title: Generation of highly selective VPAC2 receptor agonists by high throughput mutagenesis of vasoactive intestinal peptide and pituitary adenylate cyclase-activating peptide.
abstract: Pituitary adenylate cyclase-activating peptide (PACAP) has a specific receptor PAC1 and shares two receptors VPAC1 and VPAC2 with vasoactive intestinal peptide (VIP). VPAC2 activation enhances glucose-induced insulin release while VPAC1 activation elevates glucose output. To generate a large pool of VPAC2 selective agonists for the treatment of type 2 diabetes, structure-activity relationship studies were performed on PACAP, VIP, and a VPAC2 selective VIP analog. Chemical modifications on this analog that prevent recombinant expression were sequentially removed to show that a recombinant peptide would retain VPAC2 selectivity. An efficient recombinant expression system was then developed to produce and screen hundreds of mutant peptides. The 11 mutations found on the VIP analog were systematically replaced with VIP or PACAP sequences. Three of these mutations, V19A, L27K, and N28K, were sufficient to provide most of the VPAC2 selectivity. C-terminal extension with the KRY sequence from PACAP38 led to potent VPAC2 agonists with improved selectivity (100-1000-fold). Saturation mutagenesis at positions 19, 27, 29, and 30 of VIP and charge-scanning mutagenesis of PACAP27 generated additional VPAC2 selective agonists. We have generated the first set of recombinant VPAC2 selective agonists described, which exhibit activity profiles that suggest therapeutic utility in the treatment of diabetes.
authors.affiliation: Department of Molecular Technologies and Analytics and Formulation, Biotechnology, Bayer Corp., Berkeley, California 94701, USA.
authors.forename: Stephanie L
authors.initials: SL
authors.lastname: Yung
authors.forename: Fernando
authors.initials: F
authors.lastname: Dela Cruz
authors.forename: Sarah
authors.initials: S
authors.lastname: Hamren
authors.forename: Jian
authors.initials: J
authors.lastname: Zhu
authors.forename: Manami
authors.initials: M
authors.lastname: Tsutsumi
authors.forename: James W
authors.initials: JW
authors.lastname: Bloom
authors.forename: Margaret
authors.initials: M
authors.lastname: Caudle
authors.forename: Steve
authors.initials: S
authors.lastname: Roczniak
authors.forename: Tracey
authors.initials: T
authors.lastname: Todd
authors.forename: Lynn
authors.initials: L
authors.lastname: Lemoine
authors.forename: Margit
authors.initials: M
authors.lastname: MacDougall
authors.forename: Armen B
authors.initials: AB
authors.lastname: Shanafelt
authors.forename: Clark Q
authors.initials: CQ
authors.lastname: Pan
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009479:Neuropeptides / Q000737:chemistry / Q000494:pharmacology*; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide; D018005:Receptors, Vasoactive Intestinal Peptide / Q000819:agonists*; D051239:Receptors, Vasoactive Intestinal Peptide, Type II; D011994:Recombinant Proteins / Q000494:pharmacology; D013329:Structure-Activity Relationship; D014660:Vasoactive Intestinal Peptide / Q000737:chemistry / Q000494:pharmacology*
chemical_list: D009479:Neuropeptides; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide; D018005:Receptors, Vasoactive Intestinal Peptide; D051239:Receptors, Vasoactive Intestinal Peptide, Type II; D011994:Recombinant Proteins; D014660:Vasoactive Intestinal Peptide