Targeting Gαi/s Proteins with Peptidyl Nucleotide Exchange Modulators.

Chemical probes that specifically modulate the activity of heterotrimeric G proteins provide excellent tools for investigating G protein-mediated cell signaling. Herein, we report a family of selective peptidyl Gαi/s modulators derived from peptide library screening and optimization. Conjugation to a cell-penetrating peptide rendered the peptides cell-permeable and biologically active in cell-based assays. The peptides exhibit potent guanine-nucleotide exchange modulator-like activity toward Gαi and Gαs. Molecular docking and dynamic simulations revealed the molecular basis of the protein-ligand interactions and their effects on GDP binding. This study demonstrates the feasibility of developing direct Gαi/s modulators and provides a novel chemical probe for investigating cell signaling through GPCRs/G proteins.

Tetrahydroimidazo[1,2-a]pyrazine Derivatives: Synthesis and Evaluation as Gαq -Protein Ligands.

The 5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine derivative BIM-46174 and its dimeric form BIM-46187 (1) are heterocyclized dipeptides that belong to the very few cell-permeable compounds known to preferentially silence Gαq proteins. To explore the chemical space of Gαq inhibitors of the BIM chemotype, a combinatorial approach was conducted towards a library of BIM molecules. This library was evaluated in a second messenger-based fluorescence assay to analyze the activity of Gαq proteins through the determination of intracellular myo-inositol 1-phosphate. Structure-activity relationships were deduced and structural requirements for biological activity obtained, which were (i) a redox reactive thiol/disulfane substructure, (ii) an N-terminal basic amino group, (iii) a cyclohexylalanine moiety, and (iv) a bicyclic skeleton. Active compounds exhibited cellular toxicity, which was investigated in detail for the prototypical inhibitor 1. This compound affects the structural cytoskeletal dynamics in a Gαq/11 -independent manner.

Heterotrimeric Gq proteins as therapeutic targets?

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.

Direct targeting of Gαq and Gα11 oncoproteins in cancer cells.

Somatic gain-of-function mutations of GNAQ and GNA11, which encode α subunits of heterotrimeric Gαq/11 proteins, occur in about 85% of cases of uveal melanoma (UM), the most common cancer of the adult eye. Molecular therapies to directly target these oncoproteins are lacking, and current treatment options rely on radiation, surgery, or inhibition of effector molecules downstream of these G proteins. A hallmark feature of oncogenic Gαq/11 proteins is their reduced intrinsic rate of hydrolysis of guanosine triphosphate (GTP), which results in their accumulation in the GTP-bound, active state. Here, we report that the cyclic depsipeptide FR900359 (FR) directly interacted with GTPase-deficient Gαq/11 proteins and preferentially inhibited mitogenic ERK signaling rather than canonical phospholipase Cß (PLCß) signaling driven by these oncogenes. Thereby, FR suppressed the proliferation of melanoma cells in culture and inhibited the growth of Gαq-driven UM mouse xenografts in vivo. In contrast, FR did not affect tumor growth when xenografts carried mutated B-RafV600E as the oncogenic driver. Because FR enabled suppression of malignant traits in cancer cells that are driven by activating mutations at codon 209 in Gαq/11 proteins, we envision that similar approaches could be taken to blunt the signaling of non-Gαq/11 G proteins.

Rational design of a heterotrimeric G protein α subunit with artificial inhibitor sensitivity.

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.

BIM-46174 fragments as potential ligands of G proteins.

The 5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine derivative BIM-46174 has received attention as Gαq inhibitor. We conducted structural reductions to monocyclic and bicyclic substructures to explore the chemical space of BIM fragments and to gain insights into the pharmacophore of BIM-type Gαq inhibitors. Two piperazin-2-one-containing fragments and a small library of bicyclic lactams featuring fused pyrazine and diazepine rings were synthesized and evaluated. The results of a second messenger-based cellular assay indicate that the entire BIM structure is required for efficient Gαq inhibition.

Applying Molecular Networking for the Detection of Natural Sources and Analogues of the Selective Gq Protein Inhibitor FR900359.

The cyclic depsipeptide FR900359 (FR), isolated from the traditional Chinese medicine plant Ardisia crenata, is a potent Gq protein inhibitor and thus a valuable tool to study Gq-mediated signaling of G protein-coupled receptors. Two new FR analogues (3 and 4) were isolated from A. crenata together with the known analogues 1 and 2. The structures of compounds 3 and 4 were established by NMR spectroscopic data and MS-based molecular networking followed by in-depth LCMS2 analysis. The latter approach led to the annotation of further FR analogues 5-9. Comparative bioactivity tests of compounds 1-4 along with the parent molecule FR showed high-affinity binding to Gq proteins in the low nanomolar range (IC50 = 2.3-16.8 nM) for all analogues as well as equipotent inhibition of Gq signaling, which gives important SAR insights into this valuable natural product. Additionally, FR was detected from leaves of five other Ardisia species, among them the non-nodulated leaves of Ardisia lucida, implying a much broader distribution of FR than originally anticipated.

Deciphering Specificity Determinants for FR900359-Derived Gq α Inhibitors Based on Computational and Structure-Activity Studies.

Direct targeting of intracellular Gα subunits of G protein-coupled receptors by chemical tools is a challenging task in current pharmacological studies and in the development of novel therapeutic approaches. In this study we analyzed novel FR900359-based analogs from natural sources, synthetic cyclic peptides, as well as all so-far known Gq α inhibitors in a comprehensive study to devise a strategy for the elucidation of characteristics that determine interactions with and inhibition of Gq in the specific FR/YM-binding pocket. Using 2D NMR spectroscopy and molecular docking we identified unique features in the macrocyclic structures responsible for binding to the target protein correlating with inhibitory activity. While all novel compounds were devoid of effects on Gi and Gs proteins, no inhibitor surpassed the biological activity of FR. This raises the question of whether depsipeptides such as FR already represent valuable chemical tools for specific inhibition of Gq and, at the same time, are suitable natural lead structures for the development of novel compounds to target Gα subunits other than Gq .

Targeted inhibition of Gq signaling induces airway relaxation in mouse models of asthma.

Obstructive lung diseases are common causes of disability and death worldwide. A hallmark feature is aberrant activation of Gq protein-dependent signaling cascades. Currently, drugs targeting single G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptors (GPCRs) are used to reduce airway tone. However, therapeutic efficacy is often limited, because various GPCRs contribute to bronchoconstriction, and chronic exposure to receptor-activating medications results in desensitization. We therefore hypothesized that pharmacological Gq inhibition could serve as a central mechanism to achieve efficient therapeutic bronchorelaxation. We found that the compound FR900359 (FR), a membrane-permeable inhibitor of Gq, was effective in silencing Gq signaling in murine and human airway smooth muscle cells. Moreover, FR both prevented bronchoconstrictor responses and triggered sustained airway relaxation in mouse, pig, and human airway tissue ex vivo. Inhalation of FR in healthy wild-type mice resulted in high local concentrations of the compound in the lungs and prevented airway constriction without acute effects on blood pressure and heart rate. FR administration also protected against airway hyperreactivity in murine models of allergen sensitization using ovalbumin and house dust mite as allergens. Our findings establish FR as a selective Gq inhibitor when applied locally to the airways of mice in vivo and suggest that pharmacological blockade of Gq proteins may be a useful therapeutic strategy to achieve bronchorelaxation in asthmatic lung disease.

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

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

Lack of Gαi2 leads to dilative cardiomyopathy and increased mortality in ß1-adrenoceptor overexpressing mice.

AIMS: Inhibitory G (Gi) proteins have been proposed to be cardioprotective. We investigated effects of Gαi2 knockout on cardiac function and survival in a murine heart failure model of cardiac ß1-adrenoceptor overexpression. METHODS AND RESULTS: ß1-transgenic mice lacking Gαi2 (ß1-tg/Gαi2 (-/-)) were compared with wild-type mice and littermates either overexpressing cardiac ß1-adrenoceptors (ß1-tg) or lacking Gαi2 (Gαi2 (-/-)). At 300 days, mortality of mice only lacking Gαi2 was already higher compared with wild-type or ß1-tg, but similar to ß1-tg/Gαi2 (-/-), mice. Beyond 300 days, mortality of ß1-tg/Gαi2 (-/-) mice was enhanced compared with all other genotypes (mean survival time: 363 ± 21 days). At 300 days of age, echocardiography revealed similar cardiac function of wild-type, ß1-tg, and Gαi2 (-/-) mice, but significant impairment for ß1-tg/Gαi2 (-/-) mice (e.g. ejection fraction 14 ± 2 vs. 40 ± 4% in wild-type mice). Significantly increased ventricle-to-body weight ratio (0.71 ± 0.06 vs. 0.48 ± 0.02% in wild-type mice), left ventricular size (length 0.82 ± 0.04 vs. 0.66 ± 0.03 cm in wild types), and atrial natriuretic peptide and brain natriuretic peptide expression (mRNA: 2819 and 495% of wild-type mice, respectively) indicated hypertrophy. Gαi3 was significantly up-regulated in Gαi2 knockout mice (protein compared with wild type: 340 ± 90% in Gαi2 (-/-) and 394 ± 80% in ß1-tg/Gαi2 (-/-), respectively). CONCLUSIONS: Gαi2 deficiency combined with cardiac ß1-adrenoceptor overexpression strongly impaired survival and cardiac function. At 300 days of age, ß1-adrenoceptor overexpression alone had not induced cardiac hypertrophy or dysfunction while there was overt cardiomyopathy in mice additionally lacking Gαi2. We propose an enhanced effect of increased ß1-adrenergic drive by the lack of protection via Gαi2. Gαi3 up-regulation was not sufficient to compensate for Gαi2 deficiency, suggesting an isoform-specific or a concentration-dependent mechanism.