Binding and functional potency of neurokinin A analogues in the rat fundus: A structure-activity study.

Structure-activity relationships of neurokinin A (NKA) and the two analogues NKA(4-10) and [Nle10]NKA(4-10) were investigated at the rat fundus NK-2 receptor, using selected amino acid substitutions. Both radioligand binding with [125I][Lys5,Tyr(I2)7,MeLeu9, Nle10] NKA(4-10) and functional studies were performed and correlated. In membrane binding experiments loss of His1 and Lys2, or replacement of Lys2 with Ala did not substantially alter binding affinity of NKA. NKA(4-10) free acid was unable to compete with the radioligand. [Nle10]NKA(4-10) binding affinity to rat fundus membrane preparations was decreased when substituting Asp4 with Gln or Asn, or Val7 with either Tyr or Ile. Replacement of Ser5 with the negatively charged Glu also decreased the binding affinity, but substitution with the positively charged Lys substantially increased the affinity of [Nle10] NKA(4-10) for the NK-2 receptor. Lengthening NKA(4-10) or [Nle10]NKA(4-10) with Ala11 or Nle11, respectively, decreased the binding affinity of the peptide. In both binding and functional studies, replacement of any of the residues of NKA(4-10), except for Ser5, with alanine decreased the affinity of the peptide for the NK-2 receptor. Ala substitutions at positions 4, 6, and very obviously at 8, 9 and 10 of NKA(4-10) yielded peptides unable to achieve a maximum contractile response, although they did not demonstrate antagonist activity. These data confirm the importance of the NKA carboxyl terminus, and the requirement for Phe6, Val7, Gly8, Leu9 and Met10 integrity for interaction with the NK-2 receptor. They also suggest that Ser5 is a good site to target modifications leading to the design of new potential drugs.