DEVELOPMENT OF A HIGH-THROUGHPUT FUNCTIONAL ASSAY FOR STRUCTURE-ACTIVITY STUDIES OF NEUROKININ-A ANALOGS

A great variety of functional assays are in use to evaluate novel liga nds of tachykinin receptors, These techniques, however, are generally labor and time consuming and hardly suitable for high throughput scree ning. Using human recombinant NK-2 receptors expressed in CHO cells, w e have developed an assay based on direct measurement of guanine nucle otide exchange on G-proteins, [S-35]GTP gamma S, which cannot be hydro lyzed by the intrinsic GTPase activity of the alpha-subunit of heterot rimeric G-protein, was employed. Six ligands of NK-2 receptors includi ng both natural neurokinins and neurokinin A analogs were used for the assay validation. The assay was shown to discriminate successfully be tween partial and full agonism, as well as between agonism and antagon ism of the peptides. Besides the exquisite specificity and high throug hput, the assay obviates the need for laboratory animals and cumbersom e muscle-bath techniques for conventional tachykinin pharmacology, The functional GTP gamma S assay is a specific and reliable high throughp ut screening tool for the investigation of pharmacologic properties of neurokinin A analogs. The assay has multiple advantages over existing techniques and is favored for studies on structure-activity relations hips in peptides.