RESUMEN
Epstein-Barr virus-induced gene 2 (EBI2, also known as GPR183) is a G-protein-coupled receptor that is required for humoral immune responses; polymorphisms in the receptor have been associated with inflammatory autoimmune diseases. The natural ligand for EBI2 has been unknown. Here we describe the identification of 7α,25-dihydroxycholesterol (also called 7α,25-OHC or 5-cholesten-3ß,7α,25-triol) as a potent and selective agonist of EBI2. Functional activation of human EBI2 by 7α,25-OHC and closely related oxysterols was verified by monitoring second messenger readouts and saturable, high-affinity radioligand binding. Furthermore, we find that 7α,25-OHC and closely related oxysterols act as chemoattractants for immune cells expressing EBI2 by directing cell migration in vitro and in vivo. A critical enzyme required for the generation of 7α,25-OHC is cholesterol 25-hydroxylase (CH25H). Similar to EBI2 receptor knockout mice, mice deficient in CH25H fail to position activated B cells within the spleen to the outer follicle and mount a reduced plasma cell response after an immune challenge. This demonstrates that CH25H generates EBI2 biological activity in vivo and indicates that the EBI2-oxysterol signalling pathway has an important role in the adaptive immune response.
Asunto(s)
Hidroxicolesteroles/farmacología , Receptores de Superficie Celular/inmunología , Animales , Formación de Anticuerpos/inmunología , Linfocitos B , Línea Celular , Movimiento Celular/efectos de los fármacos , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/inmunología , Humanos , Hidroxicolesteroles/química , Hígado/química , Ratones , Ratones Noqueados , Receptores Acoplados a Proteínas G , Ovinos , Linfocitos T/inmunologíaRESUMEN
Several small molecule antagonists for Smoothened (Smo) have been developed, and achieved promising preclinical efficacy in cancers that are dependent on Hedgehog (Hh) signaling. However, in a recent clinical study, a drug-resistant D473H SMO mutant was identified that is thought to be responsible for cancer relapse in a patient with medulloblastoma. Here, we report two Smo antagonists that bind to distinct sites, as compared to known antagonists and agonists, and inhibit both wild-type and mutant Smo. These findings provide an insight of the ligand-binding sites of Smo and a basis for the development of potential therapeutics for tumors with drug-resistant Smo mutations.
Asunto(s)
Resistencia a Antineoplásicos/genética , Proteínas Mutantes/antagonistas & inhibidores , Mutación , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Línea Celular , Evaluación Preclínica de Medicamentos , Proteínas Hedgehog/antagonistas & inhibidores , Humanos , Ratones , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptor SmoothenedRESUMEN
Here we report the design and evaluation of a bifunctional, small molecule switch that induces a targeted immune response against tumors in vivo. A high affinity ligand for prostate specific membrane antigen (PSMA) was conjugated to a hapten that binds dinitrophenyl (DNP)-specific antibodies. When introduced into hu-PBL-NOD/SCID mice previously immunized with a KLH-DNP immunogen, this conjugate induced a targeted antibody-dependent cellular cytotoxicity (ADCC) response to PSMA-expressing tumor cells in a mouse xenograft model. The ability to create a small molecule inducible antibody response against self-antigens using endogenous non-autoreactive antibodies may provide advantages over the autologous immune response generated by conventional vaccines in certain therapeutic settings.