Structure–activity studies at the rat tachykinin NK2 receptor: effect of substitution at position 5 of neurokinin A

A series of analogues of neurokinin A(4–10) was synthesized using solid phase techniques with Chiron pins, and purified by HPLC. The potencies of 10 peptides with substitution at Ser⁵ were assessed at rat fundus NK2 receptors. In membrane binding studies with ¹²⁵I]-Lys⁵,Tyr(I₂)⁷,MeLeu⁹,Nle¹⁰]-NKA(4–10), all compounds except Asp⁵]NKA(4–10) showed reasonable affinity, and analogues with Lys and Arg substitutions were five-fold more potent than NKA(4–10). In functional studies, all peptides were able to contract the rat isolated fundus strips. Analogues with Phe, His and Asn substitutions were substantially weaker in functional than in binding studies, whereas there was an excellent correlation (r = 0.95) between binding and functional potency for the remaining seven peptides. Phe⁵]NKA(4–10) is in fact neurokinin B(4–10) and this residue may be critical in determining selectivity between NK2 and NK3 receptors. Analogues with a basic residue (Lys, Arg) at position 5 showed both increased affinity and functional potency, whereas the neutral Asn⁵]NKA(4–10) was equally as weak in contractile studies as the acidic Asp⁵]NKA(4–10). However, Glu⁵]NKA(4–10) and Gln⁵]NKA(4–10) were no different from NKA(4–10). Our results could indicate the presence of a negative charge on the NK2 receptor, close to position 5 of NKA. This would facilitate interaction with positively charged side chains and impede interaction with negatively charged side chains, particularly the inflexible side chain of aspartic acid. Thus, not only the charge, but also the length of the side chain of the residue at position 5, seems to be important for interaction with the rat NK2 receptor.