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

Abstract

Pituitaryadenylate 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 chargescanning 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.Pituitary adenylate cyclase-activating polypeptide (PACAP), 1 originally isolated from ovine hypothalamus (1) by following pituitary adenylate cyclase activation, belongs to the secretin/glucagon/vasoactive intestinal peptide (VIP) family of peptides (2). These peptides are expressed as part of larger proteins that are processed by proteolysis followed by Cterminal amidation to generate the mature amidated peptides (Fig. 1). PACAP exists as a 38-residue form (PACAP38), and as a shorter form corresponding to the N-terminal 27 amino acids of PACAP38 (PACAP27). Both forms of PACAP bind to and activate the G-protein-coupled receptors PAC1, VPAC1, and VPAC2, whereas the related 28-mer peptide VIP only recognizes VPAC1 and VPAC2 (3). Activation of multiple receptors by PACAP or VIP has broad physiological effects on nervous, endocrine, cardiovascular, reproductive, muscular, and immune systems (4). Thus, clinical applications will require selective activation of a particular receptor to minimize potential side effects mediated by the other receptors. For example, we have previously demonstrated that VPAC2 activation induces glucose-dependent insulin secretion without the undesired side effects mediated by VPAC1 such as watery diarrhea (5). Therefore, to provide a potential therapy for type 2 diabetes, VPAC2 selectivity is an absolute requirement.We sought to identify key determinants of VPAC2 selectivity and generate a peptide with the minimum number of mutations. However, structure-function relationship studies on VIP and PACAP (6 -15) have been restricted by the number of peptides that can be tested due to limitations of peptide synth...

Engineering Novel VPAC2-Selective Agonists with Improved Stability and Glucose-Lowering Activity in Vivo

“…To prolong the in vivo duration of action of BAY 55-9837, site-specific PEGylation was attempted by introducing a unique cysteine. Structural activity relationship studies of VPAC2-selective activation suggests the C terminus of VPAC2 can be modified without significantly affecting activity (Yung et al, 2003). Thus, a single cysteine was added to the C terminus of BAY 55-9837 to create BAY(C32) ( Table 1).…”

“…We have demonstrated recently that activation of VPAC2 also induces insulin secretion from ␤ cells in a glucose-dependent fashion (Tsutsumi et al, 2002). We engineered BAY 55-9837, a highly selective and potent VPAC2 peptide agonist, that was efficacious in murine models yet did not cause gastrointestinal side effects associated with VPAC1 (Yung et al, 2003). Two mutations, M17V and N24Q, were included in BAY 55-9837 in an effort to minimize potential peptide instability due to either oxidation or deamidation, respectively.…”

“…Therefore, we postulated that a VPAC2-selective agonist would enhance pancreatic ␤ cell insulin release without causing increased glucose production by the liver and would thereby lead to increased glucose disposal. To test this hypothesis, a highly VPAC2-selective agonist BAY 55-9837 was engineered through several rounds of site-directed mutagenesis (Yung et al, 2003). BAY 55-9837 stimulated glucose-dependent insulin secretion in isolated human pancreatic islets and caused a dose-dependent increase in plasma insulin levels in fasted rats.…”

A novel selective VPAC2 agonist peptide-conjugated chitosan modified selenium nanoparticles with enhanced anti-type 2 diabetes synergy effects

“…[12][13][14] BAY55-9837 is a reported specific VPAC2 agonist constructed through site-directed mutagenesis, and it can increase plasma insulin levels in a dose-dependent manner, but not leading to any hypoglycemia in rats. 15,16 Nevertheless, its therapeutic application has been hampered by its short half-life (~5 min) and limited bioavailability in vivo. 17 Mainly, dipeptidyl peptidase IV-mediated enzymolysis leads to the H-S lack of BAY55-9837 at the N-terminus, which may cause it cannot activate VPAC2.…”

Chitosan-decorated selenium nanoparticles as protein carriers to improve the in vivo half-life of the peptide therapeutic BAY 55-9837 for type 2 diabetes mellitus

“…5 PACAP is a member of the VIP-glucagon-growth hormone-releasing factor secretin superfamily. 6 PACAP exists in two molecular forms: PAC-AP-27 and PACAP-38.…”