Development of a high throughput functional assay for structure-activity studies of neurokinin a analogs

Dmitry S. Gembitsky, Sándor Lovas, Richard F. Murphy

Research output: Contribution to journalArticle

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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.

Original languageEnglish
Pages (from-to)183-188
Number of pages6
JournalJournal of Biomolecular Screening
Volume3
Issue number3
StatePublished - 1998

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Neurokinin-2 Receptors
Neurokinin A
Assays
Throughput
Tachykinin Receptors
Ligands
Heterotrimeric GTP-Binding Proteins
Tachykinins
Peptides
Guanine Nucleotides
CHO Cells
GTP Phosphohydrolases
Laboratory Animals
Structure-Activity Relationship
Baths
GTP-Binding Proteins
Pharmacology
Muscles
Screening
Muscle

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Clinical Biochemistry
  • Analytical Chemistry

Cite this

Development of a high throughput functional assay for structure-activity studies of neurokinin a analogs. / Gembitsky, Dmitry S.; Lovas, Sándor; Murphy, Richard F.

In: Journal of Biomolecular Screening, Vol. 3, No. 3, 1998, p. 183-188.

Research output: Contribution to journalArticle

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