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

Case Study of HELLP Syndrome in a pregnant woman of African Descent in Jos, North Central Nigeria

No Abstrac

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

Поводковый центр

Поводковий центр, який включав корпус, кришку, підпружинений базуючий центр, поводкові штирі та пристрій для вирівнювання навантажень між штирями, який відрізняється тим, що пристрій для вирівнювання навантажень між штирями виконаний у вигляді шариків, розміщених у кільцевій канавці, утвореній виточками корпусу та кришки, контактуючих із поводковими штирями через плунжери, розташовані всередині поліуретанових втулок, які встановлені в отворах кришки

Myc and cell cycle control

Soon after the discovery of the Myc gene (c-Myc), it became clear thatMyc expression levels tightly correlate to cell proliferation. The entry in cell cycle of quiescent cells upon Myc enforced expression has been described in manymodels. Also, the downregulation or inactivation ofMyc results in the impairment of cell cycle progression. Given the frequent deregulation of Myc oncogene in human cancer it is important to dissect out the mechanisms underlying the role ofMyc on cell cycle control. Several parallel mechanisms account forMyc-mediated stimulation of the cell cycle. First,most of the critical positive cell cycle regulators are encoded by genes induced byMyc. These Myc target genes include Cdks, cyclins and E2F transcription factors. Apart from its direct effects on the transcription, Myc is able to hyperactivate cyclin/Cdk complexes through the induction of Cdk activating kinase (CAK) and Cdc25 phosphatases. Moreover, Myc antagonizes the activity of cell cycle inhibitors as p21 and p27 through different mechanisms. Thus, Myc is able to block p21 transcription or to induce Skp2, a protein involved in p27 degradation. Finally, Myc induces DNA replication by binding to replication origins and by upregulating genes encoding proteins required for replication initiation. Myc also regulates genes involved in the mitotic control. A promising approach to treat tumors with deregulated Myc is the synthetic lethality based on the inhibition of Cdks. Thus, the knowledge of the Myc-dependent cell cycle regulatory mechanisms will help to discover new therapeutic approaches directed against malignancies with deregulated Myc. This article is part of a Special Issue entitled: Myc proteins in cell biology and pathology.The work in the laboratory of the authors is funded by grants SAF11-23796 from Spanish Ministry of Industry and Innovation, and ISCIII-RETIC RD12/0036/0033 from Spanish Ministry of Health to JL, and FIS 11/00397 to MDD. GB is recipient of a fellowship form the FPI Program. We apologize to colleagues whose work has not been cited in the form of their original papers but in reviews and whose work has not been discussed due to space limitations or unintentional omission