Identification of Key Residues for Interaction of Vasoactive Intestinal Peptide with Human VPAC1 and VPAC2Receptors and Development of a Highly Selective VPAC1Receptor Agonist

Abstract

The widespread neuropeptide vasoactive intestinal peptide (VIP) has two receptors VPAC 1 and VPAC 2 . Solid-phase syntheses of VIP analogs in which each amino acid has been changed to alanine (Ala scan) or glycine was achieved and each analog was tested for: (i) threedimensional structure by ab initio molecular modeling; (ii) ability to inhibit 125 ]VIP analog which constitutes the first highly selective (>1,000-fold) human VPAC 1 receptor agonist derived from VIP ever described. The vasoactive intestinal peptide (VIP)1 is a prominent neuropeptide with wide distribution in both peripheral and central nervous systems and a large spectrum of biological actions in mammals (1, 2). VIP-containing nerves and VIP effects have been described in digestive tract, cardiovascular system, airways, reproductive system, immune system, endocrine glands, and brain (1). Besides its short-term actions on exocrine secretions, hormone release, muscle relaxation, and metabolism (1, 2), VIP has been also characterized as a growth regulator for fetuses and tumor cells and during embryonic brain development (3). There are recent evidences for an important role of VIP in the perception of pain (4) and suppression of inflammation (5). Finally, VIP has been involved in diseases such as the watery diarrhea syndrome and clinical applications of VIP have been already suggested in impotence, asthma, lung injury, a variety of tumors and neurodegenerative diseases (1-3).VIP belongs to a large family of structurally related peptides (2, 6, 7) that comprises VIP, pituitary adenylate cyclase-activating peptide PACAP-27, and its C-terminal extended form PACAP-38, secretin, glucagon, and glucagon-like peptides-1 and -2, gastric inhibitory polypeptide, peptide histidine methionine amide, growth hormone-releasing factor (GRF), and peptides isolated from the venom of the Gila Monster. VIP and PACAP are the most closely related peptides in terms of structure and function (2, 6). They share two common receptors, VPAC 1 and VPAC 2 , which display high affinity for both VIP and PACAP (2,8). These receptors together with receptors for VIP-related peptides (see above) clearly constitute an original subfamily within the superfamily of G protein-coupled receptors (2, 9, 10). This subfamily referred to as class II (2) also comprises receptors for parathyroid hormone, calcitonin, corticotropin-releasing factor, and the so called EGF-TM7 receptors (11). Class II family of receptors for peptides display several common properties including large N-terminal extracellular domains containing highly conserved cystein residues, N-terminal leader sequences, and complex gene organization with many introns (2).Although the structure-function relationship of VIP receptors, including VPAC 1 and VPAC 2 , has been recently documented (2,9,(12)(13)(14)(15)(16)(17)(18)(19)(20), the structure-function relationship of VIP itself is still poorly understood. Some old studies carried out before the characterization and cloning of VIP receptor subtypes (21-23) indicated that: (i) th...

Generation of Highly Selective VPAC2 Receptor Agonists by High Throughput Mutagenesis of Vasoactive Intestinal Peptide and Pituitary Adenylate Cyclase-activating Peptide

“…Consistent with the results from VIP alaninescanning mutagenesis (13)(14)(15), our results from the chargescanning mutagenesis of PACAP27 indicate that the N-terminal region of the peptide is critical for binding to all three receptors. Since charge substitutions typically introduce more dramatic changes to the physical properties of peptides than alanine substitutions, most of the charge mutations within the midregion of PACAP27, which affected binding, can be explained on this basis.…”

Stapled Vasoactive Intestinal Peptide (VIP) Derivatives Improve VPAC2 Agonism and Glucose-Dependent Insulin Secretion

“…The mutations involved are N 24 X and N 28 X, implying that the polarity of N 24 and N 28 are useful in the random coiled region of the peptide at its native C-terminus. 33 This result also indicates that constraining the peptide in this region disturbs its ability to agonize VPAC 2 . Overall, hydrocarbon-stapled peptide 11 afforded the most potent VPAC 2 receptor agonist evaluated in this study (EC 50 , 0.049 (±0.02) nM), a significant improvement over both I 17 -VIP-GK (∼3-fold) and wild-type VIP (∼4-fold) ( Table 1).…”

Diffuse Pharmacophoric Domains of Vasoactive Intestinal Peptide (VIP) and Further Insights into the Interaction of VIP with the N-terminal Ectodomain of Human VPAC1 Receptor by Photoaffinity Labeling with [Bpa6]-VIP

“…It is well known that VIP has diffuse pharmacophoric domains, with the amino acid residues important for biological activity being distributed along the whole 28-amino acid peptide chain (9). In this context, we further explored the contact sites between VIP and the hVPAC1 receptor by incorporating a photoactivable benzophenone group on the side chain at position 6 of VIP.…”

“…(ii) The substitution of Bpa for phenylalanine keeps an aromatic residue, and we expected that, even though Phe 6 is important for biological activity, the [Bpa 6 ]-VIP probe should keep reasonable affinity for the receptor. (iii) Position 6 and the previously explored position 22 (8) are at the two ends of the central ␣-helical domain of VIP (9,10 position 6 of VIP is in the environment of the short sequence 104 -108 within the N-terminal ectodomain of the receptor when the photoaffinity probe is bound to the receptor.…”

“…This was done by substituting para-benzoyl-L-Phe (Bpa) for phenylalanine 6. This site of incorporation was selected for several reasons: (i) Phe 6 is important for the biological activity of VIP (9) and is strictly conserved in all natural hormones structurally related to VIP (1). (ii) The substitution of Bpa for phenylalanine keeps an aromatic residue, and we expected that, even though Phe 6 is important for biological activity, the [Bpa 6 ]-VIP probe should keep reasonable affinity for the receptor.…”