Macrocyclic Gq Protein Inhibitors FR900359 and/or YM-254890-Fit for Translation?

Guanine nucleotide-binding proteins (G proteins) transduce extracellular signals received by G protein-coupled receptors (GPCRs) to intracellular signaling cascades. While GPCRs represent the largest class of drug targets, G protein inhibition has only recently been recognized as a novel strategy for treating complex diseases such as asthma, inflammation, and cancer. The structurally similar macrocyclic depsipeptides FR900359 (FR) and YM-254890 (YM) are potent selective inhibitors of the Gq subfamily of G proteins. FR and YM differ in two positions, FR being more lipophilic than YM. Both compounds are utilized as pharmacological tools to block Gq proteins in vitro and in vivo. However, no detailed characterization of FR and YM has been performed, which is a prerequisite for the compounds' translation into clinical application. Here, we performed a thorough study of both compounds' physicochemical, pharmacokinetic, and pharmacological properties. Chemical stability was high across a large range of pH values, with FR being somewhat more stable than YM. Oral bioavailability and brain penetration of both depsipeptides were low. FR showed lower plasma protein binding and was metabolized significantly faster than YM by human and mouse liver microsomes. FR accumulated in lung after chronic intratracheal or intraperitoneal application, while YM was more distributed to other organs. Most strikingly, the previously observed longer residence time of FR resulted in a significantly prolonged pharmacologic effect as compared to YM in a methacholine-induced bronchoconstriction mouse model. These results prove that changes within a molecule which seem marginal compared to its structural complexity can lead to crucial pharmacological differences.

Sensitive LC-MS/MS Method for the Quantification of Macrocyclic Gαq Protein Inhibitors in Biological Samples.

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.

Cell-permeable high-affinity tracers for Gq proteins provide structural insights, reveal distinct binding kinetics and identify small molecule inhibitors.

BACKGROUND AND PURPOSE: G proteins are intracellular switches that transduce and amplify extracellular signals from GPCRs. The Gq protein subtypes, which are coupled to PLC activation, can act as oncogenes, and their expression was reported to be up-regulated in cancer and inflammatory diseases. Gq inhibition may be an efficient therapeutic strategy constituting a new level of intervention. However, diagnostic tools and therapeutic drugs for Gq proteins are lacking. EXPERIMENTAL APPROACH: We have now developed Gq -specific, cell-permeable 3 H-labelled high-affinity probes based on the macrocyclic depsipeptides FR900359 (FR) and YM-254890 (YM). The tracers served to specifically label and quantify Gq proteins in their native conformation in cells and tissues with high accuracy. KEY RESULTS: FR and YM displayed low nanomolar affinity for Gαq , Gα11 and Gα14 expressed in CRISPR/Cas9 Gαq -knockout cells, but not for Gα15 . The two structurally very similar tracers showed strikingly different dissociation kinetics, which is predicted to result in divergent biological effects. Computational studies suggested a "dowel" effect of the pseudoirreversibly binding FR. A high-throughput binding assay led to the discovery of novel Gq inhibitors, which inhibited Gq signalling in recombinant cells and primary murine brown adipocytes, resulting in enhanced differentiation. CONCLUSIONS AND IMPLICATIONS: The Gq protein inhibitors YM and FR are pharmacologically different despite similar structures. The new versatile tools and powerful assays will contribute to the advancement of the rising field of G protein research.

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.

Heterologous Expression, Biosynthetic Studies, and Ecological Function of the Selective Gq-Signaling Inhibitor FR900359.

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.

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.