Binding and Functional Potency of Neurokinin A Analogues in the Rat Fundus: A Structure-Activity Study

Abstract

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.