Entwicklung, Charakterisierung, Analytik und Anwendung von Tracern und Tool-Verbindungen für Gq-Proteine

G-Protein-gekoppelte Rezeptoren (GPCRs), die wichtige Arzneimittelziele sind, aktivieren intrazelluläre G-Proteine, die als Relais zur Steuerung intrazellulärer Signalwege dienen. Die Blockade von Gq-Proteinen könnte eine neue Strategie für die Behandlung komplexer Krankheiten wie Krebs, chronische Lungenerkrankungen und Störungen des Immunsystems sein. Gegenwärtig sind keine Werkzeuge oder Methoden verfügbar, um Gq-Proteine in ihrer nativen Konformation direkt zu markieren, was für eine Anzahl von biologischen und klinischen Anwendungen nützlich wäre. In der durchgeführten Arbeit wurden Gq-spezifische chemische Sonden durch Tritiierung der makrocyclischen Naturstoffe FR900359 (FR) und YM-254890 (YM) entwickelt, die zu wichtigen Werkzeugen für die Analyse von GPCR-Funktionen geworden sind. Die Tracer erlaubten uns, ihre direkten Wechselwirkungen mit Gq-Proteinen zu messen, Affinitäten von Gq-Inhibitoren zu bestimmen und Bindungskinetiken zu messen. Wir konnten zeigen, dass beide Tracer tatsächlich auffallend verschieden sind und extrem unterschiedliche Bindungskinetiken aufweisen, wobei die FR-abgeleitete Sonde eine extrem niedrige Dissoziation zeigt. Ein "Dübel-Effekt", der durch Computerstudien aufgrund lipophiler Griffe, die nur in FR, aber nicht in YM vorhanden sind, entdeckt wurde, liefert eine plausible molekulare Erklärung. Es wird erwartet, dass diese Unterschiede zu Disparitäten in den pharmakologischen Wirkungen von FR und YM führen. Unter Verwendung von CRISPR / Cas9 Gq-Knockout-Zellen, die mit den verschiedenen Gq-Subtypen transfiziert wurden, zeigten wir, dass FR und YM eine ähnlich hohe Affinität für Gq, G11, G14, aber nicht für G15 / 16 aufweisen. Die Tracer dienten dazu, Gq-Proteine in einer Vielzahl von Organen, Zellen und Geweben mit hoher Genauigkeit nachzuweisen und zu quantifizieren. Darüber hinaus haben wir Tests etabliert, die zur Entdeckung von kleinen Molekülen Gq-Protein-Inhibitoren geführt haben.G protein-coupled receptors (GPCRs), which are major drug targets, activate intracellular G proteins, that act as relays to control intracellular signaling pathways. The blockade of Gq proteins may be a new strategy for the treatment of complex diseases, such as cancer, chronic pulmonary diseases, and disorders of the immune system. Currently, no tools or methods are available for directly labeling Gq proteins in their native conformation, which would be useful for a number of biological and clinical applications. In the present study we developed Gq-specific chemical probes by tritiation of the macrocyclic natural products FR900359 (FR) and YM-254890 (YM), which have become important tools for dissecting GPCR functions. The tracers allowed us to measure their direct interactions with Gq proteins, to determine affinities of Gq inhibitors, and to measure binding kinetics. We could show that both tracers are in fact strikingly different possessing extremely divergent binding kinetics, the FR-derived probe showing extremely low dissociation. A "dowel effect" discovered by computational studies due to lipophilic handles present only in FR, but not in YM, provides a plausible molecular explanation. These differences are anticipated to translate into disparity in the pharmacological actions of FR and YM. Using CRISPR/Cas9 Gq-knock-out cells transfected with the different Gq subtypes we showed that FR and YM display similarly high affinity for Gq, G11, G14, but not for G15/16. The tracers served to detect and quantify Gq proteins in a variety of organs, cells and tissues with high accuracy. Moreover, we established assays that have led to the discovery of small molecule Gq protein inhibitors

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

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 <i>Ardisia crenata</i>, 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 (<b>3</b> and <b>4</b>) were isolated from <i>A. crenata</i> together with the known analogues <b>1</b> and <b>2</b>. The structures of compounds <b>3</b> and <b>4</b> 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 <b>5</b>–<b>9</b>. Comparative bioactivity tests of compounds <b>1</b>–<b>4</b> 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 <i>Ardisia</i> species, among them the non-nodulated leaves of <i>Ardisia lucida</i>, implying a much broader distribution of FR than originally anticipated

Heterologe Expression, Biosynthese und ökologische Funktion des selektiven Gq‐Signaltransduktionsinhibitors FR900359

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

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