RESUMEN
Tryptophan hydroxylases catalyze the first and rate-limiting step in the synthesis of serotonin. Serotonin is a key neurotransmitter in the central nervous system and, in the periphery, functions as a local hormone with multiple physiological functions. Studies in genetically altered mouse models have shown that dysregulation of peripheral serotonin levels leads to metabolic, inflammatory, and fibrotic diseases. Overproduction of serotonin by tumor cells causes severe symptoms typical for the carcinoid syndrome, and tryptophan hydroxylase inhibitors are already in clinical use for patients suffering from this disease. Here, we describe a novel class of potent tryptophan hydroxylase inhibitors, characterized by spanning all active binding sites important for catalysis, specifically those of the cosubstrate pterin, the substrate tryptophan as well as directly chelating the catalytic iron ion. The inhibitors were designed to efficiently reduce serotonin in the periphery while not passing the blood-brain barrier, thus preserving serotonin levels in the brain.
Asunto(s)
Bencimidazoles , Serotonina , Triptófano Hidroxilasa , Xantina , Animales , Bencimidazoles/química , Bencimidazoles/farmacología , Ratones , Triptófano Hidroxilasa/antagonistas & inhibidores , Xantina/química , Xantina/farmacologíaRESUMEN
Despite its essential role in the (patho)physiology of several diseases, CB2R tissue expression profiles and signaling mechanisms are not yet fully understood. We report the development of a highly potent, fluorescent CB2R agonist probe employing structure-based reverse design. It commences with a highly potent, preclinically validated ligand, which is conjugated to a silicon-rhodamine fluorophore, enabling cell permeability. The probe is the first to preserve interspecies affinity and selectivity for both mouse and human CB2R. Extensive cross-validation (FACS, TR-FRET and confocal microscopy) set the stage for CB2R detection in endogenously expressing living cells along with zebrafish larvae. Together, these findings will benefit clinical translatability of CB2R based drugs.
RESUMEN
Pharmacological modulation of cannabinoid type 2 receptor (CB2R) holds promise for the treatment of numerous conditions, including inflammatory diseases, autoimmune disorders, pain, and cancer. Despite the significance of this receptor, researchers lack reliable tools to address questions concerning the expression and complex mechanism of CB2R signaling, especially in cell-type and tissue-dependent contexts. Herein, we report for the first time a versatile ligand platform for the modular design of a collection of highly specific CB2R fluorescent probes, used successfully across applications, species, and cell types. These include flow cytometry of endogenously expressing cells, real-time confocal microscopy of mouse splenocytes and human macrophages, as well as FRET-based kinetic and equilibrium binding assays. High CB2R specificity was demonstrated by competition experiments in living cells expressing CB2R at native levels. The probes were effectively applied to FACS analysis of microglial cells derived from a mouse model relevant to Alzheimer's disease.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Colorantes Fluorescentes/química , Microglía/metabolismo , Receptor Cannabinoide CB2/análisis , Animales , Células CHO , Cricetulus , Modelos Animales de Enfermedad , Citometría de Flujo , Transferencia Resonante de Energía de Fluorescencia , Humanos , Ligandos , Ratones , Simulación del Acoplamiento Molecular , Sondas Moleculares/química , Imagen Óptica , Sensibilidad y Especificidad , Transducción de SeñalRESUMEN
Militarinone C and fumosorinone A, 3-oligoenoyltetramic acids produced by insect pathogenic fungi, were synthesized for the first time. The pyrrolidine-2,4-dione ring was closed through a late-stage Dieckmann condensation of N-(ß-ketoacyl) derivatives of tyrosine, obtained by its acylation with either thioesters or Meldrum's acid derivatives bearing the all- trans-polyene side chain. The latter was built up from ( S)-citronellol via an Evans methylation and Wittig or HWE olefinations.