.
      • et al.
      Heterologous expression, biosynthetic studies, and ecological function of the selective Gq-signaling inhibitor FR900359.
      ,
      • Gao Z.-G.
      • Inoue A.
      • Jacobson K.A.
      On the G protein-coupling selectivity of the native A2B adenosine receptor.
      ,
      • 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.
      ,
      • Lian X.
      • Beer-Hammer S.
      • König G.M.
      • Kostenis E.
      • Nürnberg B.
      • Gollasch M.
      RXFP1 receptor activation by relaxin-2 induces vascular relaxation in mice via a Gαi2-protein/PI3Kβ/γ/nitric oxide-coupled pathway.
      ,
      • Cervantes-Villagrana R.D.
      • Adame-García S.R.
      • García-Jiménez I.
      • Color-Aparicio V.M.
      • Beltrán-Navarro Y.M.
      • König G.M.
      • Kostenis E.
      • Reyes-Cruz G.
      • Gutkind J.S.
      • Vázquez-Prado J.
      Gβγ signaling to the chemotactic effector P-REX1 and mammalian cell migration is directly regulated by Gαq and Gα13 proteins.
      ,
      • Lapadula D.
      • Farias E.
      • Randolph C.E.
      • Purwin T.J.
      • McGrath D.
      • Charpentier T.H.
      • Zhang L.
      • Wu S.
      • Terai M.
      • Sato T.
      • Tall G.G.
      • Zhou N.
      • Wedegaertner P.B.
      • Aplin A.E.
      • Aguirre-Ghiso J.
      • Benovic J.L.
      Effects of oncogenic Gαq and Gα11 inhibition by FR900359 in uveal melanoma.
      ,
      • Kienitz M.-C.
      • Niemeyer A.
      • König G.M.
      • Kostenis E.
      • Pott L.
      • Rinne A.
      Biased signaling of Ca2+-sensing receptors in cardiac myocytes regulates GIRK channel activity.
      ,
      • Ebner J.K.
      • König G.M.
      • Kostenis E.
      • Siegert P.
      • Aktories K.
      • Orth J.H.C.
      Activation of Gq signaling by Pasteurella multocida toxin inhibits the osteoblastogenic-like actions of Activin A in C2C12 myoblasts, a cell model of fibrodysplasia ossificans progressiva.
      ,
      • Grundmann M.
      • Merten N.
      • Malfacini D.
      • Inoue A.
      • Preis P.
      • Simon K.
      • Rüttiger N.
      • Ziegler N.
      • Benkel T.
      • Schmitt N.K.
      • Ishida S.
      • Müller I.
      • Reher R.
      • Kawakami K.
      • Inoue A.
      • et al.
      Lack of β-arrestin signaling in the absence of active G proteins.
      ,
      • Coombs C.
      • Georgantzoglou A.
      • Walker H.A.
      • Patt J.
      • Merten N.
      • Poplimont H.
      • Busch-Nentwich E.M.
      • Williams S.
      • Kotsi C.
      • Kostenis E.
      • Sarris M.
      Chemokine receptor trafficking coordinates neutrophil clustering and dispersal at wounds in zebrafish.
      ,
      • Olianas M.C.
      • Dedoni S.
      • Onali P.
      Protection from interferon-β-induced neuronal apoptosis through stimulation of muscarinic acetylcholine receptors coupled to ERK1/2 activation.
      ,
      • Roszko K.L.
      • Bi R.
      • Gorvin C.M.
      • Bräuner-Osborne H.
      • Xiong X.-F.
      • Inoue A.
      • Thakker R.V.
      • Strømgaard K.
      • Gardella T.
      • Mannstadt M.
      Knockin mouse with mutant Gα11 mimics human inherited hypocalcemia and is rescued by pharmacologic inhibitors.
      ,
      • Lorenzen E.
      • Ceraudo E.
      • Berchiche Y.A.
      • Rico C.A.
      • Fürstenberg A.
      • Sakmar T.P.
      • Huber T.
      G protein subtype-specific signaling bias in a series of CCR5 chemokine analogs.
      ), and (v) provided experimental evidence that Gq inhibition may qualify as an effective postreceptor strategy to target oncogenic signaling in cancer cells with elevated Gq activity.
      Figure thumbnail gr3
      Figure 3Google scholar hits for Gq inhibitors FR and YM. 1988: Isolation and structure elucidation of FR; biology and mechanism of action unknown (
      • Fujioka M.
      • Koda S.
      • Morimoto Y.
      • Biemann K.
      Structure of FR900359, a cyclic depsipeptide from Ardisia crenata sims.
      ). 2004: Discovery of the structurally close analog YM (
      • Takasaki J.
      • Saito T.
      • Taniguchi M.
      • Kawasaki T.
      • Moritani Y.
      • Hayashi K.
      • Kobori M.
      A novel Gαq/11-selective inhibitor.
      ) by Yamanouchi Pharmaceutical Co., later combined in a merger with Fujisawa to form Astellas Pharma, which chose to provide YM to the scientific community in a rather restrictive manner. Until commercialization (see below), YM was available for a small number of researchers only. 2010: Rediscovery of FR, code-named “362-63-08,” from a plant extract library as inhibitor of the Gq-coupled cholecystokinin CCK1 receptor (
      • Nesterov A.
      • Hong M.
      • Hertel C.
      • Jiao P.
      • Brownell L.
      • Cannon E.
      Screening a plant extract library for inhibitors of cholecystokinin receptor CCK1 pathways.
      ). 2015: Resurrection of FR by in-depth characterization of its in vitro specificity and mechanism of action by a concerted effort of members of the signal transduction community (
      • 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.
      • et al.
      The experimental power of FR900359 to study Gq-regulated biological processes.
      ). 2016: Commercialization of YM by Fujifilm Wako Chemicals, as well as total synthesis of YM and FR (
      • 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.
      ). Coincidentally, worldwide awareness of and interest in FR and YM has risen steeply. During a short period of time, FR was commercialized under the code name “UBO-QIC” (University of Bonn–Gq-inhibiting component), which indicated market potential and, in turn, encouraged commercialization of the competing molecule YM.

      FR suppresses oncogenic signaling in melanoma cells with elevated Gq activity

      The first signs for FR efficacy in cancer treatment were obtained when exposing a panel of skin melanoma cells to FR in cell culture (
      • 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.
      • et al.
      The experimental power of FR900359 to study Gq-regulated biological processes.
      ). Interestingly, despite an intrinsically activated Gq cascade in a number of these lines, and despite potent suppression by FR of Gq-mediated inositol phosphate accumulation across all of these, proliferation, cell cycle progression, and mitogenic signaling were abolished in all but MZ7 cells. MZ7 cells harbor the constitutively active GαqR183C variant, considered susceptible to FR treatment (
      • Takasaki J.
      • Saito T.
      • Taniguchi M.
      • Kawasaki T.
      • Moritani Y.
      • Hayashi K.
      • Kobori M.
      A novel Gαq/11-selective inhibitor.
      ,
      • 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.
      ). These data provided the first hint that aberrant Gq activity per se does not suffice to instruct MZ7 cancer cells to proliferate. Apparently, an overactive Gq system is required but not sufficient to define the molecular subtype of melanoma that responds to FR treatment or else to forecast therapeutic efficacy of Gq-inhibiting agents. Given the rich mutational landscape of skin melanoma and the high frequency of mutations in the BRAF, NRAS, CDK4, PTK2B, and ERBB4 genes (
      • Dutton-Regester K.
      • Irwin D.
      • Hunt P.
      • Aoude L.G.
      • Tembe V.
      • Pupo G.M.
      • Lanagan C.
      • Carter C.D.
      • O'Connor L.
      • O'Rourke M.
      • Scolyer R.A.
      • Mann G.J.
      • Schmidt C.W.
      • Herington A.
      • Hayward N.K.
      A high-throughput panel for identifying clinically relevant mutation profiles in melanoma.
      ,
      • Davies H.
      • Bignell G.R.
      • Cox C.
      • Stephens P.
      • Edkins S.
      • Clegg S.
      • Teague J.
      • Woffendin H.
      • Garnett M.J.
      • Bottomley W.
      • Davis N.
      • Dicks E.
      • Ewing R.
      • Floyd Y.
      • Gray K.
      • et al.
      Mutations of the BRAF gene in human cancer.
      ), along with the notion that MZ7 cells also harbor the constitutively active BRAFV600E allele, the findings argue that BRAFV600E but not Gα11R183C must act as the dominant oncogenic driver and that the occurrence of R183C may merely be a consequence of the general mutational burden in this melanoma cell line. Indeed, mitogenic signaling in MZ7 cells is completely blunted by the BRAF inhibitors vemurafenib and trametinib (
      • 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.
      • et al.
      The experimental power of FR900359 to study Gq-regulated biological processes.
      ). Regardless, Gq inhibition with FR provided the proof of principle for a novel route to reprogram a range of skin melanoma cells—those that are instructed by Gq to proliferate—to a less aggressive phenotype (
      • 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.
      • et al.
      The experimental power of FR900359 to study Gq-regulated biological processes.
      ). Because mutant Gαq or Gα11 proteins are found in only 4% of skin melanoma but in 90% of uveal melanoma, it was not surprising to observe researchers turn to the study of FR in cell lines from uveal melanoma tumors: four independent studies on similar subject matter emerged within just a 6-month time frame (
      • 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.
      ,
      • Lapadula D.
      • Farias E.
      • Randolph C.E.
      • Purwin T.J.
      • McGrath D.
      • Charpentier T.H.
      • Zhang L.
      • Wu S.
      • Terai M.
      • Sato T.
      • Tall G.G.
      • Zhou N.
      • Wedegaertner P.B.
      • Aplin A.E.
      • Aguirre-Ghiso J.
      • Benovic J.L.
      Effects of oncogenic Gαq and Gα11 inhibition by FR900359 in uveal melanoma.
      ,
      • 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.
      • et al.
      Direct targeting of Gαq and Gα11 oncoproteins in cancer cells.
      ,
      • Feng X.
      • Arang N.
      • Rigiracciolo D.C.
      • Lee J.S.
      • Yeerna H.
      • Wang Z.
      • Lubrano S.
      • Kishore A.
      • Pachter J.A.
      • König G.M.
      • Maggiolini M.
      • Kostenis E.
      • Schlaepfer D.D.
      • Tamayo P.
      • Chen Q.
      • et al.
      A platform of synthetic lethal gene interaction networks reveals that the GNAQ uveal melanoma oncogene controls the Hippo pathway through FAK.
      ).

      FR inhibition of uveal melanoma Gα oncoproteins: A mechanistic surprise?

      Uveal melanoma is the most common cancer of the adult eye, originating from melanocytes in the choroid, iris, or ciliary body (
      • Sisley K.
      • Doherty R.
      • Cross N.A.
      What hope for the future? GNAQ and uveal melanoma.
      ,
      • Bastian B.C.
      The molecular pathology of melanoma: an integrated taxonomy of melanocytic neoplasia.
      ,
      • Luke J.J.
      • Triozzi P.L.
      • McKenna K.C.
      • Van Meir E.G.
      • Gershenwald J.E.
      • Bastian B.C.
      • Gutkind J.S.
      • Bowcock A.M.
      • Streicher H.Z.
      • Patel P.M.
      • Sato T.
      • Sossman J.A.
      • Sznol M.
      • Welch J.
      • Thurin M.
      • et al.
      Biology of advanced uveal melanoma and next steps for clinical therapeutics.
      ,
      • Singh A.D.
      • Turell M.E.
      • Topham A.K.
      Uveal melanoma: trends in incidence, treatment, and survival.
      ,
      • Carvajal R.D.
      • Schwartz G.K.
      • Tezel T.
      • Marr B.
      • Francis J.H.
      • Nathan P.D.
      Metastatic disease from uveal melanoma: treatment options and future prospects.
      ). The genetic signature and evolution of this particularly lethal form of melanoma is distinct from skin melanoma in that mutations within a Gq signaling module comprising the gene loci for GNAQ, GNA11, their downstream effector PLCB4, or the upstream activating CYSLTR2 occur in a mutually exclusive fashion (
      • Lamba S.
      • Felicioni L.
      • Buttitta F.
      • Bleeker F.E.
      • Malatesta S.
      • Corbo V.
      • Scarpa A.
      • Rodolfo M.
      • Knowles M.
      • Frattini M.
      • Marchetti A.
      • Bardelli A.
      Mutational profile of GNAQQ209 in human tumors.
      ,
      • Van Raamsdonk C.D.
      • Bezrookove V.
      • Green G.
      • Bauer J.
      • Gaugler L.
      • O'Brien J.M.
      • Simpson E.M.
      • Barsh G.S.
      • Bastian B.C.
      Frequent somatic mutations of GNAQ in uveal melanoma and blue naevi.
      ,
      • 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.
      • et al.
      Mutations in GNA11 in uveal melanoma.
      ,
      • Moore A.R.
      • Ceraudo E.
      • Sher J.J.
      • Guan Y.
      • Shoushtari A.N.
      • Chang M.T.
      • Zhang J.Q.
      • Walczak E.G.
      • Kazmi M.A.
      • Taylor B.S.
      • Huber T.
      • Chi P.
      • Sakmar T.P.
      • Chen Y.
      Recurrent activating mutations of G-protein-coupled receptor CYSLTR2 in uveal melanoma.
      ,
      • Robertson A.G.
      • Shih J.
      • Yau C.
      • Gibb E.A.
      • Oba J.
      • Mungall K.L.
      • Hess J.M.
      • Uzunangelov V.
      • Walter V.
      • Danilova L.
      • Lichtenberg T.M.
      • Kucherlapati M.
      • Kimes P.K.
      • Tang M.
      • Penson A.
      • et al.
      Integrative analysis identifies four molecular and clinical subsets in uveal melanoma.
      ). Particularly predominant are gain-of-function mutations within the two highly homologous G protein α subunits, Gαq and Gα11, at the recurrent hotspots Gln-209 and Arg-183 (
      • Lamba S.
      • Felicioni L.
      • Buttitta F.
      • Bleeker F.E.
      • Malatesta S.
      • Corbo V.
      • Scarpa A.
      • Rodolfo M.
      • Knowles M.
      • Frattini M.
      • Marchetti A.
      • Bardelli A.
      Mutational profile of GNAQQ209 in human tumors.
      ,
      • Van Raamsdonk C.D.
      • Bezrookove V.
      • Green G.
      • Bauer J.
      • Gaugler L.
      • O'Brien J.M.
      • Simpson E.M.
      • Barsh G.S.
      • Bastian B.C.
      Frequent somatic mutations of GNAQ in uveal melanoma and blue naevi.
      ,
      • 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.
      • et al.
      Mutations in GNA11 in uveal melanoma.
      ), with mutations at Gln-209 being 13 times more frequent than those at Arg-183 (
      • 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.
      • et al.
      Mutations in GNA11 in uveal melanoma.
      ). Both mutation hotspots are located in the GTPase domain (Fig. 4A) and are catalytically important for the GTPase turn-off reaction by stabilizing the transition state for GTP hydrolysis. Gln-209 of Gαq and Gα11 is analogous to Gln-204 within Gαi, Gln-227 within Gαs, and Gln-61 within the small GTPase Ras, the latter mutated in multiple human cancers (
      • Forbes S.A.
      • Bhamra G.
      • Bamford S.
      • Dawson E.
      • Kok C.
      • Clements J.
      • Menzies A.
      • Teague J.W.
      • Futreal P.A.
      • Stratton M.R.
      The Catalogue of Somatic Mutations in Cancer (COSMIC).
      ,
      • Slebos R.J.
      • Kibbelaar R.E.
      • Dalesio O.
      • Kooistra A.
      • Stam J.
      • Meijer C.J.
      • Wagenaar S.S.
      • Vanderschueren R.G.
      • van Zandwijk N.
      • Mooi W.J.
      K-ras oncogene activation as a prognostic marker in adenocarcinoma of the lung.
      ). If altered by mutation, Gαq/11 deactivation is disturbed, driving inappropriate proliferative signaling, yet different in extent for each of the two hotspots: the Gln-209 mutations (GαqQ209L/P or Gα11Q209L/P) cripple the GTPase activity to create persistently active Gα subunits (as inferred from pioneering X-ray crystallographic studies with Gαi (
      • Kleuss C.
      • Raw A.S.
      • Lee E.
      • Sprang S.R.
      • Gilman A.G.
      Mechanism of GTP hydrolysis by G-protein alpha subunits.
      ,
      • Coleman D.E.
      • Berghuis A.M.
      • Lee E.
      • Linder M.E.
      • Gilman A.G.
      • Sprang S.R.
      Structures of active conformations of Giα1 and the mechanism of GTP hydrolysis.
      ) and recent biochemical investigations (
      • Maziarz M.
      • Leyme A.
      • Marivin A.
      • Luebbers A.
      • Patel P.P.
      • Chen Z.
      • Sprang S.R.
      • Garcia-Marcos M.
      Atypical activation of the G protein Gαq by the oncogenic mutation Q209P.
      ), whereas Arg-183 mutants (GαqR183C or Gα11R183C) retain the capacity to hydrolyze GTP, albeit at a reduced catalytic rate (Fig. 4A) (
      • Sprang S.R.
      Invited review: activation of G proteins by GTP and the mechanism of Gα-catalyzed GTP hydrolysis.
      ). Thus, both mutants differ in their oncogenic properties because R183C prefers GTP over GDP yet still responds to receptor stimulation, whereas Q209L/P is largely, if not entirely, uncoupled from activation by upstream acting GPCRs (
      • Takasaki J.
      • Saito T.
      • Taniguchi M.
      • Kawasaki T.
      • Moritani Y.
      • Hayashi K.
      • Kobori M.
      A novel Gαq/11-selective inhibitor.
      ,
      • 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.
      • et al.
      Direct targeting of Gαq and Gα11 oncoproteins in cancer cells.
      ,
      • Sprang S.R.
      Invited review: activation of G proteins by GTP and the mechanism of Gα-catalyzed GTP hydrolysis.
      ,
      • Wu D.
      • Katz A.
      • Lee C.H.
      • Simon M.I.
      Activation of phospholipase C by α1-adrenergic receptors is mediated by the α subunits of Gq family.
      ,
      • Kalinec G.
      • Nazarali A.J.
      • Hermouet S.
      • Xu N.
      • Gutkind J.S.
      Mutated α subunit of the Gq protein induces malignant transformation in NIH 3T3 cells.
      ) (Fig. 4A). This mechanistic difference explains why Gαq/11Q209L/P but not Gαq/11R183C mutants were long considered unresponsive to inhibitors of receptor-mediated nucleotide exchange (so-called GDIs). FR and YM are precisely such GDIs, viewed as unsuited for manipulating the oncogenic signaling driven by GTPase-deficient Gαq proteins for experimental or therapeutic purposes. However, FR in particular has shown convincing efficacy against UM cancer cells as brought to focus by four independent studies (
      • 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.
      ,
      • Lapadula D.
      • Farias E.
      • Randolph C.E.
      • Purwin T.J.
      • McGrath D.
      • Charpentier T.H.
      • Zhang L.
      • Wu S.
      • Terai M.
      • Sato T.
      • Tall G.G.
      • Zhou N.
      • Wedegaertner P.B.
      • Aplin A.E.
      • Aguirre-Ghiso J.
      • Benovic J.L.
      Effects of oncogenic Gαq and Gα11 inhibition by FR900359 in uveal melanoma.
      ,
      • 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.
      • et al.
      Direct targeting of Gαq and Gα11 oncoproteins in cancer cells.
      ,
      • Feng X.
      • Arang N.
      • Rigiracciolo D.C.
      • Lee J.S.
      • Yeerna H.
      • Wang Z.
      • Lubrano S.
      • Kishore A.
      • Pachter J.A.
      • König G.M.
      • Maggiolini M.
      • Kostenis E.
      • Schlaepfer D.D.
      • Tamayo P.
      • Chen Q.
      • et al.
      A platform of synthetic lethal gene interaction networks reveals that the GNAQ uveal melanoma oncogene controls the Hippo pathway through FAK.
      ). How come?
      Figure thumbnail gr4
      Figure 4Tertiary structure and signaling phenotypes of WT Gαq and GTPase-inactivating mutations R183C and Q209L. A, ribbon drawings of WT and mutant Gαq subunits bound to GDP and FR and based on the atomic coordinates of the Gαi/q-YM-inhibitor complex crystal structure (Protein Data Bank entry 3AH8 (
      • 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.
      )). The GTPase-inactivating mutational hotspots R183C and Q209L are shown as space-filling models, and Gq inhibitor FR is illustrated as a stick model located in the interdomain cleft between the GTPase and the helical domain. B, schematic showing intrinsic properties of WT and mutationally activated Gαq oncoproteins. Curved arrows indicate rates of nucleotide exchange (top) or GTP hydrolysis (bottom), with thin arrows depicting rate-limiting reactions and thick arrows representing non-rate-limiting reactions: for Gαqwt, for example, nucleotide exchange is rate-limiting, but GTP hydrolysis is not, placing Gαqwt under upstream control of a GEF, the GPCR.

      Experimental efficacy of FR in UM cancer cells: Solving an apparent paradox

      G protein signaling requires both activation and deactivation. In normal cells, deactivation is an intrinsic property of the Gα subunit and is not rate-limiting (Fig. 4B). Mammalian Gα proteins typically deactivate by hydrolyzing GTP to GDP at catalytic rates kcat between 0.01 and 3.5 min−1 (
      • Sprang S.R.
      Invited review: activation of G proteins by GTP and the mechanism of Gα-catalyzed GTP hydrolysis.
      ). Because GTP hydrolysis is faster than GDP release, the steady-state pool of activated Gα subunits is tightly linked to the amount of agonist-occupied GPCRs (Fig. 4B). In this way, G protein signaling is largely controlled by and dependent on catalytic input from the upstream acting receptors. However, in Gαq/11Q209L/P mutant cells, the inherent hydrolysis rate is far too slow to reset GDP-Gα (Fig. 4B). It follows that the nucleotide state of Gαq/11Q209L/P becomes more dependent on nucleotide affinity and concentration. Because GTP is in molar excess over GDP in living cells (
      • Traut T.W.
      Physiological concentrations of purines and pyrimidines.
      ) and because GTP dissociates an order of magnitude slower than GDP (
      • Chidiac P.
      • Markin V.S.
      • Ross E.M.
      Kinetic control of guanine nucleotide binding to soluble Gαq.
      ), GTPase-deficient mutants predominantly exist in the GTP-bound state (Fig. 5). However, inhibitors of nucleotide dissociation may shift the nucleotide preference to enrich the fraction of inactive GαqGDP-βγ heterotrimers over time (Fig. 5). Their onset of action will depend on the rate of nucleotide exchange and/or the rate of GTP hydrolysis in a given cellular environment. Let us pause for a moment to reiterate this point: For a GTPase-deficient Gq to become GDP-bound at a relatively fast pace, it must either exchange nucleotides in cells at rates much faster than those believed to occur in in vitro experiments and/or hydrolyze GTP better than predicted from in vitro studies. It may therefore be advisable to revisit the molecular details underlying these quintessential processes of nucleotide exchange and GTP hydrolysis in the living cell context. This does not only appear timely but may also be technically feasible, given the availability of CRISPR-Cas9 genome-edited cells depleted of multiple G protein α subunits (
      • Grundmann M.
      • Merten N.
      • Malfacini D.
      • Inoue A.
      • Preis P.
      • Simon K.
      • Rüttiger N.
      • Ziegler N.
      • Benkel T.
      • Schmitt N.K.
      • Ishida S.
      • Müller I.
      • Reher R.
      • Kawakami K.
      • Inoue A.
      • et al.
      Lack of β-arrestin signaling in the absence of active G proteins.
      ). So far, only FR (and not YM) has shown efficacy in the UM context. It is conceivable that this efficacy is in keeping with the kinetic parameters recently determined for direct interaction between tritiated FR and Gq; unlike YM, FR dissociates from Gq with a remarkably slow off rate (t½diss(FR) ∼92 min versus t½diss(YM) ∼4 min (
      • Kuschak M.
      • Namasivayam V.
      • Rafehi M.
      • Voss J.H.
      • Garg J.
      • Schlegel J.G.
      • Abdelrahman A.
      • Kehraus K.
      • Reher R.
      • Küppers J.
      • Sylvester K.
      • Hinz S.
      • Matthey M.
      • Wenzel D.
      • Fleischmann B.K.
      • et al.
      Cell-permeable high-affinity tracers for Gq proteins provide structural insights, reveal distinct binding kinetics and identify small molecule inhibitors.
      )), suggesting interaction in a pseudo-irreversible manner. Long Gq residence times may therefore be decisively advantageous for duration of action as well as experimental and therapeutic efficacy of Gq inhibitors in UM. Regardless of the kinetic differences, inhibitors of guanine nucleotide dissociation diminish the signaling of GTP-bound Gα in an indirect manner, clearly illustrating their dual value to blunt signaling not only of WT GTPases but also of mutationally activated GTPase-deficient oncogenes.
      Figure thumbnail gr5
      Figure 5Schematic for FR inhibition of oncogenic Gαq/11 GTPases. A, oncogenic Gln-209 mutations result in functional activation of Gαq/11 family proteins by impairing GTP hydrolysis. With diminished regulation by GTPase activity (GTP hydrolysis is rate-limiting), the nucleotide state of mutant Gq becomes more dependent on nucleotide affinity and concentration. Because GTP is in higher abundance than GDP in cells, GαGTP freed from its Gβγ binding partner is the major nucleotide-bound form of GTPase-deficient Gln-209 mutants. Inhibitors of nucleotide dissociation, such as FR, shift the equilibrium toward GαGDP-βγ heterotrimers over time, thereby enriching the fraction of G proteins in a signaling-incompetent state. Subversion of the nucleotide preference of Gln-209 mutants to favor GDP over GTP is an allosteric mechanism whereby FR gains control over aberrant signaling of oncogenic GTPase-deficient Gαq/11 proteins. B, schematic, overall structural fold and detailed view of the heterotrimeric G protein Gαq subunit (Protein Data Bank entry 3AH8) in its inactive, GDP-bound form; Q209L is visualized with a space-filling model. FR does not directly interact with Gln-209 but allosterically stabilizes the GDP-bound fraction of the oncoprotein, a conformation that cannot be maintained when Gαq is GTP-bound (
      • 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.
      ).

      Heterotrimeric Gα subunits as drug targets?

      Inhibition of GαGTP and, thereby, its downstream signaling repertoire, may be relevant to treat pathologies that are driven by overactive G proteins as is the case in various types of human cancers (
      • O'Hayre M.
      • Degese M.S.
      • Gutkind J.S.
      Novel insights into G protein and G protein-coupled receptor signaling in cancer.
      ,
      • O'Hayre M.
      • Vázquez-Prado J.
      • Kufareva I.
      • Stawiski E.W.
      • Handel T.M.
      • Seshagiri S.
      • Gutkind J.S.
      The emerging mutational landscape of G-proteins and G-protein coupled receptors in cancer.
      ,
      • Spiegel A.M.
      • Weinstein L.S.
      Inherited diseases involving G proteins and G protein-coupled receptors.
      ). Provided that targeting of heterotrimeric G proteins in a subfamily- or even isoform-specific manner will be expanded beyond the Gq/11 branch, the issue of ubiquitous Gα expression will still remain a perceived safety concern for potential medications. One possibility to overcome systemic toxicity is local drug application. For FR treatment of ocular melanoma, this may be achieved by local delivery directly into the eye just as established for a number of clinically used intravitreal therapeutics. Topical application, for the avoidance of systemic adverse effects, has already proven successful for FR inhibition of Gq-GPCR signaling in the airways using various in vivo models for acute and chronic lung diseases (
      • 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.
      ). Whereas the pulmonary administration route of an FR aerosol effectively suppressed Gq signaling, as evidenced by remarkable bronchodilation, systemic side effects that would directly result from Gq inhibition, such as blood pressure or heart rate alterations, were not detected (
      • 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.
      ). Long-term toxicity studies will be required to assess whether FR accumulates in certain cells, tissues, or organs to judge its potential to be administered to humans.
      In the current absence of precision pharmacological targeting for mutationally activated Gα proteins, one can only speculate about possible advantages of targeted GαGTP therapeutics. Such a strategy does spring to mind as an attempt to preferentially diminish the aberrant Gα activity in cancer cells only, akin to therapies targeting mutationally activated BRAFV600E in metastatic melanoma. Yet, mutation-specific inhibitors for active Gα have not been reported to date, and, moreover, GαGTP antagonizing agents will likely also block the signaling of WT GTPases in that only a low dosage might afford a therapeutic window for targeted (preferential) inhibition of the oncogenic over the WT GαGTP pool. In light of these considerations and the current absence of X-ray structural information on GTPase-deficient Gαq, the recent successes to target mutationally activated Gq with FR in uveal melanoma must be viewed as a considerable breakthrough (
      • 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.
      ,
      • 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.
      • et al.
      Direct targeting of Gαq and Gα11 oncoproteins in cancer cells.
      ,
      • Feng X.
      • Arang N.
      • Rigiracciolo D.C.
      • Lee J.S.
      • Yeerna H.
      • Wang Z.
      • Lubrano S.
      • Kishore A.
      • Pachter J.A.
      • König G.M.
      • Maggiolini M.
      • Kostenis E.
      • Schlaepfer D.D.
      • Tamayo P.
      • Chen Q.
      • et al.
      A platform of synthetic lethal gene interaction networks reveals that the GNAQ uveal melanoma oncogene controls the Hippo pathway through FAK.
      ). Guanine nucleotide dissociation inhibitors of heterotrimeric G proteins such as FR may therefore evolve to be cornerstones of “anti-GαGTP therapies,” given their proven capacity to shift the nucleotide preference of Gα proteins toward the GDP-bound inactive state (Fig. 5). If combined with tissue- or cell-specific targeting, such as antibody-drug conjugates, systemic side effects may be kept at a minimum or even be spared. In such a scenario, concomitant inhibition of both mutationally activated and WT Gα may even be of advantage to harm the aberrant cells.

      Conclusions and outlook

      It has been known for decades that GTPase-inactivating point mutations in Gα proteins are important causative factors in many human cancers. However, there have been few attempts to establish approaches for inhibition of Gα oncoproteins (
      • 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.
      ,
      • Lapadula D.
      • Farias E.
      • Randolph C.E.
      • Purwin T.J.
      • McGrath D.
      • Charpentier T.H.
      • Zhang L.
      • Wu S.
      • Terai M.
      • Sato T.
      • Tall G.G.
      • Zhou N.
      • Wedegaertner P.B.
      • Aplin A.E.
      • Aguirre-Ghiso J.
      • Benovic J.L.
      Effects of oncogenic Gαq and Gα11 inhibition by FR900359 in uveal melanoma.
      ,
      • 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.
      • et al.
      Direct targeting of Gαq and Gα11 oncoproteins in cancer cells.
      ,
      • Feng X.
      • Arang N.
      • Rigiracciolo D.C.
      • Lee J.S.
      • Yeerna H.
      • Wang Z.
      • Lubrano S.
      • Kishore A.
      • Pachter J.A.
      • König G.M.
      • Maggiolini M.
      • Kostenis E.
      • Schlaepfer D.D.
      • Tamayo P.
      • Chen Q.
      • et al.
      A platform of synthetic lethal gene interaction networks reveals that the GNAQ uveal melanoma oncogene controls the Hippo pathway through FAK.
      ). One possible daunting challenge may have been that G protein–targeted pharmacological agents must enter the cell to exert their desired biological effect. However, molecules like FR or YM are beginning to bring this goal within reach. As far as pharmacological strategies are concerned, direct competition with GTP binding, in analogy to kinase inhibitors that compete with ATP binding, has not been seriously considered. This is because of the extremely high affinities of GTP and GDP for their nucleotide-binding pockets along with their micromolar abundance in cells, meaning that nucleotide binding to the catalytic site is very hard to overcome by any competitive inhibitor (
      • John J.
      • Sohmen R.
      • Feuerstein J.
      • Linke R.
      • Wittinghofer A.
      • Goody R.S.
      Kinetics of interaction of nucleotides with nucleotide-free H-ras p21.
      ). What other strategies do come to mind to hinder constitutively active Gα proteins from aberrant signaling? Pharmacological reactivation of deficient Gα-GTPase activity may be a way to go (
      • Ja W.W.
      • Wiser O.
      • Austin R.J.
      • Jan L.Y.
      • Roberts R.W.
      Turning G proteins on and off using peptide ligands.
      ), but conceivably very hard to implement. Thus, in the current absence of pharmacological agents to directly antagonize persistently active Gα, targeting nucleotide exchange, for long viewed ineffective for this purpose, appears particularly straightforward. In this respect, the re-emergence of FR, a highly specific Gαq-directed inhibitor of GDP/GTP exchange and cellular signaling, has not only revitalized the idea of targeting G protein oncogenes but also provided proof of principle in vitro (
      • 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.
      ,
      • Lapadula D.
      • Farias E.
      • Randolph C.E.
      • Purwin T.J.
      • McGrath D.
      • Charpentier T.H.
      • Zhang L.
      • Wu S.
      • Terai M.
      • Sato T.
      • Tall G.G.
      • Zhou N.
      • Wedegaertner P.B.
      • Aplin A.E.
      • Aguirre-Ghiso J.
      • Benovic J.L.
      Effects of oncogenic Gαq and Gα11 inhibition by FR900359 in uveal melanoma.
      ,
      • 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.
      • et al.
      Direct targeting of Gαq and Gα11 oncoproteins in cancer cells.
      ,
      • Feng X.
      • Arang N.
      • Rigiracciolo D.C.
      • Lee J.S.
      • Yeerna H.
      • Wang Z.
      • Lubrano S.
      • Kishore A.
      • Pachter J.A.
      • König G.M.
      • Maggiolini M.
      • Kostenis E.
      • Schlaepfer D.D.
      • Tamayo P.
      • Chen Q.
      • et al.
      A platform of synthetic lethal gene interaction networks reveals that the GNAQ uveal melanoma oncogene controls the Hippo pathway through FAK.
      ) and in vivo (
      • 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.
      • et al.
      Direct targeting of Gαq and Gα11 oncoproteins in cancer cells.
      ) that this is indeed experimentally feasible.

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