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ENDOCRINE AND DIABETES
Bayer Healthcare, Biotechnology, Berkeley, California (C.Q.P., I.T., W.W., M.D., W.F., S.L.Y., S.R., Y.J.W.); and Bayer Healthcare, Pharmaceuticals, Department of Metabolic Disease Research, West Haven, Connecticut (F.L., Y.L., T.H.C., J.P.W.)
Received August 9, 2006; accepted November 15, 2006.
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Abstract |
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). PACAP exists as either a 38-amino acid (PACAP38) or 27-amino acid (PACAP27) peptide. PACAP27 has 68% structural homology to the 28-amino acid VIP peptide. Both forms of PACAP bind to and activate the G-protein-coupled receptors, PAC1, VPAC1, and VPAC2. On the other hand, VIP only binds to and activates VPAC1 and VPAC2 (Laburthe and Couvineau, 2002). These receptors have wide tissue distribution, and both PACAP and VIP play important roles in a number of physiological processes, including effects on the nervous, endocrine, cardiovascular, reproductive, and immune systems (for review, see Vaudry et al., 2000).
PAC1 and VPAC2 are expressed in pancreatic islets, and both PACAP and VIP have been shown to increase insulin secretion from the pancreas (Filipsson et al., 1998b). However, the role of PACAP in control of glucose homeostasis is complex because it also plays a role in increasing glucagon and catecholamine secretion, which acts to increase glucose output from the liver (Yokota et al., 1995; Filipsson et al., 1997; Hamelink et al., 2002).
The expression pattern of VPAC1, coupled with the glycogenolytic activity of PACAP, suggests that activation of this receptor contributes to an increase in hepatic glucose production (Sekiguchi et al., 1994; Yokota et al., 1995; Wei and Mojsov, 1996). The administration of PACAP27 to either mice or humans has been reported to increase plasma insulin levels without affecting plasma glucose levels (Filipsson et al., 1997, 1998a). Because PACAP27 activates both VPAC1 and VPAC2, the increase in glucose production may offset the increase in insulin secretion. 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. Continuous i.v. or s.c. infusion of the peptide reduced the glucose area under the curve after an i.p. glucose tolerance test (Tsutsumi et al., 2002).
A number of GLP-1 receptor agonists promote glucose-dependent insulin secretion and have demonstrated glucose-lowering activity in clinical studies (for review, see Riddle and Drucker, 2006). However, nausea and vomiting are common side effects associated with the inhibition of gastrointestinal motility by GLP-1 (Arulmozhi and Portha, 2006). A VPAC2-selective agonist has the potential to lower blood glucose without causing nausea and vomiting. In addition, VPAC receptor agonists have been reported to have potential anti-inflammatory activity that could be beneficial in the setting of type 2 diabetes (for review, see Pozo, 2003). Unlike GLP-1 receptor agonists, there is no evidence to suggest that VPAC receptor agonists promote weight loss.
Development of BAY 55-9837 as a potential peptide therapeutic for the treatment of type 2 diabetes was limited by poor peptide stability in aqueous solution and a very short half-life in vivo, most likely due to kidney clearance of the peptide. To overcome these limitations, we sought to identify a more stable form of the peptide and to prolong its activity in vivo. Analysis of BAY 55-9837 stored at 40°C over several weeks revealed both N-terminal degradation and deamidation (W. Wang, S. Martin-Moe, C. Pan, L. Musza, and J. Wang, unpublished data). Peptide half-life in vivo can be increased through attachment of polyethylene glycol (PEG) to the peptide, thereby reducing clearance of the peptide by the kidney and decreasing protease degradation of the peptide (Harris et al., 2001). In the current report, we engineered seven novel analogs of BAY 55-9837 and tested them for improved stability in aqueous buffer and retention of VPAC2-selective binding and activation. The peptide with optimal stability in aqueous solution was further engineered to determine the site of PEGylation and the size of the PEG that would allow retention of VPAC2 binding and activation in vitro. This led to the identification of stable PEGylated VPAC2 agonist peptides with prolonged glucose-lowering activity in vivo.
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Materials and Methods |
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Stability Determination. The peptide samples formulated in 20 mM sodium phosphate and 150 mM sodium chloride, pH 8.0, buffer were placed into a constant temperature stability chamber at 40°C. Samples were removed periodically for analysis by capillary electrophoresis, a rapid and sensitive method to detect degradation of polypeptide in these formulations. The area of various peaks was summed, and the peak area of the parent polypeptide was divided by the total peak area. The quotient is the percentage purity. Because there are impurities present in the fresh polypeptide, the purity change was normalized by dividing the purity at different time points by the initial purity.
Neutral Capillary Zone Electrophoresis. The method employs a neutral capillary to reduce the adsorption of peptides or proteins onto the capillary surface during electrophoresis. The silanol groups on silica capillary wall were deactivated, thereby reducing the electrostatic interactions between the proteins and the capillary wall. Thus, the electro-osmotic flow of the coated capillary was greatly reduced. Buffers with a pH less than 6.0 can be used for the separation of peptides and proteins with isoelectric points greater than 4.0. The sample was introduced into the capillary by hydrostatic injection and then was run in a 20 mM sodium citrate and 6.67 mM MES buffer, pH 4.5. The separation step was carried out at 13.5 kV with a 0.5-min ramp time. A UV monitor set at 214 nm monitored the migration of the peptide.
Competition Binding Assay. Membranes were prepared from CHO cells expressing human PAC1, VPAC1, or VPAC2. Cells were washed with phosphate-buffered saline (PBS), scraped in homogenization buffer (10 mM Tris, pH 7.4, 2 mM EDTA, 5 mM MgCl2, 1 mM phenylmethylsulfonyl fluoride), followed by centrifugation at 4000g for 10 min at 4°C. The cell pellet was resuspended in homogenization buffer and homogenized using a Polytron. Membranes were collected by centrifugation at 30,000g for 30 min at 4°C, resuspended in homogenization buffer, and stored at –80°C until use. To measure binding of peptides, 10 µg of membrane was incubated with 0.1 nM 125I-PACAP27 (PerkinElmer Life and Analytical Sciences, Wellesley, MA) in the presence of increasing concentrations of peptide in a total volume of 100 µl of reaction buffer (20 mM HEPES, pH 7.4, 150 mM NaCl, 0.5% BSA, 2 mM MgCl2, and 0.1 mg/ml Bacitracin). After incubating at 37°C for 20 min, bound ligand was collected on GF/C filters pretreated with 0.1% polyethylenimine. The filters were washed with cold 25 mM NaPO4 containing 1% BSA and counted in a gamma counter. All reagents were purchased from Sigma unless otherwise indicated.
cAMP Scintillation Proximity Assay. CHO cells expressing human PAC1, VPAC1, or VPAC2 were plated in 96-well plates (Corning Life Sciences, Acton, MA) at 2 x 104 cell/well and grown at 37°C for 24 h in 
minimal essential medium + nucleosides + glutamine (Invitrogen, Carlsbad, CA), 10% fetal bovine serum, 100 µg/ml Pen/Strep, 0.3 mg/ml glutamine, and 1 mM HEPES, 0.5 mg/ml Geneticin (Invitrogen). RINm5F rat insulinoma cell line (American Type Culture Collection, Manassas, VA) was grown in RPMI 1640 (Invitrogen) supplemented with 5% fetal bovine serum (Invitrogen), penicillin (100 U/ml), and streptomycin (100 µg/ml). The media were removed, and the plates were washed with PBS. The cells were incubated with compounds in HEPES-PBS-BSA (1%) with 0.4 mg/ml soybean trypsin inhibitor, 0.5 mg/ml bacitracin, and 100 µM 3-isobutyl-1-methylxanthine for 15 min at 37°C. Cyclic AMP in the cell extracts was quantitated using a commercial cAMP scintillation proximity assay (SPA) screening assay system (GE Healthcare, Little Chalfont, Buckinghamshire, UK).
Peptide PEGylation and Purification. Peptides were reacted with either a 22-kDa linear mPEG-MAL or a 43-kDa branched mPEG2-MAL (Nektar Therapeutics, Huntsville, AL). PEGylation was performed with 3 to 15 mg of peptide for in vitro assays. One-half molar excess of 22-kDa mPEG-MAL or 43-kDa mPEG2-MAL reagent (750 µM) was reacted with peptide (500 µM) dissolved in buffer containing 0.1 M sodium phosphate, pH 6.5. After incubation at room temperature for 0.5 h, the reaction was terminated with 2-fold molar excess of cysteine (1.5 mM) over PEG reagent. The reaction mixture was incubated with SP-Sepharose resin (GE Healthcare) at a ratio of 1 mg of peptide/ml resin for 4 h at 4°C with shaking. The resin was pre-equilibrated with 10 mM sodium phosphate, pH 6.5 (buffer A); after binding, it was washed with 5 column volumes of buffer A to remove nonspecific binding. The PEGylated peptide was eluted with buffer A + 0.5 M NaCl. Residual peptide was removed by dialysis against 4 liters of phosphate-buffered saline (Invitrogen) using a Slide-A-Lyzer cassette (Pierce, Rockford, IL) with a mol. wt. cut-off of 7000. PEGylation and purification were monitored by SDS-polyacrylamide gel electrophoresis (PAGE). Final concentration of PEGylated peptides was determined by amino acid analysis (Commonwealth Biotechnologies, Inc., Richmond VA). Endotoxin concentration of samples was determined by the Chromogenic Limulus Amebocyte Lysate Test according to the manufacturer's protocol (Cambrex Bio Science Baltimore, Inc., Baltimore, MD).
SDS-PAGE. Sodium dodecyl sulfate-PAGE was conducted under reducing conditions using supplies and reagents supplied by Invitrogen. The gels were NuPAGE 4 to 12% Bis-Tris run with MES buffer. The gels were blue-stained using GelCode (Pierce).
Animals. Male BALB/c mice (20–25g) were purchased from Charles River Breeding Laboratories (Portage, MI). All animals were maintained on standard laboratory rodent chow ad libitum for at least 5 days before being used in an experiment. All procedures were approved by the Bayer Animal Care and Use Committee, and all experiments were performed in accordance with relevant guidelines and regulations.
Intraperitoneal Glucose Tolerance Test. The i.p. glucose tolerance test (IPGTT) was performed in male Wistar rats as described previously (Pan et al., 2006). In brief, at the appropriate time after dosing, the fasting blood glucose level was measured from tail-tip blood using a Glucometer (Bayer HealthCare, Mishawaka, IN), and the animals were given 2 g/kg glucose by i.p. injection. Blood glucose was measured again after 15, 30, and 60 min. The area under the glucose curve (AUC) was calculated using the trapezoidal method, and the effect of the peptide on the AUC was expressed as a percentage of the AUC for the vehicle-treated group.
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The seven newly designed peptides were also tested together with the starting peptide, BAY 55-9837, for stability at 40°C in 20 mM sodium phosphate and 150 mM sodium chloride, pH 8.0, aqueous buffer or dimethylsulfoxide (DMSO). After 4 weeks at 40°C, the main peptide peak for BAY 55-9837 and BAY(T2E3) was greatly diminished or disappeared, and two later migrating peaks emerged, probably as a result of peptide degradation (Fig. 1A). On the other hand, BAY(Q9Q28) exhibited dramatic improvement in stability, losing only 7% (normalized by the percentage purity at zero time) of the main peak during the 4-week incubation at 40°C. The 2- and 4-week stability data showed that any peptide with the double N to Q mutations, as in BAY(Q9Q28), BAY(T2E3Q9Q28), and AcBAY(T2Q9Q28), were much more stable than those without [BAY 55-9837, AcBAY, BAY(T2), BAY(E3), and BAY(T2E3)] (Fig. 1, B and C). Overall stability in DMSO was somewhat better than aqueous buffer. Of the peptides with the N9Q/N28Q mutations, BAY(Q9Q28) displayed the best selectivity/activity profile. BAY(Q9Q28) maintained VPAC2 activation (EC50 = 0.3 nM) and selectivity over VPAC1 and PAC1 (>300-fold). Thus, this peptide was chosen as the peptide sequence for further engineering.
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). Thus, a single cysteine was added to the C terminus of BAY 55-9837 to create BAY(C32) (Table 1). Because the PEG moiety is much larger than the peptide and has the potential to affect receptor binding, a two-amino acid spacer ("SW") between the C terminus and the cysteine was also tested [BAY(S32W33C34)]. The addition of the cysteine residue or spacer plus cysteine residue to BAY 55-9837 affected peptide potency, with BAY(S32W33C34) slightly less potent than BAY(C32) (Table 2). Thus, the C terminus without spacer addition was chosen as the site of PEGylation. Introducing the C-terminal cysteine to BAY(Q9Q28), the peptide optimized for in vitro stability and functional activity, led to BAY(Q9Q28C32), which is a potent activator of VPAC2 (EC50 = 0.38 nM) and has no significant activity at either the PAC1 or VPAC1 receptors (Table 2).
PEGylated VPAC2 Agonists. BAY(C32), BAY(S32W33C34), and BAY(Q9Q28C32) were PEGylated with a linear 22-kDa or a branched 43-kDa PEG. The extent of modification was characterized by Coomassie Blue-stained SDS-polyacrylamide gels (Fig. 2). PEGylation of BAY(Q9Q28C32) yielded mainly monomodified products BAY(Q9Q28C32)PEG22 and BAY(Q9Q28C32)PEG43 (22- and 43-kDa PEGylated, respectively), migrating at approximately 48 and 75 kDa, respectively. A minor component was observed at 
4 kDa that represented residual unmodified peptide. PEGylated peptides, which carry net positive charges at pH values < 10, were separated from uncharged free PEG by cation exchange chromatography. Residual unmodified BAY(Q9Q28C32) was partially removed by dialysis. The final products contained greater than 90% monomodified peptides. Modification of peptides with a linear 22-kDa PEG had less effects on VPAC2 binding and activation of VPAC2 overexpressed on the CHO cells (Table 2) or endogenous VPAC2 from RINm5F cells (Table 3) as compared with conjugation with the larger 43-kDa branched PEG.
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In Vitro Profile of PEGylated BAY(Q9Q28C32). The PEGylated products, BAY(Q9Q28C32)PEG22 and BAY(Q9Q28C32)PEG43, bind to the human VPAC2 with an IC50 of 126 and 200 nM, respectively. This is higher than that of PACAP27 and the non-PEGylated peptide BAY(Q9Q28C32), which have IC50 values of 12 and 20 nM, respectively (Table 2; Fig. 3A). It is noteworthy that the PEGylated peptides bind with a high degree of selectivity to VPAC2 with IC50 values > 10 µM for both PAC1 and VPAC1 (Table 2). BAY(Q9Q28C32)PEG22 and BAY(Q9Q28C32)PEG43 activate the VPAC2 receptor overexpressed on the CHO cells with EC50 of 1.3 and 18 nM, respectively (Table 2; Fig. 3B) and RINm5F cells that express the endogenous VPAC2 with an EC50 of 68 and 187 nM, respectively (Table 3).
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Discussion |
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cells (for review, see Riddle and Drucker, 2006). However, nausea and vomiting have been associated with GLP-1 receptor activation (Arulmozhi and Portha, 2006). 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. However, BAY 55-9837 was not stable in liquid formulation over a 1-week period as evidenced by N-terminal deletions and further deamidations (Wang et al., unpublished data). Furthermore, BAY 55-9837 had a very short duration of action in vivo and required continuous infusion to demonstrate prolonged efficacy in vivo (Tsutsumi et al., 2002).
Aqueous buffer instability of VIP has been attributed to hydrolysis (Mody et al., 1994). BAY 55-9837 and VIP share the N-terminal His-Ser-Asp sequence that contains the necessary amino acids to make up a potential catalytic triad for serine proteases. However, mutating these residues did not significantly improve liquid stability as exemplified by BAY(T2), BAY(E3), and BAY(T2E3). Furthermore, the use of a proteinase inhibitor, phenylmethylsulfonyl fluoride, did not improve stability of BAY 55-9837 either (data not shown). Therefore, the instability of BAY 55-9837 in liquid formulation is unlikely to be due to self-hydrolysis. Instead, the major degradation pathway appeared to be dimerization initiated through dehydration at aspartic acid residues or deamidation at asparagine residues, with concurrent formation of a cyclic imide, followed by a nucleophilic addition of a basic amino acid such as lysine from another molecule. Our current data with mutated asparagine peptides suggest that the deamidation at asparagine may be the dominant determinant for instability because the removal of this amino acid results in peptides such as BAY(Q9Q28), BAY(T2E3Q9Q28), and AcBAY(T2Q9Q28) that appear to be stable for nearly 1 month at 40°C in liquid formulation.
Alanine scanning mutagenesis has been performed to determine the role of individual amino acids within VIP responsible for VPAC2 activation (O'Donnell et al., 1991; Nicole et al., 2000; Igarashi et al., 2002). Alanine substitution at position 3 but not position 2 greatly reduces VPAC2 binding and activation. In the context of BAY 55-9837, which has seven mutations from VIP, making the highly conservative change from Asp3 to Glu3 led to a peptide with lower potency as shown by BAY(E3). However, a conservative mutation at position 2 of BAY 55-9837 also reduced potency. The N-terminal region of VIP adopts a flexible structure even in partially organic solution that mimics membrane interface (Theriault et al., 1991). Perhaps the replacement of the native Ser at position 2 with the bulkier Thr instead of Ala, as in BAY(T2), reduces the flexibility of the N-terminal region, leading to lower potency at VPAC2. Alternatively, BAY 55-9837 may be more sensitive to position 2 substitution than VIP. Consistent with previous alanine scanning mutagenesis data, conservative substitutions at positions 9 and 28 did not negatively impact potency as shown by BAY(Q9Q28). Thus, our current mutagenesis data are generally consistent with previously published structure activity studies on VIP. It is noteworthy that none of the current mutations affected VPAC2 selectivity, consistent with our previous work (Yung et al., 2003).
PEGylation is one method used to increase the life span of a peptide or protein through the covalent attachment of long-chained polyethylene glycol or PEG molecules. PEGylation protects proteins from protease digestion and keeps the material out of the kidney filtrate (for review, see Harris and Chess, 2003). In addition, PEGylation may also increase the overall stability and solubility of the protein. Finally and importantly, the sustained plasma concentration of PEGylated peptides can reduce the extent of adverse side effects by reducing the trough to peak levels of the drug. However, success in peptide PEGylation has been limited by the propensity of the large polymers to interfere with peptide function. Recently, we site-directly PEGylated a GLP-1 agonist/glucagon antagonist hybrid peptide at its C terminus (Pan et al., 2006). Although significant in vitro potency was lost, especially with the larger 43-kDa branched PEG, the loss was more than compensated by the greatly extended in vivo activity. In the current work, we also PEGylated the VIP analogs at the C terminus because VIP, GLP-1, and glucagons belong to the same family of peptides. We were able to mostly preserve in vitro functional activity with the smaller 22-kDa PEG but realized some loss with the larger 43-kDa PEG. However, the 22- and 43-kDa PEGylated peptides, BAY(Q9Q28C32)PEG22 and BAY(Q9Q28C32)PEG43, are active in vivo for at least 3 and 6 h post-s.c. injection, respectively.
In conclusion, we have identified the cause of BAY 55-9837 instability to be predominantly deamidation and successfully removed two potential deamidation sites without lowering functional activities. The VPAC2 agonist BAY(Q9Q28) is more stable than BAY 55-9837 and shows comparable VPAC2 selective activation. We have further identified the optimal site for PEGylation of the VPAC2 agonists and generated PEGylated peptides that retain significant in vitro VPAC2 activity and selectivity, as well as prolonged in vivo activity. Therefore, the current work demonstrates a fully integrated peptide engineering approach to drug development. Functional properties such as VPAC2 binding, activation, and selectivity were optimized simultaneously with physical stability. Further studies will be required to explore the glucose-lowering activity of the PEGylated VPAC2 agonist peptides in diabetic animal models.
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Acknowledgements |
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Footnotes |
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ABBREVIATIONS: VIP, vasoactive intestinal peptide; PACAP, pituitary adenylate cyclase-activating polypeptide; GLP-1, glucagon-like peptide-1; PEG, polyethylene glycol; CHO, Chinese hamster ovary; PBS, phosphate-buffered saline; BSA, bovine serum albumin; SPA, scintillation proximity assay; PAGE, polyacrylamide gel electrophoresis; IPGTT, i.p. glucose tolerance test; AUC, area under the glucose curve; DMSO, dimethyl sulfoxide; BAY 55-9837, peptide HSDAVFTDNYTRLRKQVAAKKYLQSIKNKRY.
Address correspondence to: James P. Whelan, Department of Metabolic Disease Research, Bayer HealthCare, Pharmaceuticals, West Haven, CT 06516. E-mail: james.whelan.b{at}bayer.com
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References |
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Harris JM and Chess RB (2003) Effect of pegylation on pharmaceuticals. Nat Rev Drug Discov 2: 214–221.[CrossRef][Medline]
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Igarashi H, Ito T, Pradhan TK, Mantey SA, Hou W, Coy DH, and Jensen RT (2002) Elucidation of the vasoactive intestinal peptide pharmacophore for VPAC(2) receptors in human and rat and comparison to the pharmacophore for VPAC(1) receptors. J Pharmacol Exp Ther 303: 445–460.
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