PROBING THE FUNCTIONAL CONFORMATION OF NEUROPEPTIDE-Y THROUGH THE DESIGN AND STUDY OF CYCLIC ANALOGS

The functional importance of the PP-fold conformation in neuropeptide Y (NPY) was investigated. NPY and N(alpha)-Ac-NPY(10-36), and corresponding cyclic analogues cyclo18,22-[Lys18,Asp22]-NPY and N(alpha)-Ac-cyclo18,22-[Lys18,Asp22]-NPY(10-36), were synthesized. Strategies for synthesis of the cyclic analogues included the use of the Kaiser oxime resin and a segment condensation approach. Circular dichroism studies in phosphate buffer, pH 5.0, indicated self-association of all four peptides at low micromolar concentrations. Monomeric N(alpha)-Ac-NPY(10-36) showed only 13% alpha-helix, compared to 32% alpha-helix for monomeric NPY, demonstrating a helix-stabilizing effect of residues 1-9 that is consistent with the PP fold. The [Lys18,Lys22] lactam bridge stabilized the helical conformation in N(alpha)-Ac-NPY(10-36) (51% alpha-helix), but was helix destabilizing in NPY (21% alpha-helix). In rat brain receptor binding assays, the cyclic and linear N(alpha)-Ac-NPY(10-36) analogues were equipotent (IC50 = 13 nM for I-125-BH-NPY displacement), although the cyclic analogue was twice as potent in rat vas deferens assays. NPY was more potent than its cyclic analogue in the brain receptor binding assays (IC50 = 0.07 and 0.25 nM, respectively), but these peptides were equipotent in the vas deferens assays. These results support a functional role for the PP fold in NPY and correlate with the solution conformations of the monomeric peptides.