Structure-activity relationship of neurokinin A(4-10) at the human tachykinin NK2 receptor:: the effect of amino acid substitutions on receptor affinity and function

A structure-activity study of the neurokinin A (NKA) fragment NKA(4-10) was performed to investigate the importance of amino acid residues for receptor efficacy, potency and affinity at the NK2 receptor in human colon circular muscle. Fourteen analogs of NKA(4-10) were produced with substitutions at positions 4, 5, 7, 9 and/or 10 of NKA. Their potencies were determined by in vitro contractile responses and affinities by radioligand binding using [I-125]NKA. Functional potency was enhanced 8-fold by single amino acid substitutions with Lys(5) and MeLeu(9) but not significantly altered by substitutions Glu(4), Arg(5), His(5) and Nle(10). The multiply-substituted analogs [MeLeu9, Nle(10)]NKA(4-10), [Lys(5),MeLeu(9),Nle(10)]NKA(4-10) and [Lys(5),(Tyr(7)),MeLeu(9),Nle(10)]NKA(4-10) displayed 6-9-fold increase in potency. Although [Arg(5),Nle(10)]NKA(4-1 0) was similar in potency to NKA(4-1 0), it was the only analog to show significantly reduced efficacy. All analogs were able to compete fully for [I-125]NKA binding. [Lys(5),MeLeu9JNKA (4-10), [MeLeu(9),Nle(10)]NKA(4-1 0), [Lys(5),Nle(10)]NKA(4-10) and analogs containing single substitutions with Glu(4),Arg(5),Lys(5) and MeLeu(9) displayed significantly higher affinity, whereas those with Nle(10) and [Glu(4),Nle(10)] substitutions showed significantly lower affinity than NKA(4-10). There was a positive correlation (r = 0.63) between binding affinity and functional potency, which was markedly improved (r = 0.95) by removal of three analogs: [Lys(5),MeLeu(9),Nle(10)]NKA(4-10), Lys(5),Tyr(7),MeLeu(9),Nle(10)]NKA(4-10) and [Lys(5),Tyr(I-2)(7),MeLeu(9),Nle(10)]NKA(4-10). These exhibited similar binding affinities to that of NKA(4-10) but were more potent in functional studies, possibly indicating a different mechanism of receptor interaction. In conclusion, substitution of Ser(5) with Lys, and/or N-methylation of Leu(9), were the most effective changes to increase functional and binding potency of NKA(4-10) at the human colon NK2 receptor. (C) 2002 Elsevier Science Inc. All rights reserved.