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The cyclic depsipeptide FR900359 (FR) is derived from the soil bacterium Chromobacterium vaccinii and known to bind Gq proteins of mammals and insects, thereby abolishing the signal transduction of their Gq protein-coupled receptors, a process that leads to severe physiological consequences. Due to their highly conserved structure, Gq family of proteins are a superior ecological target for FR producing organisms, resulting in a defense towards a broad range of harmful organisms. Here, we focus on the question whether bacteria like C. vaccinii are important factors in soil in that their secondary metabolites impair, e.g., plant harming organisms like nematodes. We prove that the Gq inhibitor FR is produced under soil-like conditions. Furthermore, FR inhibits heterologously expressed Gαq proteins of the nematodes Caenorhabditis elegans and Heterodera schachtii in the micromolar range. Additionally, in vivo experiments with C. elegans and the plant parasitic cyst nematode H. schachtii demonstrated that FR reduces locomotion of C. elegans and H. schachtii. Finally, egg-laying of C. elegans and hatching of juvenile stage 2 of H. schachtii from its cysts is inhibited by FR, suggesting that FR might reduce nematode dispersion and proliferation. This study supports the idea that C. vaccinii and its excreted metabolome in the soil might contribute to an ecological equilibrium, maintaining and establishing the successful growth of plants. [ABSTRACT FROM AUTHOR]

G protein-coupled receptors stimulate Rho guanine nucleotide exchange factors that promote mammalian cell migration. Rac and Rho GTPases exert opposing effects on cell morphology and are stimulated downstream of Gβγ and Gα12/13 or Gαq, respectively. These Gα subunits might in turn favor Rho pathways by preventing Gβγ signaling to Rac. Here, we investigated whether Gβγ signaling to phosphatidylinositol 3,4,5-trisphosphate-dependent Rac exchange factor 1 (P-REX1), a key Gβγ chemotactic effector, is directly controlled by Rho-activating Gα subunits. We show that pharmacological inhibition of Gαq makes P-REX1 activation by Gq/Gi-coupled lysophosphatidic acid receptors more effective. Moreover, chemogenetic control of Gi and Gq by designer receptors exclusively activated by designer drugs (DREADDs) confirmed that Gi differentially activates P-REX1. GTPase-deficient GαqQL and Gα13QL variants formed stable complexes with Gβγ, impairing its interaction with P-REX1. The N-terminal regions of these variants were essential for stable interaction with Gβγ. Pulldown assays revealed that chimeric Gα13-i2QL interacts with Gβγ unlike to Gαi2-13QL, the reciprocal chimera, which similarly to Gαi2QL could not interact with Gβγ. Moreover, Gβγ was part of tetrameric Gβγ-GαqQL-RGS2 and Gβγ-Gα13-i2QL-RGS4 complexes, whereas Gα13QL dissociated from Gβγ to interact with the PDZ-RhoGEF-RGS domain. Consistent with an integrated response, Gβγ and AKT kinase were associated with active SDF-1/CXCL12-stimulated P-REX1. This pathway was inhibited by GαqQL and Gα13QL, which also prevented CXCR4-dependent cell migration. We conclude that a coordinated mechanism prioritizes Gαq- and Gα13-mediated signaling to Rho over a Gβγ-dependent Rac pathway, attributed to heterotrimeric Gi proteins.

G protein-coupled receptors (GPCRs) transfer extracellular signals across cell membranes by activating intracellular heterotrimeric G proteins. Several studies suggested G proteins as novel drug targets for the treatment of complex diseases, e.g., asthma and cancer. Recently, we developed specific radiotracers, [³H]PSB-15900-FR and [³H]PSB-16254-YM, for the Gαq family of G proteins by tritiation of the macrocyclic natural products FR900359 (FR) and YM-254890 (YM). In the present study, we utilized these potent radioligands to perform autoradiography studies in tissues of healthy mice, mouse models of disease, and human tissues. Specific binding was high, while non-specific binding was extraordinarily low, giving nearly identical results for both radioligands. High expression levels of Gαq proteins were detected in healthy mouse organs showing the following rank order of potency: kidney > liver > brain > pancreas > lung > spleen, while expression in the heart was low. Organ sub-structures, e.g., of mouse brain and lung, were clearly distinguishable. Whereas an acute asthma model in mice did not result in altered Gαq protein expressions as compared to control animals, a cutaneous melanoma model displayed significantly increased expression in comparison to healthy skin. These results suggest the future development of Gαq-protein-binding radio-tracers as novel diagnostics. [ABSTRACT FROM AUTHOR]

The biological activity of natural products YM-254890 (YM) and FR900359 (FR) has led to significant interest in both their synthesis and the construction of more simplified analogs. While the simplified analogs lose much of the potency of the natural products, they are of interest in their own right, and their synthesis has revealed synthetic barriers to the family of molecules that need to be addressed if a scalable synthesis of YM and FR analogs is to be constructed. In the work described here, a synthetic route to simplified analogs of YM is examined and strategies for circumventing some of the challenges inherent to constructing the molecules are forwarded. [ABSTRACT FROM AUTHOR]

The natural product FR900359 (FR) has generated significant attention lately, due to its characteristics as potent and selective inhibitor of G q/11 mediated signal transduction of associated G protein-coupled receptors (GPCRs). This makes FR both a widely used pharmacological tool compound and a lead molecule for targeted cancer therapy. The exploration of structure-activity-relationship (SAR) of the scaffold by total synthesis has been complicated by its structural complexity and its incompatibility with standard approaches of solid-phase peptide synthesis. Options for late-stage functionalization of FR are limited due to a lack of tractable functional groups. Here we present a mixed approach combining (i) genetic engineering of the FR-assembly line in Chromobacterium vaccinii, to obtain a novel FR analog featuring a primary amine, with (ii) its subsequent synthetic modification and biological profiling for further SAR exploration of the FR scaffold., (© 2024 Wiley-VCH GmbH.)

Access to the cyclic depsipeptide FR900359 (FR), a selective Gq/11 protein inhibitor of high pharmacological interest and a potential lead molecule for targeted therapy of cancers with oncogenic GNAQ or GNA11 mutations (encoding Gq and G11 respectively), has been challenging ever since its initial discovery more than three decades ago. The recent discovery of Chromobacterium vaccinii as a cultivable FR producer enables the development of approaches leading to a high‐yielding, scalable and sustainable biotechnological process for production of FR, thereby removing this bottleneck. Here we characterize different promoters in exchange of the native promoter of the FR assembly line, resulting in an overexpression mutant with significantly increased production of FR. Thereby, the isolation and structure elucidation of novel FR analogs of low abundance is enabled. Further, we explore the antiproliferative activities of fifteen chromodepsins against uveal melanoma cell lines harboring Gq/11 mutations and characterize the major metabolite of FR formed in plasma. [ABSTRACT FROM AUTHOR]

G protein-coupled receptors (GPCRs) transfer extracellular signals across cell membranes by activating intracellular heterotrimeric G proteins. Several studies suggested G proteins as novel drug targets for the treatment of complex diseases, e.g., asthma and cancer. Recently, we developed specific radiotracers, [³H]PSB-15900-FR and [³H]PSB-16254-YM, for the Gαq family of G proteins by tritiation of the macrocyclic natural products FR900359 (FR) and YM-254890 (YM). In the present study, we utilized these potent radioligands to perform autoradiography studies in tissues of healthy mice, mouse models of disease, and human tissues. Specific binding was high, while non-specific binding was extraordinarily low, giving nearly identical results for both radioligands. High expression levels of Gαq proteins were detected in healthy mouse organs showing the following rank order of potency: kidney > liver > brain > pancreas > lung > spleen, while expression in the heart was low. Organ sub-structures, e.g., of mouse brain and lung, were clearly distinguishable. Whereas an acute asthma model in mice did not result in altered Gαq protein expressions as compared to control animals, a cutaneous melanoma model displayed significantly increased expression in comparison to healthy skin. These results suggest the future development of Gαq-protein-binding radio-tracers as novel diagnostics.

The cyclic depsipeptide FR900359 (FR) isolated from the plant Ardisia crenata and produced by endosymbiotic bacteria acts as a selective Gq protein inhibitor. It is a powerful tool to study G protein-coupled receptor signaling, and has potential as a novel drug for the treatment of pulmonary diseases and cancer. For pharmacokinetic studies, sensitive quantitative measurements of drug levels are required. In the present study we established an LC-MS/MS method to detect nanomolar concentrations of FR and the structurally related natural product YM-254890 (YM) in biological samples. HPLC separation coupled to ESI-QTOF-MS and UV-VIS detection was applied. For identification and quantification, the extract ion chromatogram (EIC) of M+1 was evaluated. Limits of detection (LOD) of 0.53–0.55 nM and limits of quantification (LOQ) of 1.6–1.7 nM were achieved for both FR and YM. This protocol was subsequently applied to determine FR concentrations in mouse organs and tissues after peroral application of the drug. A three-step liquid-liquid extraction protocol was established, which resulted in adequate recovery rates of typically around 50%. The results indicated low peroral absorption of FR. Besides the gut, highest concentrations were determined in eye and kidney. The developed analytical method will be useful for preclinical studies to evaluate these potent Gq protein inhibitors, which may have potential as future drugs for complex diseases.

G proteins are key mediators of G protein‐coupled receptor (GPCR) signaling, facilitating a plethora of important physiological processes. The role of G proteins is much less understood than other aspects of GPCR function, which is largely due to the shortage of potent and selective G protein inhibitors. The natural cyclic depsipeptides YM‐254890 and FR900359 are two of the very few known selective inhibitors of the Gq subfamily, and are used as unique pharmacological tools in the study of G q‐mediated signaling. Moreover, a peptide‐based G protein antagonist‐2A (GP‐2A), a 27‐residue peptide (27mer(I860A)) derived from phospholipase C‐β3 (PLC‐β3), and the small molecule BIM‐46187 have also been characterized as selective G q inhibitors within the past 5 years. In this review, we highlight the recent development in chemical syntheses, characterization, and mechanism of action of these selective G q inhibitors. The development and application of G q‐selective inhibitors will expand our knowledge of the structure and function of G protein‐mediated signaling, shed light on the development of inhibitors for other G protein classes, and feed in to drug discovery for diseases where G proteins are implicated, including various forms of cancer. [ABSTRACT FROM AUTHOR]

Abstract: Das cyclische Depsipeptid FR900359 (FR), isoliert aus der tropischen Pflanze Ardisia crenata, zeigt starke und selektive Inhibierung von Gq‐Proteinen. Dadurch ist es sowohl für die Erforschung Gq‐abhängiger Prozesse interessant als auch ein vielversprechender Arzneimittelkandidat. Gq‐Inhibierung ist ein neuer Wirkmechanismus für Abwehrstoffe und entscheidend für die ökologische Funktion von FR, wie durch In‐vivo‐Experimente an Mäusen, Affinität zu Gq‐Proteinen von Insekten und Toxizitätsstudien an Insekten gezeigt wurde. Die Sequenzierung des unkultivierten Endosymbionten von A. crenata führte zur Entdeckung des nichtribosomalen Peptidsynthetasegenclusters von FR (frs). Wir präsentieren hier ein Modell der Biosynthese von FR, unterstützt durch bioinformatische und In‐vitro‐Enzymstudien und das neue Derivat AC‐1, welches Flexibilität der Starter‐Kondensierungsdomänen von FR beweist. Expression der frs‐Gene in E. coli führte erstmals zu heterologen Produktion von FR in einem kultivierbaren bakteriellen Wirt. [ABSTRACT FROM AUTHOR]

Abstract: The cyclic depsipeptide FR900359 (FR), isolated from the tropical plant Ardisia crenata, is a strong and selective inhibitor of Gq proteins, making it an indispensable pharmacological tool to study Gq‐related processes, as well as a promising drug candidate. Gq inhibition is a novel mode of action for defense chemicals and crucial for the ecological function of FR, as shown by in vivo experiments in mice, its affinity to insect Gq proteins, and insect toxicity studies. The uncultured endosymbiont of A. crenata was sequenced, revealing the FR nonribosomal peptide synthetase (frs) gene cluster. We here provide a detailed model of FR biosynthesis, supported by in vitro enzymatic and bioinformatic studies, and the novel analogue AC‐1, which demonstrates the flexibility of the FR starter condensation domains. Finally, expression of the frs genes in E. coli led to heterologous FR production in a cultivable, bacterial host for the first time. [ABSTRACT FROM AUTHOR]

Gα q inhibitor UBO-QIC (FR900359) is becoming an important pharmacological tool, but its selectivity against other G proteins and their subunits, especially βγ, has not been well characterized. We examined UBO-QIC’s effect on diverse signaling pathways mediated via various G protein-coupled receptors (GPCRs) and G protein subunits by comparison with known Gα i inhibitor pertussis toxin. As expected, UBO-QIC inhibited Gα q signaling in all assay systems examined. However, other non-Gα q -events, e.g. Gβγ-mediated intracellular calcium release and inositol phosphate production, following activation of G i -coupled A 1 adenosine and M 2 muscarinic acetylcholine receptors, were also blocked by low concentrations of UBO-QIC, indicating that its effect is not limited to Gα q . Thus, UBO-QIC also inhibits Gβγ-mediated signaling similarly to pertussis toxin, although UBO-QIC does not affect Gα i -mediated inhibition or Gα s -mediated stimulation of adenylyl cyclase activity. However, the blockade by UBO-QIC of GPCR signaling, such as carbachol- or adenosine-mediated calcium or inositol phosphate increases, does not always indicate inhibition of Gα q -mediated events, as the βγ subunits released from G i proteins following the activation of G i -coupled receptors, e.g. M 2 and A 1 Rs, may produce similar signaling events. Furthermore, UBO-QIC completely inhibited Akt signaling, but only partially blocked ERK1/2 activity stimulated by the Gq-coupled P2Y 1 R. Thus, we have revealed new aspects of the pharmacological interactions of UBO-QIC. [ABSTRACT FROM AUTHOR]

G protein-coupled receptors (GPCRs) transfer extracellular signals across cell membranes by activating intracellular heterotrimeric G proteins. Several studies suggested G proteins as novel drug targets for the treatment of complex diseases, e.g., asthma and cancer. Recently, we developed specific radiotracers, [³H]PSB-15900-FR and [³H]PSB-16254-YM, for the Gαq family of G proteins by tritiation of the macrocyclic natural products FR900359 (FR) and YM-254890 (YM). In the present study, we utilized these potent radioligands to perform autoradiography studies in tissues of healthy mice, mouse models of disease, and human tissues. Specific binding was high, while non-specific binding was extraordinarily low, giving nearly identical results for both radioligands. High expression levels of Gαq proteins were detected in healthy mouse organs showing the following rank order of potency: kidney > liver > brain > pancreas > lung > spleen, while expression in the heart was low. Organ sub-structures, e.g., of mouse brain and lung, were clearly distinguishable. Whereas an acute asthma model in mice did not result in altered Gαq protein expressions as compared to control animals, a cutaneous melanoma model displayed significantly increased expression in comparison to healthy skin. These results suggest the future development of Gαq-protein-binding radio-tracers as novel diagnostics.

A cyclic depsipeptide, FR900359, isolated from Ardisia crenata was evaluated for vasorelaxant effects on rat aortic arteries. FR900359 caused concentration-dependent relaxation (1 nM-10 μM) in phenylephrine-precontracted endothelium-intact aortic rings, which was inhibited by addition of l-NMMA, a NOS inhibitor. In endothelium-denuded rings, the relaxant effect of low concentrations of FR900359 was diminished, but remained at high concentrations. In endothelium-denuded rings, FR900359 at 0.1 μM significantly attenuated high-K-induced contractions and completely inhibited Ca-induced contractions. These results suggest that the vasorelaxant effect of FR900359 is mediated through the increased release of NO from endothelial cells at low concentrations, and can be attributed to inhibitory effects on voltage-dependent Ca channel- and receptor-operated Ca channel-dependent Ca influx at high concentrations. [ABSTRACT FROM AUTHOR]

G proteins represent intracellular switches that transduce signals relayed from G protein-coupled receptors. The structurally related macrocyclic depsipeptides FR900359 (FR) and YM-254890 (YM) are potent, selective inhibitors of the Gα q protein family. We recently discovered that radiolabeled FR and YM display strongly divergent residence times, which translates into significantly longer antiasthmatic effects of FR. The present study is aimed at investigating the molecular basis for this observed disparity. Based on docking studies, we mutated amino acid residues of the Gα q protein predicted to interact with FR or YM, and recombinantly expressed the mutated Gα q proteins in cells in which the native Gα q proteins had been knocked out by CRISPR-Cas9. Both radioligands showed similar association kinetics, and their binding followed a conformational selection mechanism, which was rationalized by molecular dynamics simulation studies. Several mutations of amino acid residues near the putative binding site of the "lipophilic anchors" of FR, especially those predicted to interact with the isopropyl group present in FR but not in YM, led to dramatically accelerated dissociation kinetics. Our data indicate that the long residence time of FR depends on lipophilic interactions within its binding site. The observed structure-kinetic relationships point to a complex binding mechanism of FR, which likely involves snap-lock- or dowel-like conformational changes of either ligand or protein, or both. These experimental data will be useful for the design of compounds with a desired residence time, a parameter that has now been recognized to be of utmost importance in drug development. [Display omitted] [ABSTRACT FROM AUTHOR]

G protein-coupled receptors are the largest protein family in the human body and represent the most important class of drug targets. They receive extracellular signals and transduce them into the cytosol. The guanine nucleotide-binding Gα proteins represent the main relays by which GPCRs induce intracellular effects. More than 800 different GPCRs interact with 16 Gα proteins belonging to 4 families, Gα i , Gα s , Gα q , and Gα 12/13 . The direct inhibition of Gα protein subunits rather than the modulation of GPCR subtypes has been proposed as a novel strategy for the treatment of complex diseases, including inflammation and cancer. This mini-review presents an introduction to G protein structure and function and describes achievements in the development of peptidic and peptide-derived Gα protein inhibitors. They have become indispensable pharmacological tools, and some of them exhibit significant potential as future drugs., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Mechanisms that control mobilization of cytosolic calcium [Ca2+] i are key for regulation of numerous eukaryotic cell functions. One such paradigmatic mechanism involves activation of phospholipase Cβ (PLCβ) enzymes by G protein βγ subunits from activated Gα i -Gβγ heterotrimers. Here, we report identification of a master switch to enable this control for PLCβ enzymes in living cells. We find that the Gα i -Gβγ-PLCβ-Ca2+ signaling module is entirely dependent on the presence of active Gα q. If Gα q is pharmacologically inhibited or genetically ablated, Gβγ can bind to PLCβ but does not elicit Ca2+ signals. Removal of an auto-inhibitory linker that occludes the active site of the enzyme is required and sufficient to empower "stand-alone control" of PLCβ by Gβγ. This dependence of Gi-Gβγ-Ca2+ on Gα q places an entire signaling branch of G-protein-coupled receptors (GPCRs) under hierarchical control of Gq and changes our understanding of how Gi-GPCRs trigger [Ca2+] i via PLCβ enzymes. • Newly delineated mechanism of Ca2+ signaling by Gi-G-protein-coupled receptors • Paradigmatic Gi-Gβγ-Ca2+ signals require Gα q subunit in living cells • Relieving auto-inhibition of PLCβ empowers Gβγ-Ca2+ signaling in the absence of Gα q Calcium mobilization by Gi-coupled GPCRs has been perceived as a stand-alone signaling mechanism for almost 30 years. Here, Pfeil et al. report the unexpected discovery that this quintessential biological process does not occur without, and totally depends on, the heterotrimeric G protein subunit Gαq in the living cell context. [ABSTRACT FROM AUTHOR]

Heterotrimeric G protein subunits Gαq and Gα11 are inhibited by two cyclic depsipeptides, FR900359 (FR) and YM-254890 (YM), both of which are being used widely to implicate Gq/11 proteins in the regulation of diverse biological processes. An emerging major research question therefore is whether the cellular effects of both inhibitors are on-target, that is, mediated via specific inhibition of Gq/11 proteins, or off-target, that is, the result of nonspecific interactions with other proteins. Here we introduce a versatile experimental strategy to discriminate between these possibilities. We developed a Gαq variant with preserved catalytic activity, but refractory to FR/YM inhibition. A minimum of two amino acid changes were required and sufficient to achieve complete inhibitor resistance. We characterized the novel mutant in HEK293 cells depleted by CRISPR-Cas9 of endogenous Gαq and Gα11 to ensure precise control over the Gα-dependent cellular signaling route. Using a battery of cellular outcomes with known and concealed Gq contribution, we found that FR/YM specifically inhibited cellular signals after Gαq introduction via transient transfection. Conversely, both inhibitors were inert across all assays in cells expressing the drug-resistant variant. These findings eliminate the possibility that inhibition of non-Gq proteins contributes to the cellular effects of the two depsipeptides. We conclude that combined application of FR or YM along with the drug-resistant Gαq variant is a powerful in vitro strategy to discern on-target Gq against off-target non-Gq action. Consequently, it should be of high value for uncovering Gq input to complex biological processes with high accuracy and the requisite specificity., Competing Interests: Conflict of interest The authors declare that they have no conflict of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Heterotrimeric G proteins are essential mediators of intracellular signaling of G protein-coupled receptors. The G q/11 subfamily consists of G q , G 11 , G 14 , and G 16 proteins, of which all but G 16 are inhibited by the structurally related natural products YM-254890 and FR900359. These inhibitors act by preventing the GDP/GTP exchange, which is necessary for activation of all G proteins. A homologous putative binding site for YM-254890/FR900359 can also be found in members of the other three G protein families, G s , G i/o , and G 12/13 , but none of the published analogs of YM-254890/FR900359 have shown any inhibitory activity for any of these. To explain why the YM-254890/FR900359 scaffold only inhibits G q/11/14 , the present study delineated the molecular selectivity determinants by exchanging amino acid residues in the YM-254890/FR900359-binding site in G q and G s We found that the activity of a G s mutant with a G q -like binding site for YM-254890/FR900359 can be inhibited by FR900359, and a minimum of three mutations are necessary to introduce inhibition in G s In all, this suggests that although the YM-254890/FR900359 scaffold has proven unsuccessful to derive G s , G i/o , and G 12/13 inhibitors, the mechanism of inhibition between families of G proteins is conserved, opening up the possibility of targeting by other, novel inhibitor scaffolds. In lack of a selective Gα s inhibitor, FR900359-sensitive Gα s mutants may prove useful in studies where delicate control over Gα s signaling would be of the essence., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Boesgaard et al.)

The cyclic depsipeptide FR900359 (FR) isolated from the plant Ardisia crenata and produced by endosymbiotic bacteria acts as a selective Gq protein inhibitor. It is a powerful tool to study G protein-coupled receptor signaling, and has potential as a novel drug for the treatment of pulmonary diseases and cancer. For pharmacokinetic studies, sensitive quantitative measurements of drug levels are required. In the present study we established an LC-MS/MS method to detect nanomolar concentrations of FR and the structurally related natural product YM-254890 (YM) in biological samples. HPLC separation coupled to ESI-QTOF-MS and UV-VIS detection was applied. For identification and quantification, the extract ion chromatogram (EIC) of M+1 was evaluated. Limits of detection (LOD) of 0.53-0.55 nM and limits of quantification (LOQ) of 1.6-1.7 nM were achieved for both FR and YM. This protocol was subsequently applied to determine FR concentrations in mouse organs and tissues after peroral application of the drug. A three-step liquid-liquid extraction protocol was established, which resulted in adequate recovery rates of typically around 50%. The results indicated low peroral absorption of FR. Besides the gut, highest concentrations were determined in eye and kidney. The developed analytical method will be useful for preclinical studies to evaluate these potent Gq protein inhibitors, which may have potential as future drugs for complex diseases., (Copyright © 2020 Kuschak, Schlegel, Schneider, Kehraus, Voss, Seidinger, Matthey, Wenzel, Fleischmann, König and Müller.)

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 G q family-specific inhibitor FR900359 (FR) is unexpectedly efficacious in silencing the signaling of G q oncoproteins, mutant G q 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 G q 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 G q protein oncogenes as well as broaden our mechanistic understanding of G q 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., Competing Interests: The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Kostenis et al.)

G proteins are key mediators of G protein-coupled receptor (GPCR) signaling, facilitating a plethora of important physiological processes. The role of G proteins is much less understood than other aspects of GPCR function, which is largely due to the shortage of potent and selective G protein inhibitors. The natural cyclic depsipeptides YM-254890 and FR900359 are two of the very few known selective inhibitors of the G q subfamily, and are used as unique pharmacological tools in the study of G q -mediated signaling. Moreover, a peptide-based G protein antagonist-2A (GP-2A), a 27-residue peptide (27mer(I860A)) derived from phospholipase C-β3 (PLC-β3), and the small molecule BIM-46187 have also been characterized as selective G q inhibitors within the past 5 years. In this review, we highlight the recent development in chemical syntheses, characterization, and mechanism of action of these selective G q inhibitors. The development and application of G q -selective inhibitors will expand our knowledge of the structure and function of G protein-mediated signaling, shed light on the development of inhibitors for other G protein classes, and feed in to drug discovery for diseases where G proteins are implicated, including various forms of cancer., (© 2019 Wiley Periodicals, Inc.)

Transmembrane signals initiated by a range of extracellular stimuli converge on members of the Gq family of heterotrimeric G proteins, which relay these signals in target cells. Gq family G proteins comprise Gq, G11, G14, and G16, which upon activation mediate their cellular effects via inositol lipid-dependent and -independent signaling to control fundamental processes in mammalian physiology. To date, highly specific inhibition of Gq/11/14 signaling can be achieved only with FR900359 (FR) and YM-254890 (YM), two naturally occurring cyclic depsipeptides. To further development of FR or YM mimics for other Gα subunits, we here set out to rationally design Gα16 proteins with artificial FR/YM sensitivity by introducing an engineered depsipeptide-binding site. Thereby we permit control of G16 function through ligands that are inactive on the WT protein. Using CRISPR/Cas9-generated Gαq/Gα11-null cells and loss- and gain-of-function mutagenesis along with label-free whole-cell biosensing, we determined the molecular coordinates for FR/YM inhibition of Gq and transplanted these to FR/YM-insensitive G16. Intriguingly, despite having close structural similarity, FR and YM yielded biologically distinct activities: it was more difficult to perturb Gq inhibition by FR and easier to install FR inhibition onto G16 than perturb or install inhibition with YM. A unique hydrophobic network utilized by FR accounted for these unexpected discrepancies. Our results suggest that non-Gq/11/14 proteins should be amenable to inhibition by FR scaffold-based inhibitors, provided that these inhibitors mimic the interaction of FR with Gα proteins harboring engineered FR-binding sites., (© 2019 Malfacini et al.)

G protein-coupled receptors stimulate Rho guanine nucleotide exchange factors that promote mammalian cell migration. Rac and Rho GTPases exert opposing effects on cell morphology and are stimulated downstream of Gβγ and Gα 12/13 or Gα q , respectively. These Gα subunits might in turn favor Rho pathways by preventing Gβγ signaling to Rac. Here, we investigated whether Gβγ signaling to phosphatidylinositol 3,4,5-trisphosphate-dependent Rac exchange factor 1 (P-REX1), a key Gβγ chemotactic effector, is directly controlled by Rho-activating Gα subunits. We show that pharmacological inhibition of Gα q makes P-REX1 activation by G q /G i -coupled lysophosphatidic acid receptors more effective. Moreover, chemogenetic control of G i and G q by designer receptors exclusively activated by designer drugs (DREADDs) confirmed that G i differentially activates P-REX1. GTPase-deficient Gα q QL and Gα 13 QL variants formed stable complexes with Gβγ, impairing its interaction with P-REX1. The N-terminal regions of these variants were essential for stable interaction with Gβγ. Pulldown assays revealed that chimeric Gα 13-i2 QL interacts with Gβγ unlike to Gα i2-13 QL, the reciprocal chimera, which similarly to Gα i2 QL could not interact with Gβγ. Moreover, Gβγ was part of tetrameric Gβγ-Gα q QL-RGS2 and Gβγ-Gα 13-i2 QL-RGS4 complexes, whereas Gα 13 QL dissociated from Gβγ to interact with the PDZ-RhoGEF-RGS domain. Consistent with an integrated response, Gβγ and AKT kinase were associated with active SDF-1/CXCL12-stimulated P-REX1. This pathway was inhibited by Gα q QL and Gα 13 QL, which also prevented CXCR4-dependent cell migration. We conclude that a coordinated mechanism prioritizes Gα q - and Gα 13 -mediated signaling to Rho over a Gβγ-dependent Rac pathway, attributed to heterotrimeric G i proteins., (© 2019 Cervantes-Villagrana et al.)