Affinity, Potency, Efficacy, And Selectivity Of Neurokinin A Analogs At Human Recombinant Nk2 And Nk1 Receptors

A series of peptide NK2 receptor agonists was evaluated for affinity, potency, efficacy, and selectivity at human recombinant NK2 and NK1 receptors expressed in CHO cells to identify compounds with the greatest separation between NK2 and NK1 receptor agonist activity. Binding studies were performed using displacement of [I-12(5)]-NKA binding to NK2 receptors and displacement of [H-3]-Septide binding to NK1 receptors expressed in CHO cells. Functional studies examining the increase in intracellular calcium levels and cyclic AMP stimulation were performed using the same cell lines. A correlation was demonstrated between binding affinities (Ki) and potency to increase intracellular calcium (EC50) for NK2 and NK1 receptors. Ranking compounds by their relative affinity (Ki) or potency (EC50) at NK2 or NK1 receptors indicated that the most selective NK2 agonists tested were [Lys(5),MeLeu(9),Nle(10)]-NKA(4-10) (NK1/NK2 Ki ratio = 674; NK1/NK2 EC50 ratio = 105) and [Arg(5),MeLeu(9),Nle(10)]-NKA(4-10) (NK1/NK2 Ki ratio = 561; NK1/NK2 EC50 ratio = 70). The endogenous peptide, NKA, lacked selectivity with an NK1/NK2 Ki ratio = 20 and NK1/NK2 EC50 ratio = 1. Of the compounds selected for evaluation in cyclic AMP stimulation assays, [beta-Ala(8)]-NKA(4-10) had the greatest selectivity for activation of NK2 over NK1 receptors (NK1/NK2 EC50 ratio = 244), followed by [Lys(5),MeLeu(9),Nle(10)]-NKA(4-10) (ratio = 74), and NKA exhibited marginal selectivity (ratio = 2.8).