Asunto(s)
Descubrimiento de Drogas , Neoplasias/tratamiento farmacológico , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/agonistas , Secuencia de Aminoácidos , Cromatografía Líquida de Alta Presión , Humanos , Espectrometría de Masas , Imitación Molecular , Datos de Secuencia MolecularRESUMEN
The pituitary glycoprotein hormones, luteinizing hormone and follicle-stimulating hormone (FSH), act through their cognate receptors to initiate a series of coordinated physiological events that results in germ cell maturation. Given the importance of FSH in regulating folliculogenesis and fertility, the development of FSH mimetics has been sought to treat infertility. Currently, purified and recombinant human FSH are the only FSH receptor (FSH-R) agonists available for infertility treatment. By screening unbiased combinatorial chemistry libraries, using a cAMP-responsive luciferase reporter assay, we discovered thiazolidinone agonists (EC50's = 20 microm) of the human FSH-R. Subsequent analog library screening and parallel synthesis optimization resulted in the identification of a potent agonist (EC50 = 2 nm) with full efficacy compared with FSH that was FSH-R-selective and -dependent. The compound mediated progesterone production in Y1 cells transfected with the human FSH-R (EC50 = 980 nm) and estradiol production from primary rat ovarian granulosa cells (EC50 = 10.5 nm). This and related compounds did not compete with FSH for binding to the FSH-R. Use of human FSH/thyroid-stimulating hormone (TSH) receptor chimeras suggested a novel mechanism for receptor activation through a binding site independent of the natural hormone binding site. This study is the first report of a high affinity small molecule agonist that activates a glycoprotein hormone receptor through an allosteric mechanism. The small molecule FSH receptor agonists described here could lead to an oral alternative to the current parenteral FSH treatments used clinically to induce ovarian stimulation for both in vivo and in vitro fertilization therapy.
Asunto(s)
Receptores de HFE/agonistas , Tiazolidinedionas/química , Tiazolidinedionas/farmacología , Regulación Alostérica/efectos de los fármacos , Animales , Línea Celular , Técnicas Químicas Combinatorias , Cricetinae , Estradiol/metabolismo , Femenino , Hormona Folículo Estimulante/farmacología , Células de la Granulosa/efectos de los fármacos , Células de la Granulosa/metabolismo , Humanos , Ratones , Estructura Molecular , Progesterona/metabolismo , Ratas , Receptores de HFE/metabolismo , Relación Estructura-ActividadRESUMEN
We have developed a class of binding proteins, called avimers, to overcome the limitations of antibodies and other immunoglobulin-based therapeutic proteins. Avimers are evolved from a large family of human extracellular receptor domains by in vitro exon shuffling and phage display, generating multidomain proteins with binding and inhibitory properties. Linking multiple independent binding domains creates avidity and results in improved affinity and specificity compared with conventional single-epitope binding proteins. Other potential advantages over immunoglobulin domains include simple and efficient production of multitarget-specific molecules in Escherichia coli, improved thermostability and resistance to proteases. Avimers with sub-nM affinities were obtained against five targets. An avimer that inhibits interleukin 6 with 0.8 pM IC50 in cell-based assays is biologically active in two animal models.