Development of a High Throughput Functional Assay for Structure-Activity Studies of Neurokinin A Analogs

Abstract

A great variety of functional assays are in use to evaluate novel ligands of tachykinin receptors. These techniques, however, are generally labor and time consuming and hardly suitable for high throughput screening. Using human recombinant NK-2 receptors expressed in CHO cells, we have developed an assay based on direct measurement of guanine nucleotide exchange on G-proteins. [35S]GTPγS, which cannot be hydrolyzed by the intrinsic GTPase activity of the α-subunit of heterotrimeric G-protein, was employed. Six ligands of NK-2 receptors including both natural neurokinins and neurokinin A analogs were used for the assay validation. The assay was shown to discriminate successfully between partial and full agonism, as well as between agonism and antagonism of the peptides. Besides the exquisite specificity and high throughput, the assay obviates the need for laboratory animals and cumbersome muscle-bath techniques for conventional tachykinin pharmacology. The functional GTPγS assay is a specific and reliable high throughput 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 relationships in peptides.