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1.
Chem Sci ; 12(11): 4057-4062, 2021 Feb 04.
Artículo en Inglés | MEDLINE | ID: mdl-34163676

RESUMEN

Vasopressin (VP) and oxytocin (OT) are cyclic neuropeptides that regulate fundamental physiological functions via four G protein-coupled receptors, V1aR, V1bR, V2R, and OTR. Ligand development remains challenging for these receptors due to complex structure-activity relationships. Here, we investigated dimerization as a strategy for developing ligands with novel pharmacology. We regioselectively synthesised and systematically studied parallel, antiparallel and N- to C-terminal cyclized homo- and heterodimer constructs of VP, OT and dVDAVP (1-deamino-4-valine-8-d-arginine-VP). All disulfide-linked dimers, except for the head-to-tail cyclized constructs, retained nanomolar potency despite the structural implications of dimerization. Our results support a single chain interaction for receptor activation. Dimer orientation had little impact on activity, except for the dVDAVP homodimers, where an antagonist to agonist switch was observed at the V1aR. This study provides novel insights into the structural requirements of VP/OT receptor activation and spotlights dimerization as a strategy to modulate pharmacology, a concept also frequently observed in nature.

2.
J Mol Med (Berl) ; 97(2): 201-213, 2019 02.
Artículo en Inglés | MEDLINE | ID: mdl-30535954

RESUMEN

Sinusoidal obstruction syndrome (SOS) is a major complication after hematopoietic stem cell transplantation and belongs to a group of diseases increasingly identified as transplant-related systemic endothelial disease. Administration of defibrotide affords some protection against SOS, but the effect is modest. Hence, there is unmet medical need justifying the preclinical search for alternative approaches. Prostaglandins exert protective actions on endothelial cells of various vascular beds. Here, we explored the therapeutic potential of the prostacyclin analog treprostinil to prevent SOS. Treprostinil acts via stimulation of IP, EP2, and EP4 receptors, which we detected in murine liver sinusoidal endothelial cells (LSECs). Busulfan-induced cell death was reduced when pretreated with treprostinil in vitro. In a murine in vivo model of SOS, concomitantly administered treprostinil caused lower liver weight-to-body weight ratios indicating liver protection. Histopathological changes were scored to assess damage to liver sinusoidal endothelial cells, to hepatocytes, and to the incipient fibrotic reaction. Treprostinil indeed reduced sinusoidal endothelial cell injury, but this did not translate into reduced liver cell necrosis or fibrosis. In summary, our observations provide evidence for a beneficial effect of treprostinil on damage to LSECs but unexpectedly treprostinil was revealed as a double-edged sword in SOS. KEY MESSAGES: Murine liver sinusoidal endothelial cells (LSECs) express prostanoid receptors. Treprostinil reduces busulfan-induced cell death in vitro. Treprostinil lowers liver weight-to-body weight ratios in mice. Treprostinil positively affects LSECs in mice but not hepatic necrosis/fibrosis.


Asunto(s)
Células Endoteliales/efectos de los fármacos , Epoprostenol/análogos & derivados , Enfermedad Veno-Oclusiva Hepática/tratamiento farmacológico , Sustancias Protectoras/uso terapéutico , Animales , Muerte Celular/efectos de los fármacos , Citoprotección/efectos de los fármacos , Células Endoteliales/patología , Epoprostenol/uso terapéutico , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Enfermedad Veno-Oclusiva Hepática/etiología , Enfermedad Veno-Oclusiva Hepática/patología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL
3.
Sci Signal ; 10(508)2017 Dec 05.
Artículo en Inglés | MEDLINE | ID: mdl-29208680

RESUMEN

Oxytocin and vasopressin mediate various physiological functions that are important for osmoregulation, reproduction, cardiovascular function, social behavior, memory, and learning through four G protein-coupled receptors that are also implicated in high-profile disorders. Targeting these receptors is challenging because of the difficulty in obtaining ligands that retain selectivity across rodents and humans for translational studies. We identified a selective and more stable oxytocin receptor (OTR) agonist by subtly modifying the pharmacophore framework of human oxytocin and vasopressin. [Se-Se]-oxytocin-OH displayed similar potency to oxytocin but improved selectivity for OTR, an effect that was retained in mice. Centrally infused [Se-Se]-oxytocin-OH potently reversed social fear in mice, confirming that this action was mediated by OTR and not by V1a or V1b vasopressin receptors. In addition, [Se-Se]-oxytocin-OH produced a more regular contraction pattern than did oxytocin in a preclinical labor induction and augmentation model using myometrial strips from cesarean sections. [Se-Se]-oxytocin-OH had no activity in human cardiomyocytes, indicating a potentially improved safety profile and therapeutic window compared to those of clinically used oxytocin. In conclusion, [Se-Se]-oxytocin-OH is a novel probe for validating OTR as a therapeutic target in various biological systems and is a promising new lead for therapeutic development. Our medicinal chemistry approach may also be applicable to other peptidergic signaling systems with similar selectivity issues.


Asunto(s)
Ansiedad/tratamiento farmacológico , Receptores de Oxitocina/agonistas , Animales , Células COS , Química Farmacéutica , Chlorocebus aethiops , Condicionamiento Psicológico/efectos de los fármacos , Femenino , Células HEK293 , Humanos , Infusiones Intraventriculares , Ligandos , Masculino , Ratones , Ratas
4.
Mol Pharmacol ; 89(6): 630-44, 2016 06.
Artículo en Inglés | MEDLINE | ID: mdl-26989084

RESUMEN

Activation of Gs-coupled receptors enhances engraftment of hematopoietic stem and progenitor cells (HSPCs). We tested the hypothesis that treprostinil, a prostacyclin analog approved for the treatment of pulmonary hypertension, can be repurposed to improve hematopoietic stem cell transplantation. Murine and human HSPCs were isolated from bone marrow and umbilical cord blood, respectively. Prostanoid receptor agonists and the combination thereof with forskolin were tested for their capacity to stimulate [(3)H]cAMP accumulation in HSPCs. Three independent approaches were employed to verify the ability of agonist-activated HSPCs to reconstitute the bone marrow in lethally irradiated recipient mice. The underlying mechanism was explored in cellular migration assays and by blocking C-X-C motif chemokine receptor 4 (CXCR4). Among several prostanoid agonists tested in combination with forskolin, treprostinil was most efficacious in raising intracellular cAMP levels in murine and human HPSCs. Injection of murine and human HSPCs, which had been pretreated with treprostinil and forskolin, enhanced survival of lethally irradiated recipient mice. Survival was further improved if recipient mice were subcutaneously administered treprostinil (0.15 mg kg(-1) 8 h(-1)) for 10 days. This regimen also reduced the number of HSPCs required to rescue lethally irradiated mice. Enhanced survival of recipient mice was causally related to treprostinil-enhanced CXCR4-dependent migration of HSPCs. Treprostinil stimulates the engraftment of human and murine hematopoietic stem cells without impairing their capacity for self-renewal. The investigated dose range corresponds to the dose approved for human use. Hence, these findings may be readily translated into a clinical application.


Asunto(s)
Reposicionamiento de Medicamentos , Epoprostenol/análogos & derivados , Trasplante de Células Madre Hematopoyéticas , Animales , Médula Ósea/efectos de los fármacos , Médula Ósea/metabolismo , Trasplante de Médula Ósea , Ciclo Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Quimiocina CXCL12/farmacología , Toxina del Cólera/farmacología , Colforsina/farmacología , AMP Cíclico/metabolismo , Epoprostenol/administración & dosificación , Epoprostenol/farmacología , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/metabolismo , Humanos , Ratones Endogámicos BALB C , Receptores CXCR4/metabolismo , Receptores de Epoprostenol/metabolismo , Análisis de Supervivencia , Irradiación Corporal Total
6.
J Biol Chem ; 288(40): 28831-44, 2013 Oct 04.
Artículo en Inglés | MEDLINE | ID: mdl-23965991

RESUMEN

The adenosine A2A receptor is a prototypical rhodopsin-like G protein-coupled receptor but has several unique structural features, in particular a long C terminus (of >120 residues) devoid of a palmitoylation site. It is known to interact with several accessory proteins other than those canonically involved in signaling. However, it is evident that many more proteins must interact with the A2A receptor, if the trafficking trajectory of the receptor is taken into account from its site of synthesis in the endoplasmic reticulum (ER) to its disposal by the lysosome. Affinity-tagged versions of the A2A receptor were expressed in HEK293 cells to identify interacting partners residing in the ER by a proteomics approach based on tandem affinity purification. The receptor-protein complexes were purified in quantities sufficient for analysis by mass spectrometry. We identified molecular chaperones (heat-shock proteins HSP90α and HSP70-1A) that interact with and retain partially folded A2A receptor prior to ER exit. Complex formation between the A2A receptor and HSP90α (but not HSP90ß) and HSP70-1A was confirmed by co-affinity precipitation. HSP90 inhibitors also enhanced surface expression of the receptor in PC12 cells, which endogenously express the A2A receptor. Finally, proteins of the HSP relay machinery (e.g. HOP/HSC70-HSP90 organizing protein and P23/HSP90 co-chaperone) were recovered in complexes with the A2A receptor. These observations are consistent with the proposed chaperone/coat protein complex II exchange model. This posits that cytosolic HSP proteins are sequentially recruited to folding intermediates of the A2A receptor. Release of HSP90 is required prior to recruitment of coat protein complex II components. This prevents premature ER export of partially folded receptors.


Asunto(s)
Citoplasma/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Receptor de Adenosina A2A/metabolismo , Transducción de Señal , Animales , Citoplasma/efectos de los fármacos , Células HEK293 , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Humanos , Inmunoprecipitación , Oxidorreductasas Intramoleculares/metabolismo , Espectrometría de Masas , Células PC12 , Prostaglandina-E Sintasas , Complejo de la Endopetidasa Proteasomal/metabolismo , Inhibidores de Proteasoma/farmacología , Unión Proteica/efectos de los fármacos , Pliegue de Proteína/efectos de los fármacos , ARN Interferente Pequeño/metabolismo , Ratas , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal/efectos de los fármacos , Solubilidad , Ubiquitina-Proteína Ligasas/metabolismo
7.
Biochem Soc Trans ; 41(1): 197-204, 2013 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-23356283

RESUMEN

The design and development of selective ligands for the human OT (oxytocin) and AVP (arginine vasopressin) receptors is a big challenge since the different receptor subtypes and their native peptide ligands display great similarity. Detailed understanding of the mechanism of OT's interaction with its receptor is important and may assist in the ligand- or structure-based design of selective and potent ligands. In the present article, we compared 69 OT- and OT-like receptor sequences with regards to their molecular evolution and diversity, utilized an in silico approach to map the common ligand interaction sites of recently published G-protein-coupled receptor structures to a model of the human OTR (OT receptor) and compared these interacting residues within a selection of different OTR sequences. Our analysis suggests the existence of a binding site for OT peptides within the common transmembrane core region of the receptor, but it appears extremely difficult to identify receptor or ligand residues that could explain the selectivity of OT to its receptors. We remain confident that the presented evolutionary overview and modelling approach will aid interpretation of forthcoming OTR crystal structures.


Asunto(s)
Evolución Molecular , Receptores de Oxitocina/metabolismo , Secuencia de Aminoácidos , Humanos , Ligandos , Datos de Secuencia Molecular , Unión Proteica , Receptores de Oxitocina/química
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