Development of helix-based vasoactive intestinal peptide analogues: identification of residues required for receptor interaction

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Abstract
Several VIP analogues have been designed on the basis of the hypothesis that the region from residue 6 to residue 28 forms a .pi.-helical structure when bound to membrane receptors. An empirical approach for the design and construction of analogues based upon distribution frequency and structural homology with several sequence-related peptides is presented. Five peptides were designed, synthesized, and analyzed. One analogue, model 5, containing the native hydrophobic and an altered hydrophilic surface, was an effective VIP agonist in both binding to rat lung membrane receptors (KD1= 11 .+-. 8 pM, KD2 = 6.4 .+-. 0.2 nM; VIP KD1 = 21 .+-. 13 pM, KD2 = 1.8 .+-. 0.6 nM) and stimulation of amylase release from guinea pig pancreatic acini (ED50 = 90 pM; VIP ED50 = 27 pM). The four other analogues were considerably less potent than VIP, yet retained full intrinsic activity. Our results showed that the hydrophobic surface of this helical domain (residues 6-28) contains amino acids important for interaction with receptors, whereas amino acid residues on the hydrophilic surface do not seem to participate strongly in receptor binding or signal transduction. Furthermore, on the basis of high-affinity binding, the stimulation of amylase release in pancreatic acini appears to be coupled to the higher affinity receptors. These results suggest that an approach based on the construction of putative .pi.-helical structures can be applied to the design of biologically active analogues of VIP. Thus, we have identified several residues within the VIP sequence that are critical for receptor binding using this approach.