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GASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL
Digestive Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland (H.I., T.I., S.A.M., T.K.P., W.H., D.H.C., R.T.J.); and Peptide Research Laboratories, Department of Medicine, Tulane University Health Sciences Center, New Orleans, Louisiana (D.H.C.)
Received for publication
April 28, 2005
Accepted
June 28, 2005.
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Abstract |
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 Top Abstract Materials and MethodsResultsDiscussionReferences |
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; Gozes and Brenneman, 2000). VIP is proposed to play a role in a number of disease states (Gozes and Furman, 2004), including a role in growth of cancer cells (Moody, 1996; Moody et al., 2003; Gozes and Furman, 2004), various central nervous system disorders (Gozes and Brenneman, 2000; Dogrukol-Ak et al., 2004; Gozes and Furman, 2004), various inflammatory disorders such as rheumatoid arthritis (Gozes and Brenneman, 2000), and various immunological disorders (Delgado et al., 2004), and a role has been proposed for VIP in treatment of asthma (Groneberg et al., 2001), impotence (Sandhu et al., 1999; Kalsi et al., 2002), and for treatment of septic shock (Kalsi et al., 2002; Delgado et al., 2004), central nervous system disorders (Gozes and Brenneman, 2000; Dogrukol-Ak et al., 2004), and diabetes (Yung et al., 2003).
In almost all cases, which VIP receptor subtype is mediating the action of VIP in these various conditions, is unclear. The actions of VIP are mediated by two receptor subtypes (VPAC1 and VPAC2), which have different pharmacology and distributions (Dockray, 1994; Harmar et al., 1998). VIP has high affinity for both VIP receptor subtypes and therefore does not discriminate between the two VIP receptor subtypes (Harmar et al., 1998). Furthermore, VIP is a 28-amino acid peptide that undergoes rapid degradation in vivo with a half-life less than 1 min (Domschke et al., 1978). Therefore, simplified VIP analogs that retain high affinity and have selectivity for one VIP receptor subtype, especially if metabolically stable, could be of value in investigating VIP's roles in physiological or pathological states as well as its use as a possible therapeutic agent. Furthermore, a metabolically stable, simplified VIP analog that retained high affinity for both VIP receptor subtypes could be useful for imaging tumors overexpressing VIP receptors as well as possibly for VIP receptor-directed antitumor treatment.
Recently, we (Igarashi et al., 2002a,b) and others (Nicole et al., 2000) have performed alanine scanning as well as D-amino acid scanning of VIP (Igarashi et al., 2002a,b) to define the VIP pharmacophore for the human VPAC1 and human VPAC2 receptors. These studies (Nicole et al., 2000; Igarashi et al., 2002a,b) provided information that could be helpful in the design of a simplified VIP analog that had either selective high affinity for one VPAC or that retained high affinity for both and yet might be metabolically stable. The latter point is supported by results of a previous study that demonstrated a polyalaninated VIP analog with high affinity for VPAC1 was much more metabolically stable than VIP (Igarashi et al., 2002b). However, in that study (Igarashi et al., 2002b), the selectivity of this VIP analog for VPAC1 (analog 2 in the present study) was not determined. Therefore, in the present study we used an analysis of these study results (Nicole et al., 2000; Igarashi et al., 2002a,b) on the VIP pharmacophore to design five additional VIP analogs with multiple alanine replacements that should have high affinity retained for VPAC1 and three analogs that should have high affinity retained for VPAC2 and assessed their affinities for each VPAC, their selectivity, and metabolic stability of selected analogs.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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-,6-diphenylglycouril was from Pierce Chemical (Rockford, IL); HEPES was from Roche Diagnostics (Indianapolis, IN). Dulbecco's modified Eagle's medium (DMEM), RPMI 1640 medium, and Ham's F-12K medium were from Biofluids (Rockville, MD). The standard incubation solution contained 24.5 mM HEPES, pH 7.45, 98 mM NaCl, 6 mM KCl, 2 mM KH2PO4, 5 mM sodium pyruvate, 5 mM sodium fumarate, 5 mM sodium glutamate, 2 mM glutamine, 11.5 mM glucose, 0.5 mM CaCl2, 1 mM MgCl2, 1% (w/v) BSA, 0.2% (w/v) soybean trypsin inhibitor, 1% (v/v) amino acid mixture, and 1% (v/v) essential vitamin mixture.
Preparation of Peptides. Multiple alanine-substituted VIP analogs (VPAC-A-1 to 9; Table 1 and Fig. 1) were synthesized using standard solid phase methods as described previously (Sasaki and Coy, 1987; Igarashi et al., 2002a). Homogeneity of the peptides was assessed by thin layer chromatography and analytical reverse-phase PHLC with the purity 
97% for each peptide.
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; Igarashi et al., 2002a). Cell Culture. The hVPAC1-R/PANC1 cells and hVPAC2-R/PANC1 cells were grown in DMEM supplemented with 10% (v/v) fetal bovine serum, 1% (v/v) antibiotics, and 300 µg/ml G418. Sup T1 human lymphoblastoma cells were grown in RPMI 1640 medium supplemented with 2 mM L-glutamine, 10% (v/v) fetal bovine serum, and 1% (v/v) antibiotics, adjusted to contain 1.5 g/l sodium bicarbonate, 4.5 g/l glucose, 10 mM HEPES, and 1.0 mM sodium pyruvate as recommended by American Type Culture Collection and were maintained in incubators at 37°C in an atmosphere of 5% CO2 and 95% air. T47D human breast cancer cells were grown in RPMI 1640 medium supplemented with 2 mM L-glutamine, 10% (v/v) fetal bovine serum 1% (v/v) penicillin/streptomycin, and 0.2 IU/ml insulin and were maintained in incubators at 37°C in an atmosphere of 5% CO2 and 95% air.
Preparations of 125I-Ro 25-1553. 125I-Ro 25-1553 (a cyclic VIP analog selective for VPAC2-R) (O'Donnell et al., 1994a,b; Gourlet et al., 1997b) at a specific activity of 2200 Ci/mmol was prepared by a modification of the methods described previously (Zhou et al., 1989). In brief, 0.8 µg of 1,3,4,6-tetrachloro 3-,6-diphenylglycouril in chloroform was transferred to a vial, dried under a stream of nitrogen, and washed with 100 µl of 0.5 M KH2PO4, pH 8.0. To this vial, 20 µl of 0.5 M KH2PO4, pH 8.0, 8 µg of peptide in 4 µl of water, and 2 mCi (20 µl) of Na125I were added, mixed gently, and incubated at room temperature for 6 min. The incubation was stopped by the addition of 100 µl of distilled water. The iodination mixture was applied to a Sep-Pak C18 (Waters, Milford, MA), and free 125I was eluted with 5 ml of water followed by 5 ml of 0.1% (v/v) trifluoroacetic acid. The radiolabeled peptides were eluted with 200 µl of sequential elutions (x10) with 60% acetonitrile in 0.1% trifluoroacetic acid. The two or three fractions with the highest radioactivity were combined and purified on a reverse-phase, high-performance liquid chromatography with a Vydac C18 column (0.46 x 25 cm). The column was eluted with a linear gradient of acetonitrile in 0.1% trifluoroacetic acid (v/v) from 16 to 60% acetonitrile in 60 min, and 1-ml fractions were collected and assayed for radioactivity and receptor binding. The pH of the pooled fractions was adjusted to 7 using 0.2 M Tris, pH 9.5, and radioligands were stored in aliquots with 0.5% bovine serum albumin (w/v) at -20°C. 125I-[Ala2,8,9,11,19,24,25,27,28]VIP (VIP analog 2; Table 1; Igarashi et al., 2002a) and 125I-[Ala2,8,9,16,19,24,25]VIP (VIP analog 8; Table 1) were prepared using the same procedures.
Binding Studies. Binding of 125I-VIP to hVPAC1-R/PANC1 and T47D cells, and binding of 125I-PACAP(1-27) to hVPAC2-R/PANC1 were performed by incubation in standard incubation solution containing 0.05% (w/v) bacitracin for 60 min at room temperature and as described previously (Ito et al., 2000; Igarashi et al., 2002a,b). To assess VPAC2-R affinities in Sup T1 human lymphoblastoma cells, binding study was performed using 125I-Ro 25-1553 in standard incubation solution containing 0.05% (w/v) bacitracin for 60 min at 37°C, because 125I-VIP and 125I-PACAP(1-27) were rapidly degraded in these cells even with protease inhibitors present. The separation of bound from free radioactivity was obtained by centrifugation of cells through 2% (w/v) BSA in standard incubation solution. The tubes were washed twice with 2% (w/v) BSA in standard incubation solution and radioactivity was counted. Nonsaturable binding for 125I-VIP, 125I-PACAP(1-27), or 125I-Ro 25-1553 was less than 5% of total binding.
For all peptides, the IC50 was calculated, which was the concentration that gave half-maximal inhibition of that seen with a saturating concentration of VIP (1 µM). The IC50 was calculated using the curve-fitting program Kaleidagraph.
cAMP Assay. T47D cells (0.05 x 106 cells), hVPAC1-R/PANC1 (0.05 x 106 cells), and hVPAC2-R/PANC1 (0.05 x 106 cells) were plated on 24-well plates and incubated for 48 h at 37°C with media containing 10% FBS (v/v). The media were then replaced with media supplemented with 2% FBS (v/v) and 2 µCi/ml [2-3H]adenine. Cells were incubated for an additional 48 h at 37°C. The media were removed, and cells were incubated in the 500 µl of DMEM containing 1% (w/v) BSA and 0.5 mM IBMX with or without peptides at various concentrations for 1 h at 37°C. Reactions were terminated by the addition of 120 µl of cAMP stopping solution [2% SDS (w/v), 25 mM cAMP] followed by 1 ml of 50 mM Tris, pH 7.4. Samples were stored at -20°C until analyzed. The amount of cAMP formation was determined using a modification of a method reported previously using Dowex AG 50W-X4 anion exchange resin and alumina (Benya et al., 1994; Igarashi et al., 2002b). For studying cAMP generation in Sup T1 cells, 20 ml of medium containing 2.0 to 4.0 x 106 cells/ml, supplemented with 2% FBS (v/v) and 2 µCi/ml [2-3H]adenine were incubated for 48 h at 37°C. Then, the solution containing the cells was centrifuged, and the cell pellet was washed with DMEM twice, followed by suspended with 50 ml of DMEM containing 1% (w/v) BSA and 0.5 mM IBMX. Five hundred microliters of this cell solution was added to each tube with or without peptides at various concentrations and incubated for 1 h at 37°C. The procedure of termination of the reaction and column processing procedure were the same as described above. For all peptides, the EC50 was calculated, which was the concentration of the peptide that gave half-maximal stimulation of a maximally effective concentration of VIP (1 µM). The EC50 was calculated using the curve-fitting program Kaleidagraph.
Degradation Studies of Radiolabeled Ligands.125I-[Ala2,8,9,16,19,24,25]VIP, 125I-[Ala2,8,9,11,19,24,25,27,28]VIP, or 125I-VIP corresponding to 500,000 cpm (250 pM) was incubated in 1 ml of standard incubation solution in the absence of bacitracin, with or without hVPAC1-R/PANC cells (0.3 x 106 cells/ml) at 37°C for 7.5 min, or hVPAC2-R/PANC cells (0.4 x 106 cells/ml) at 37°C for 15 min. After incubation aliquots were centrifuged, and distribution of the radioactivity in the supernatants was analyzed by HPLC. Each supernatant (200,000 cpm) was injected onto an HPLC with a Vydac C18 column. Two-milliliter fractions were collected and radioactivity was determined.
Statistical Analysis. The results are mean ± S.E.M. of three or more experiments. IC50 and EC50 were calculated using the curvefitting program Kaleidagraph. Statistical comparisons were made using the Student's t test.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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), we demonstrated a simplified multialaninated analog of VIP (analog 2; present study) could be synthesized that retained high affinity for rat, guinea pig, and human VPAC1-R. This analog also had increased stability compared with VIP for the hVPAC1-R (Igarashi et al., 2002a). However, no studies were performed on the selectivity or stability of this analog with hVPAC2-R. In the present study, we examined the VPAC-R selectivity of this analog and synthesized additional simplified, multialaninated analogs in an attempt to develop a highly selective VIP analog for hVPAC1-R and hVPAC2-R that might also be metabolically stable for either or both receptors.
Using the results of alanine scanning of VIP to define the amino acids essential for high-affinity interaction with the hVPAC1-R (Igarashi et al., 2002a) and the hVPAC2-R (Igarashi et al., 2002b), we synthesized eight additional multialaninated VIP analogs that, based on single alanine substitutions, should retain high affinity for the hVPAC1-R or the hVPAC2-R. Five new analogs (analogs 1 and 3–6; Fig. 1) were synthesized based on the VIP pharmacophore for hVPAC1-R and three new analogs (analogs 7–9; Fig. 1) based on the VIP pharmacophore of the hVPAC2-R. In each case, the multialaninated simplified analogs were made by making combinations of analogs in which, when alanine was substituted alone in a given position, there was a <10-fold decrease in affinity for either the VPAC1 (Igarashi et al., 2002a) or VPAC2 receptor (Igarashi et al., 2002b) (Fig. 1).
Affinity of Multialanine-Substituted VIP Analogs Designed to Be hVPAC1-R Agonists for hVPAC1-R- or hVPAC2-R-Containing Cells. Multialaninated VIP analogs 1 and 3 to 6 retained high affinity for the hVPAC1-R, similar to VIP analog 2 (Fig. 2; Table 1). The simplest, multialaninated VIP analog (VIP analog 4; Fig. 1) ([Ala2,8,9,11,19,24,27]VIP) had a 6- to 7-fold decrease in the affinity compared with VIP, for the hVPAC1-R-containing cells. When two more alanine substitutions were added in positions 25 and 28, which resulted in the VIP analog 2, it had an equal affinity to VIP for the hVPAC1-R, similar to that reported previously (Igarashi et al., 2002a). An additional alanine replacement of either valine19 (analog 3) or Lys21 (analog 4), Tyr22 (analog 5) or Ile26 (analog 26) had variable effects on hVPAC1-R affinity (Fig. 2, top; Table 1). Analogs 3, 5, and 6 had a 2- to 17-fold decrease in the affinity for the hVPAC1-R compared with VIP and analog 2 (Fig. 2, top; Table 1).
To determine the possible VPAC-R selectivity of these analogs, their affinities for native hVPAC2-R on Sup T1 human lymphoblastoma cells and hVPAC2-R stably transfected PANC1 cells were determined (Fig. 2, bottom; Table 1). VIP had a high affinity for the hVPAC2-R-containing cells; however, analogs 1 to 6 had a lower affinity. Analog 1 and analog 2 specifically had a 15- to 90-fold decrease in the affinity compared with VIP for the hVPAC2-R. When additional alanine substitutions for Met17, Lys21, Tyr22, or Ile26 were made to analog 2, the affinity for hVPAC2-R became much lower, especially with the additional alanine substitution for Tyr22 and Ile26 (Fig. 2, bottom; Table 1). The resultant analogs 5 and 6 had a >4500-fold and 2500- to 3000-fold lower affinity than to VIP for hVPAC2-R. In terms of hVPAC-R selectivity, VIP had a 4-fold higher affinity for hVPAC1-R, whereas VIP analog 1 had a 36-fold higher selectivity for hVPAC1-R over hVPAC2-R and analog 2, a 150-fold selectivity for hVPAC1-R (Table 1). The additional substitution of Met17 or Lys21 resulted in analogs 3 and 4, respectively, which had a 150- to 200-fold higher selectivity for hVPAC1-R over hVPAC2-R (Table 1). Alanine substitution of Tyr22 or Ile26 in analog 2, which yielded VIP analogs 5 and 6, had the greatest selectivity for hVPAC1-R over hVPAC2-R, which were >2400- and 660-fold, respectively (Table 1).
Affinity of Multialanine-Substituted VIP Analogs Designed to Be hVPAC2-R Agonists for hVPAC1-R- or hVPAC2-R-Containing Cells. We synthesized three multialaninated VIP analogs that should retain high affinity for the hVPAC2-R (VIP analogs 7–9; Fig. 1) and determined their abilities to interact with hVPAC1-R receptors (Fig. 3, top; Table 1) and hVPAC2-R-containing cells (Fig. 3, bottom; Table 1). VIP analog 7 with six alanine substitutions (i.e., for Ser2, Asp8, Asn9, Gln16, Val19, and Asn24; Fig. 1) as well as VIP analog 8, which had an additional alanine substitution for Ser25 (Fig. 1), had a similar high affinity to VIP for the hVPAC2-R (Fig. 3, bottom; Table 1). However, when two additional alanine substitutions for Lys20 and Lys21 were added to analog 8, the resultant analog 9 had a 3- to 8-fold decrease in affinity for hVPAC2-R compared with VIP (Fig. 3, bottom; Table 1). We also determined their affinities for hVPAC1-R-containing cells (Fig. 3, top; Table 1) and found that analog 7 and analog 8 showed a 2- to 3-fold and a 5- to 7-fold decrease, respectively, in affinity for hVPAC1-R compared with VIP. In contrast, analog 9 had a 50-fold decrease in the affinity compared with VIP for hVPAC1-R (Fig. 3, top; Table 1). In terms of VPAC-R selectivity, each of these 3 analogs (7–9) had a 2- to 4-fold higher affinity for hVPAC2-R than hVPAC1-R, whereas VIP had a 3.7-fold higher selectivity for hVPAC1-R than hVPAC2-R (Table 1; Fig. 3).
Potency of Multialanine-Substituted VIP Analogs Designed to Be hVPAC1-R Agonists for hVPAC1-R or hVPAC2-R Activation. Activation of adenylate cyclase is the principal intracellular mediator of the action of VPAC1 and VPAC2 receptors (Zhou et al., 1989; Ulrich, II et al., 1998; Nicole et al., 2000). To investigate whether these multialaninated VIP analogs functioned as agonists and if so, their potency and efficacy for hVPAC1-R and hVPAC2-R activation, we first determined the ability of VIP analogs 1 to 6, which were hVPAC1-R-preferring by binding studies, to stimulate cAMP generation in hVPAC1-R- and hVPAC2-R containing cells (Fig. 4; Table 2). Each of the six analogs had similar efficacy to VIP in stimulating cAMP generation through the hVPAC1-R (Fig. 4, top). VIP had a high potency for stimulating cAMP generation in hVPAC1-R-containing cells with EC50 values of 2.5 to 2.7 nM and caused a maximal stimulation with 100 nM concentration (Table 2; Fig. 4). Except for VIP analogs 1 and 4, which showed a 2- to 4-fold decrease in potency for activating VPAC1-R compared with VIP, each of the other 4 VPAC1-R-preferring analogs (2, 3, 5, and 6) retained a high potency for the VPAC1-R (Fig. 4, top; Table 2).
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Potency of Multialanine-Substituted VIP Analogs (Analogs 7–9) Designed to Have Higher Affinity for the hVPAC2-R for hVPAC1-R or hVPAC2-R Activation. VIP analogs 7 to 9 had equal efficacy to VIP for activating either hVPAC1R (Fig. 5, top) or hVPAC2-R cells (Fig. 5, bottom). Each of these three analogs had a similar potency to VIP for stimulating cAMP generation in VPAC1-R cells (Fig. 5, top; Table 3). In contrast, none of these three analogs had higher potency than VIP for stimulating cAMP generation in hVPAC2-R cells (Fig. 5, bottom; Table 3). In terms of relative potency for VPAC-R activation, none of these three analogs had selectivity for hVPAC2-R over hVPAC1-R (Table 3).
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Stability of VIP and Multialaninated VIP Analogs Incubated with hVPAC1-R/PANC1 Cells or hVPAC2-R/PANC1 Cells. In a previous study (Igarashi et al., 2002a), we demonstrated analog 2 ([Ala2,8,9,11,19,24,25,27,28]VIP was more resistant to degradation than VIP by hVPAC1-R/PANC1 cells. However, in the present study, we demonstrate the VIP analog 2 has less than a 200-fold selectivity for the hVPAC1-R (Table 1) and thus would not be useful for in vivo receptor studies attempting to localize both hVPAC1-R and hVPAC2-R in tumors. It is unknown whether the other multialaninated analogs that retained high affinity for both hVPAC-R subtypes described in the present study (Tables 1 and 2) and could thus be useful for in vivo receptor characterization of both VPAC-R subtypes, were also resistant to degradation. To address this question, we prepared 125I-analog 8 (125I-Ala2,8,9,16,19,24,25]VIP) and 125I-analog 2 (125I-Ala2,8,9,11,19,24,25,27,28]VIP) and compared the amount of degradation during an incubation with hVPAC1-R/PANC1 cells or hVPAC2-R/PANC1 cells to that seen with 125I-VIP. As seen in Fig. 6, left, more than 70% of 125I-VIP was degraded during incubation with hVPAC1-R/PANC1 cells, whereas only 20% 125I-analog 8 was degraded. As reported previously (Igarashi et al., 2002a), no degradation of 125I-analog 2 was seen under the same conditions, suggesting that analog 8 had a greater stability than VIP incubated with hVPAC1-R/PANC1 cells, but it was less metabolically stable than analog 2. Figure 6, right, demonstrates that more than 70% of 125I-VIP was degraded during an incubation with hVPAC2-R/PANC1 cells; however, only 20% of 125I-analog 8 was degraded, showing that analog 8 also had a greater stability than VIP during incubation with hVPAC2-R/PANC1 cells.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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), we identified a simplified analog of VIP, [Ala2,8,9,11,19,24,25,27,28] (analog 2 in present study), which retained high affinity for hVPAC1 and which was synthesized after analyzing the VIP pharmacophore for VPAC1 in human, rat, and guinea pig. In the present study, we confirmed that [Ala2,8,9,11,19,24,25,27,28] has a similar high affinity to VIP for the human VPAC1 in both native hVPAC1-containing cells (T47D cells) and hVPAC1-transfected PANC1 cells; however, we further demonstrate this simplified VIP analog has relatively low selectivity (<170- fold) for hVPAC1 over hVPAC2. Therefore, [Ala2,8,9,11,19,24,25,27,28] does not fulfill our goal of identifying a highly selective agonist for either VPAC. Furthermore, even though it is metabolically stable (Igarashi et al., 2002a), its lower affinity for hVPAC2 (100–500 nM) makes it unsuitable as a high-affinity ligand for both VPACs, our second goal. In the present study, we have successfully identified simplified VIP analogs that have high selectivity and retain high affinity for the hVPAC1. Two of the five new, simplified VIP analogs, [Ala2,8,9,11,19,22,24,25,27,28]VIP (analog 5) and [Ala2,8,9,11,19,24–28]VIP (analog 6), had >2400- fold and 600-fold higher binding affinities, respectively, for hVPAC1 over hVPAC2 receptors. Furthermore, each of these two analogs was a fully efficacious agonist at the VPAC1 receptor, and analog 5 had >15,000-fold greater potency for activating the hVPAC1 compared with the hVPAC2, and analog 6 had >1200-fold greater potency for hVPAC1 over hVPAC2. This discrepancy between potency and binding affinity for the VPAC1 compared with native VIP is probably at least partially due to their greater metabolic stability, because each of these analogs has multialaninated substitutions similar to analogs 8 and 2 in the present study and analog 2 in a previous study (Igarashi et al., 2002a), each of which has enhanced metabolic stability. These results demonstrate that both the simplified VIP analogs 6 and 5 may have greater selectivity than the 65-fold selectivity of [Leu22]VIP for hVPAC1 over hVPAC2 (Gourlet et al., 1998; Bhargava et al., 2002) and at least comparable and maybe greater than the widely used VPAC1-selective agonist (Gourlet et al., 1997a) [Lys15, Arg16, Leu27]VIP(1-7)-GRF(8-27), which is reported to have a 53- to 169-fold selectivity for rVPAC1 over rVPAC2 transfected into Chinese hamster ovary cells in one study (Ito et al., 2000), but a 15,000-fold selectivity in another study (Gourlet et al., 1997a), and a 300- to 30,000-fold selectivity in hVPAC1/hVPAC2-containing cells (Gourlet et al., 1997a; Igarashi et al., 2002b).
In contrast to our results with the hVPAC1, none of the three simplified VIP analogs synthesized, based on the analysis of the VIP pharmacophore for the VPAC2 (Igarashi et al., 2002b), demonstrated selectivity for the hVPAC2. However, two of these three simplified VIP analogs [(Ala2,8,9,16,19,24)VIP (analog 7) and [(Ala2,8,9,16,19,24,25)VIP (analog 8)] retained high affinity (5.6 and 7.1 nM) for the hVPAC2, in contrast to each of the six simplified VIP analogs (analogs 1–6) designed to have high affinity for VPAC1, each of which had low affinity for hVPAC2 (468 to >30,000 nM). These results demonstrate that using the designed strategy applied in the present study, simplified analogs of VIP could be made which retained high affinity for hVPAC2. The lack of selectivity of these two high-affinity simplified VIP analogs (analogs 7 and 8) for VPAC2 is in contrast to findings in other studies using different strategies that have reported finding peptides that have selectivity for VPAC2. The VIP-related peptide, helodermin is reported to have 15-fold higher affinity for hVPAC2 over hVPAC1 (Gourlet et al., 1997b). Ro 25-1553, a cyclic analog of VIP with a lactam ring (O'Donnell et al., 1994a), is reported to have 75- to 600-fold selectivity for hVPAC2 over hVPAC1 (Gourlet et al., 1997b; Ito et al., 2000; Moreno et al., 2000; Igarashi et al., 2002a), 246- to 4300-fold for rVPAC2 over rVPAC1 (Ito et al., 2000), and hexanoyl [Ala19, Lys27,28]VIP, to have an 800-fold selectivity for hVPAC2 over hVPAC1 (Langer et al., 2004).
A review of the previous studies of the VIP pharmacophore for the VPAC1 and VPAC2 (Nicole et al., 2000; Igarashi et al., 2002a,b) provides some insights into why the approach used in the study probably resulted in selective VPAC1 but not selective VPAC2 agonists. In these previous studies (Gourlet et al., 1998; Nicole et al., 2000; Igarashi et al., 2002a,b) using alanine scanning to identify the VIP pharmacophore for hVPACs, a number of amino acids, particularly Thr11, Tyr22, Asn24, Leu27, and Asn28 were more important for VPAC2 than hVPAC1 affinity. In these studies (Nicole et al., 2000; Igarashi et al., 2002a,b) and others (Gourlet et al., 1998), the presence of Tyr22 in VIP was much more important for high affinity for VPAC2 than VPAC1, with the result that a single substitution of alanine in position 22 of VIP had the most profound effect on VIP's ability to interact with each VPAC subtype of any single alanine substitution. In our study, analog 5 ([Ala2,8,9,11,18,19,22,24,25,27,28]VIP), which had the greatest selectivity for hVPAC1, was the only simplified analog to contain this substitution. The Ala22 substitution was not included in the other five proposed VPAC1-selective agonists (analogs 1–4 and 6) because it has been shown to cause a 4-fold decrease in hVPAC1 affinity (Igarashi et al., 2002a,b), which we wanted to avoid if possible. The single substitution of alanine for Thr11 or Leu27 in VIP also caused a >15-fold decrease in VPAC2 affinity with minimal changes in VPAC1 affinity (Igarashi et al., 2002a,b). Both of these substitutions were included in each of the six proposed simplified VPAC1 selective agonists; however, they only resulted in a 35- to 196-fold selectivity in analogs 1 to 4. Furthermore, the single substitution of Ala for Met17, Lys21, Ile26, or Asn28 in VIP caused only a 2.5- to 7.3-fold decrease in VPAC2 affinity, with minimal effects on VPAC1 affinity (Igarashi et al., 2002a,b). Likewise, when they were included in the simplified proposed VPAC1-selective agonists (analogs 2–4 and 6), they generally had only a modest effect on increasing VPAC1 selectivity. In contrast to the results with VPAC1 in studies of the VIP pharmacophore for the VPAC2 (Nicole et al., 2000; Igarashi et al., 2002a,b), no single alanine substitution or D-amino acid substitution in VIP resulted in much greater decrease in affinity for the hVPAC1 than the hVPAC2. VIP itself had a 4-fold greater affinity for hVPAC1 than VPAC2. Single alanine substitution for Gln16 or Lys20 in VIP resulted in a 3- to 4-fold greater decrease in affinity for VPAC1 than VPAC2 (Igarashi et al., 2002a,b). However, inclusion of these substitutions in analogs 7 to 9 resulted in only a 3- to 4-fold decrease in affinity of the substituted VIP analogs (analogs 7–9) for VPAC1, with the result these simplified analogs had nearly equal affinity for both hVPAC1 and hVPAC2 and therefore were not selective.
The second goal of this study was to attempt to identify a simplified VIP analog that retained high affinity for each VPAC subtype and that also might be metabolically stable. Such VIP analogs could be particularly useful for imaging of tumors overexpressing VIP receptors or for VIP receptor-directed antitumor treatment. Previous studies (Bryant et al., 1976; Domschke et al., 1978) have demonstrated VIP is rapidly degraded in vivo, having a half-life less than 1 min (Bryant et al., 1976; Domschke et al., 1978) in human It has been proposed (Bryant et al., 1976; Domschke et al., 1978) that VIP is primarily degraded by being cleaved primarily at Ser25-Ile26 or at Thr7-Asp8 residues to yield the major products VIP(1-25) and VIP(26-28) and the minor products VIP(1-7) and VIP(8-28). All of these products are either inactive or have very low affinity for the VPAC receptors (Bolin et al., 1995). Furthermore, another study (O'Donnell et al., 1991) suggests an alanine replacement of Val19 in VIP may increase VIP's resistance to degradation. O'Donnell et al. (1991) investigated the effect of alanine substitutions into the VIP analog Ac-[Lys12, Nle12, Val26, Thr28]VIP on the duration of bronchodilator activity and reported that the substitution of Val19 by an Ala19 extended the duration of effect by more than 17 times. We therefore anticipated that our analogs, which all had with substitutions at positions 8, 19, 25, and/or 26 of VIP, should have enhanced stability. This possibility was further supported by our previous study (Igarashi et al., 2002a) in which we found that the multialaninated analog [Ala2,8,9,11,19,24,25,27,28]VIP (analog 2) was much more resistant than VIP to degradation. A number of our results support the conclusion that we successfully achieved the second aim in the present study. First, in binding studies both analog 7 ([Ala2,8,9,16,19,24]VIP) and analog 8 ([Ala2,8,9,16,19,24,25]VIP) retained high affinity for both VPAC1 and VPAC2. Second, both analogs 7 and 8 were fully efficacious agonists at both VPACs, and each retained high potency for activating each VPAC and stimulating adenylate cyclase activity. Third, radiolabeled analog 8 demonstrated none to minimal degradation by hVPAC1 or hVPAC2 cells, whereas 125I-VIP was degraded >70% by both, demonstrating analog 8 was metabolically stable with cells containing both VPAC subtypes. Furthermore, with VPAC1-containing cells, 125I- [Ala2,8,9,16,19,24,25]VIP (125I-analog 8) was only slightly less metabolically stable than 125I-[Ala2,8,9,11,18,19,24,25,27,28]VIP (analog 2), which was reported previously (Igarashi et al., 2002a) to be much more metabolically stable than VIP with hVPAC1-containing cells.
In conclusion, analyzing the results of studies of the VIP pharmacophore for high-affinity interaction with the hVPAC1 or hVPAC2 (Nicole et al., 2000; Igarashi et al., 2002a,b), we synthesized nine simplified, polyalaninated analogs of VIP to attempt to develop high-affinity VIP analogs that were either selective for one of the two VPAC subtypes or that functioned as high-affinity agonists for each VPAC and that would be metabolically stable. Our results demonstrate that [Ala2,8,9,11,19,22,24,25,27,28]VIP (analog 5) has high affinity and >2000-fold selectivity for hVPAC1 over hVPAC2. No selective VPAC2 agonists were identified. However, [Ala2,8,9,16,19,24,25]VIP (analog 8) had high affinity and potency for both VPAC subtypes and was much more metabolically stable than VIP in cells containing each VPAC subtype. These simplified, metabolically stable analogs should be useful for investigating the role of VPAC1 in biological and pathological processes, for enhanced imaging of tumors overexpressing VIP receptors using VIP receptor scintigraphy (Virgolini, 1997; Thakur et al., 2000, 2004; Rao et al., 2001; Bhargava et al., 2002) as well as for possible VIP receptor-directed antitumor treatment for tumors overexpressing VPACs (Gotthardt et al., 2004; Moody et al., 2004; Ou et al., 2005).
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ABBREVIATIONS: VIP, vasoactive intestinal peptide; VPAC, VIP-PACAP receptors (for nomenclature see Harmar et al., 1998); hVPAC1, human VPAC1 receptor subtype; hVPAC2, human VPAC2 receptor subtype; IBMX, 3-isobutyl-1-methyl xanthine; BSA, bovine serum albumin; PACAP, pituitary adenylate cyclase-activating peptide; DMEM, Dulbecco's modified Eagle's medium; HPLC, high-performance liquid chromatography; Ro 25-1553, Ac-His-Ser-Asp-Ala-Val-Phe-Thr-Glu-Asn-Tyr-Thr-Lys-Leu-Arg-Lys-Gln-Nle-Ala-Ala-Lys-cyclo[Lys-Tyr-Leu-Asn-Asp]-Leu-LysLysGly-Gy-Thr-NH2.
Address correspondence to: Dr. Robert T. Jensen, Digestive Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bldg. 10, Rm. 9C-103, 10 Center Dr. MSC 1804, Bethesda MD 20892-1804. E-mail: robertj{at}bdg10.niddk.nih.gov
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