Gary F. Musso

Design, Synthesis, and Analysis of Secondary Structure Based Vasoactive Intestinal Peptide Analogues

Vasoactive intestinal polypeptide (VIP) is a 28 amino acid peptide initially isolated and characterized from porcine gut tissue.’ VIP has strong sequence homology with several closely related peptides including glucagon, secretin, growth hormone releasing factor (GRF), porcine peptide histidine isoleucine (PHI), human PHI (PHM), and gastric inhibitory peptide (GIP)? It is proposed that the biologically active conformation of these peptides can be understood in terms of the “amphiphilic helical Using this approach, a series of VIP analogues have been designed, synthesized, and characterized for VIP-like properties. Five analogues, ranging from 28 to 68% homology with mammalian VIP, were designed to test this hypothesis. Model 1 was designed to be a helical peptide analogue of VIP in the region from 6-28 with minimal sequence identity to VIP. Models 2 and 3 were variants of Model 1 in which the pairs of basic residues were scrambled so as to separate them in the linear sequence, yet retain similar spatial presentation when in a helical conformation. Since the amphiphilic helical structure has two surfaces, one hydrophobic and one hydrophilic, Models 4 and 5 were designed to analyze the specific structural requirements of each domain. Model 4 was a hybrid utilizing the hydrophobic surface of Model 1 and the hydrophilic surface of VIP. In Model 5, the amino acids chosen for the respective domains were the inverse of those used in Model 4. The peptides were synthesized by solid-phase peptide synthesis and purified by preparative HPLC. Analytical HPLC and amino acid analysis confirmed the homogeneity, purity, and proper composition of the analogues. The ability of the analogues to behave as VIP agonists were assessed by their ability to interact specifically with VIP receptors on rat lung membranes (FIG. 1)’ and to stimulate amylase release in dispersed guinea pig acini (FIG. 2): Comparison of results obtained with Models 4 and 5 indicated that the integrity of the hydrophobic surface of the VIP sequence was essential for specific binding to VIP receptors with high affinity. From the data obtained for Model 1, it appeared that radically redesigned analogues could still effectively compete for specific binding to lung membrane receptors, but displayed much weaker potency in stimulating amylase secretion. Further experiments are being conducted to determine if Model 1 is a weak VIP agonist or a strong antagonist.