SYNTHESIS AND CHARACTERIZATION OF SELECTIVE FLUORESCENT LIGANDS FOR THE NEUROKININ NK2 RECEPTOR

Several fluorescent probes for the NK2 receptor were designed, synthes ized, and pharmacologically characterized. These fluorescent ligands a re analogues of the selective NK2 heptapeptide antagonist -alpha-benzo yl-Ala-Ala-D-Trp-Phe-D-Pro-Pro-Nle-NH2 (1, GR94800). They were obtaine d by substitution of 2,n-diaminoalkyl amino acid (n = 3-6) for Ala(1) and the subsequent coupling of the fluorophore NBD (7-ntrobenz-2-oxa-1 ,3-diazol-4-yl) or fluoresceinthiocarbamyl to the N-omega amino group. The fluorescent derivatives retained high binding affinities for the NK2 receptor in transfected CHO cells. In contrast, fluorescent deriva tives made by replacing the N-alpha-benzoyl group of 1 by NBD or fluor escein were considerably less active. The effect on ligand potency of varying the length of the spacer arm between the peptide moiety and th e fluorescent group was also studied. The most potent fluorescent anta gonists were oyl-Dab(gamma-NBD)-Ala-D-Trp-Phe-D-Pro-Pro-Nle-NH2 (5B), pK(i) = 8.87 for NK2; oyl-Orn(delta-NBD)-Ala-D-Trp-Phe-D-Pro-Pro-Nle-N H2 (4B), pK(i) = 8.84; and l-Lys(epsilon-NBD)-Ala-D-Trp-Phe-D-Pro-Pro- Nle-NH2 (3B), pK(i) = 8.83. These three compounds were highly selectiv e for NK2 over NK3 and NK1 receptors. We show that these fluorescent l igands are useful tools for the detection of NK2 receptor expression b y flow cytometry. Additionally, these fluorescent probes should prove valuable for fluorescence microscopy and study of ligand-receptor inte raction by spectrofluorimetry.