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 analogue s NKA(4-10) and [Nle(10)]NKA(4-10) were investigated at the rat fundus NK-2 receptor, using selected amino acid substitutions. Both radioligand bindin g with [I-125][Lys(5),Tyr(I-2)(7),MeLeu(9), Nle(10)] NKA(4-10) and function al studies were performed and correlated. In membrane binding experiments l oss of His(1) and Lys(2), or replacement of Lys2 with Ala did not substanti ally alter binding affinity of NKA. NKA(4-10) free acid was unable to compe te with the radioligand. [Nle(10)]NKA(4-10) binding affinity to rat fundus membrane preparations was decreased when substituting Asp(4) with Gin or As n, or Val(7) with either Tyr or lie. Replacement of Ser(5) with the negativ ely charged Glu also decreased the binding affinity, but substitution with the positively charged Lys substantially increased the affinity of [Nle(10) ] NKA(4-10) for the NK-2 receptor. Lengthening NKA(4-10) or [Nel(10)]NKA(4- 10) with Ala(11) or Nle(11), respectively, decreased the binding affinity o f the peptide. In both binding and functional studies, replacement of any o f the residues of NKA(4-10), except for Ser(5), with alanine decreased the affinity of the peptide for the NK-2 receptor. Ala substitutions at positio ns 4, 6, and very obviously at 8, 9 and 10 of NKA(4-10) yielded peptides un able to achieve a maximum contractile response, although they did not demon strate antagonist activity. These data confirm the importance of the NKA ca rboxyl terminus, and the requirement for Phe(6), Val(7), Gly(8), Leu(9) and Met(10) integrity for interaction with the NK-2 receptor. They also sugges t that Sers is a good site to target modifications leading to the design of new potential drugs.