A structure–function study of PACAP using conformationally restricted analogs: Identification of PAC1 receptor-selective PACAP agonists

• Published in: Peptides (New York, N.Y. : 1980) Vol. 66; pp. 26 - 42
• Main Authors: Ramos-Álvarez, Irene, Mantey, Samuel A., Nakamura, Taichi, Nuche-Berenguer, Bernardo, Moreno, Paola, Moody, Terry W., Maderdrut, Jerome L., Coy, David H., Jensen, Robert T.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.04.2015
• Subjects: Animals; Brain Injuries - metabolism; Cell Line, Tumor; Humans; Mice; Neuroprotection; NIH 3T3 Cells; PACAP; Pituitary Adenylate Cyclase-Activating Polypeptide - agonists; Pituitary Adenylate Cyclase-Activating Polypeptide - chemistry; Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide, Type I - metabolism; Receptors, Vasoactive Intestinal Peptide, Type II - metabolism; Receptors, Vasoactive Intestinal Polypeptide, Type I - metabolism; Stroke; Stroke - metabolism; Structure–function study; Traumatic brain injury; Vasoactive intestinal peptide; Vasoactive Intestinal Peptide - metabolism; Neuroprotection; Traumatic brain injury; GRP; DTT; PLC; CNS; PACAP27; cAMP; BSA; DMEM; 3T3; VPAC1-R; VPAC2-R; FBS; IC50; Ro 25-1553; EC50; Structure–function study; PBS; Stroke; PACAP; IBMX; IP; PACAP38; PAC1-R; GHRH; Vasoactive intestinal peptide; T47D cells; VIP; Sup T1 cells
• ISSN: 0196-9781; 1873-5169
• DOI: 10.1016/j.peptides.2015.01.009

•46 PACAP analogs were synthesized with conformationally restricted substitutions.•Their pharmacology (affinity and potency) was determined for VPAC1R, VPAC2R and PAC1-R.•15 peptides had 6–78-fold higher affinity [up to 103-fold potency] for PAC1-R.•A number of analogs also had marked selectivity for PAC1-R over VPAC2.•This study describes for first time, peptide agonists selective for PAC1-R. Pituitary adenylate cyclase-activating polypeptide (PACAP) has widespread physiological/pathophysiological actions and there is increased interest for its use therapeutically, especially in the CNS (neuroprotection). Unfortunately, no selective PACAP-analogs exist for PACAP-preferring PAC1-receptors, primarily because of its high sequence identity to VIP and particularly, because of the inability of structure–function studies to separate the pharmacophore of PAC1-R from VPAC1-R, which has high affinity for PACAP and VIP. The present study attempted to develop PAC1-R-selective agonists primarily by making conformationally restricted PACAP-analogs in positions important for receptor-selectivity/affinity. Forty-six PACAP-related-analogs were synthesized with substitutions in positions 1–4, 14–17, 20–22, 28, 34, 38 and receptor-selectivity determined in PAC1-R,VPAC1-R,VPAC2-R-transfected or native cells from binding or cAMP-generation experiments. Fifteen PACAP-analogs had 6–78-fold higher affinities for PAC1-R than VPAC1-R and 13 were agonists. Although binding-affinities correlated significantly with agonist potency, the degree of receptor-spareness varied markedly for the different PACAP-analogs, resulting in selective potencies for activating the PAC1 receptor over the VPAC1 receptor from 0- to 103-fold. In addition, a number of PACAP-analogs were identified that had high selectivity for PAC1-R over VPAC2-R as well as PACAP-analogs that could prove more useful therapeutically because of substitutions known to extend their half-lives (substitutions at potential sites of proteolysis and attachment of long-chain fatty acids). This study provides for the first time a separation of the pharmacophores for PAC1-R and VPAC1-R, resulting in PACAP-related analogs that are PAC1-R-preferring. Some of these analogs, or their modifications, could prove useful as therapeutic agents for various diseases.