GLYCOSYLATED PEPTIDES WITH PSEUDOPROLINE RESIDUES AND HAVING ENHANCED HALF-LIVES AND ABILITY TO CROSS THE BLOOD BRAIN BARRIER

Abstract

Glycosylated peptides with glycosyiatiou at or near the C-terminal domain of the peptide have an enhanced ability to cross the blood brain barrier (BBB) and/or enhanced half-lives. These glycosylated peptides may be used as drugs. For example, a PACAP peptide with a C-terminal glycosylation, e.g., in lieu of the terminal leucine, functions as a PAC.sub.1 agonist with enhanced ability to cross the BBB and with enhanced half-life. The peptides can have a pseudoproline residue with glycosylation at or near the C-terminal domain.

Claims

1. A glycopeptide having at least one pseudoproline residue, wherein the glycopeptide is according to formula 1: ##STR00008## wherein A is an oxygen or a sulfur, R.sub.1 is a first peptide chain, R.sub.2 is a second peptide chain or (CO)NH.sub.2, R.sub.3 is a hydrogen or methyl, and R.sub.4 is a glycoside moiety, wherein the pseudoproline residue is formed by hydrogen bonding of an amino functionality to the A being glycosidically bonded to the glycoside moiety, wherein the glycopeptide has an increased ability to cross a blood brain barrier and an increased half life as compared to a version of the glycosylated peptide lacking a glycosylation.

2. The glycopeptide of claim 1, wherein the first peptide chain or second peptide chain is an enkephalin, an endorphin, a dynorphin, a pituitary adenylate cyclase-activating polypeptide (PACAP), an endogenous neuropeptide, a secretin family peptide, an angiotensin-(1-7) peptide, alamandine, or derivatives thereof.

3. The glycopeptide of claim 1, wherein the glycoside moiety is a saccharide.

4. (canceled)

5. The glycopeptide of claim 3, wherein the saccharide is a glucose, a maltose, a xylose, a lactose or a cellobiose.

6. The glycopeptide of claim 1, wherein the glycopeptide is amphipathic.

7. The glycopeptide of claim 1, wherein the glycoside moiety effectuates crossing of the glycopeptide by adsorption and desorption from a biological membrane.

8. The glycopeptide of any of claim 1, wherein the glycopeptide is water-soluble.

9. (canceled)

10. The glycopeptide of claim 1, wherein the glycopeptide is folded along the peptide side chain, wherein charged groups of the glycopeptide are disposed toward one end of the glycopeptide.

11. The glycopeptide of claim 1, wherein the glycopeptide is helical.

12. A method for synthesizing a glycopeptide having at least one pseudoproline residue, wherein a conformation of the glycopeptide is according to formula 1: ##STR00009## wherein A is an oxygen or a sulfur, R.sub.1 is a first peptide chain, R.sub.2 is a second peptide chain or (CO)NH.sub.2, R.sub.3 is a hydrogen or methyl, and R.sub.4 is a glycoside moiety, said method comprising: a. synthesizing a glycosidic bond between a saccharide and a reactive functional group of a carrier amino acid to form a glycosylated amino acid, wherein the carrier amino acid comprises A, R.sub.2, and R.sub.3, wherein the reactive functional group comprises A, and wherein the saccharide comprises R.sub.4; b.incorporating the glycosylated amino acid into an amino acid residue of R.sub.1; and c. effecting ring closure by spontaneously forming a hydrogen bond between a hydrogen atom of an amino group of the amino acid residue and the reactive functional group, A, to form a pseudoproline, thereby forming the properly oriented glycosylated peptide.

13. The method of claim 12, wherein the carrier amino acid is serine, threonine, cysteine, or extended alkyl derivatives thereof.

14. The method of claim 12, wherein the first peptide chain or second peptide chain is an enkephalin, an endorphin, a dynorphin, a pituitary adenylate cyclase-activating polypeptide (PACAP), an endogenous neuropeptide, a secretin family peptide, an angiotensin-(1-7) peptide, alamandine, or derivatives thereof.

15. The method of claim 12, wherein the reactive functional group is an OH or SH.

16. (canceled)

17. The method of claim 12, wherein the saccharide is a glucose, a maltose, a lactose or a cellobiose.

18. The method of claim 12, wherein the glycopeptide is amphipathic.

19. The method of claim 12, wherein the saccharide effectuates crossing of the glycopeptide by adsorption and desorption from a biological membrane.

20. The method of claim 12, wherein the glycopeptide is water-soluble.

21. (canceled)

22. The method of claim 12, wherein the glycopeptide is folded along the side peptide chain such that charged groups of the glycopeptide are disposed on one end of the glycopeptide.

23. The method of claim 12, wherein the glycopeptide is helical.

24-84. (canceled)

85. A composition of a glycopeptide having at least one pseudoproline residue, said composition of the glycopeptide comprising one of the following: ##STR00010## wherein HSDGIFTDSY.sub.10SRYRKQMAVK.sub.20KYLAAV=pituitary adenylate cyclase-activating peptide (PACAP.sub.1-26) (SEQ ID NO: 10); ##STR00011## wherein FTDSY.sub.10SRYRKQMAVK.sub.20KYLAAV=pituitary adenylate cyclase-activating peptide (PACAP.sub.6-26) (SEQ ID NO: 11); ##STR00012## wherein FTDSY.sub.10SRYRKQMAVK.sub.20KYLAAV=pituitary adenylate cyclase-activating peptide (PACAP.sub.6-26) (SEQ ID NO: 11), and lactose=(--D-Glc--D-Gal); ##STR00013## wherein DRVYIH=angiotensin peptide (Ang-(1-6)) (SEQ ID NO: 12); ##STR00014## wherein DRVYIH=angiotensin peptide (Ang-(1-6)) (SEQ ID NO: 12), and lactose=(--D-Glc--D-Gal); ##STR00015## wherein ARVYIH=angiotensin peptide (Ang-(1-6)) (SEQ ID NO: 21); or ##STR00016## wherein ARVYIH=angiotensin peptide (Ang-(1-6)) (SEQ ID NO: 21), and lactose=(--D-Glc--D-Gal).

86-90. (canceled)

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The features and advantages of the present invention will become apparent from a consideration of the following detailed description presented in connection with the accompanying drawings in which:

[0021] FIG. 1 shows a non-limiting example of a pseudoproline in an Angiotensin-(1-7) drug.

[0022] FIG. 2 is a graph of showing differences in H-H exchange rates determined by temperature-dependent 1H-NMR studies.

[0023] FIGS. 3A-3B shows an exemplary schematic of membrane Hopping and BBB

[0024] Transport. As shown in FIG. 3A, glycosylation of Ang1-7 produces a highly amphipathic folded structure that was characterized in silico using published NMR data as a starting point for the final calculated structure. A Solvent Accessible Surface Area was constructed using the MOE software package and the AMBER-99 force field, and colorized red and blue to illustrate the resulting amphipathicity of the folded structure. FIG. 3B shows unfolded coil structures engaging in biousian behavior (reversible adsorption/desorption) in order to undergo transcytosis at the endothelial layer to traverse the BBB (1). Membrane hopping (2) is anticipated to lead to an efficient 2D membrane search for Mas receptors within the brain leading to binding (3) and receptor activation (4).

[0025] FIG. 4 shows how the angle of the glycoside moiety can be altered by the presence of the pseudoproline structure. The orientation of the glycoside moiety with respect to the peptide ligand and the membrane is ideally near 90, although other lesser angles may be acceptable. Some angles are clearly unacceptable, and will interfere with membrane binding and subsequent receptor binding.

[0026] FIG. 5 shows Lactoside PACAP1-26-S-L, a molecular dynamics representation (MOE) of PACAP1-27 that has been modified by replacing the terminal Leucine 27 amide with a Serine lactoside amide (Ser-O--D-Glc--D-Gal). The calculations suggest that the PACAP glycosides can adopt a largely helical conformation.

[0027] FIG. 6 shows MS Fragmentation of Glucoside PACAP.sub.1-27-S-G. Collision induced fragmentation of the 2.sup.+ ion at 1642.7 m/z from mouse brain dialysate carried out using MS/MS. This spectrum shows a clear pattern from the N-terminus in which fragmentation occurs at the aspartic acid residues resulting y.sup.2+ ions. The glycosylated y.sub.19.sup.2+ and y.sub.24.sup.2+ at 1206.0 and 1472.7 m/z respectively, are the predominant ions in the spectrum (NH.sub.3 loss omitted for clarity). -Glc cleavage gave 1560.9 m/z, matching the calculated mass for PACAP.sub.1-7 with a C-terminal amide. Loss of the glucoside along with peptide backbone cleavage resulted in y.sub.19.sup.2+ and y.sub.4.sup.2+ ions at 1124.9 and m/z 1391.6.

[0028] FIGS. 7A-7C show stability of PACAP.sub.1-27, PACAP.sub.1-26-S-G, and PACAP.sub.1-27-S-L. FIG. 7A: PACAP.sub.1-27 and its glycosylated analogues degraded over 30 min in mouse serum at 37 C., as revealed by shotgun FI-MS.sup.n analysis. Data were fitted using a single exponential decay model (R.sup.2>0.71, in all cases). FIG. 7B: Serine glucoside (PACAP.sub.1-27SG, Glc) showed a significant increase in mouse serum t.sub.1/2 in vitro compared to the native peptide PACAP.sub.1-27 and the corresponding lactoside (PACAP.sub.1-27-S-L, Lac) when compared using a 1-way analysis of variance (F2=12.91, p=0.0067, Tukey's multiple comparison Native vs GIc, q=5.760 p<0.05, Lac vs Glc, q=6.602 p<0.5). FIG. 70: CLC-MS.sup.3 allowed for rapid separation and simultaneous quantification of Native, Glc, and Lac PACAP derivatives. The MS.sup.2 fragmentation pattern (MS.sup.2 spectrum) for native PACAP, and the fragmentation patterns (MS.sup.3 spectra) of GIc and Lac are shown. Interestingly, all resulted in the same primary fragment ion at 755 mlz. The specific ion transitions observed: PACAP.sub.1-27 Native 630.fwdarw.755, PACAP.sub.1-27-S-G, GIc 657.fwdarw.625.fwdarw.755, PACAP.sub.1-27-S-L Lac 690.fwdarw.625.fwdarw.755.

[0029] FIG. 8 shows agonist activity of PACAP derivatives at PAC.sub.1. PAC.sub.1-CHO calcium flux activation was measured using FLIPR in response to 11 point concentration curves of PACAP.sub.1-27, the glucoside PACAP.sub.1-27-S-G, and the truncated derivatives (putative antagonists) of PACAP.sub.6-27. Response was measured over 10 minutes, the max-min calculated, and all data was normalized to the maximum response caused by PACAP.sub.1-27 (100%) and vehicle (0%). The meanSEM is shown, using the mean value from each independent experiment. N=3 independent experiments performed, 3 variable non-linear curve fit using Prism. Derived values reported in Table 2. PACAP.sub.1-27 and PACAP.sub.1-27-S-G display potent, efficacious agonist activity.

[0030] FIGS. 9A-9C show antagonist activity of PACAP derivatives at PAC.sub.1. The ability of PACAP.sub.6-27 derivatives to block PACAP.sub.1-27 induced calcium flux was measured using FLIPR. Derived values are reported in Table 2. FIG. 9A: Variable concentration mode antagonist experiments. Concentration curves of PACAP.sub.6-38, PACAP.sub.6-27, and PACAP.sub.6-27 derivatives were added to the cells for 2 minutes, followed by 5 nM of PACAP.sub.1-27. The max-min response was determined, and normalized to the stimulation caused by 5 nM PACAP.sub.1-27 (100%) and vehicle (0%). 3 variable non-linear curve fit (Prism), N=3-4 independent experiments, meanSEM reported. Only PACAP.sub.6-38 shows antagonism, but it is low potency. FIG. 9B: Fixed concentration mode experiments with PACAP.sub.6-38. Fixed concentrations of PACAP.sub.6-38 added to cells for 2 minutes, followed by concentration curves of PACAP.sub.1-27. The max-min response was determined, and normalized to the max response of the PACAP.sub.1-27 curve without antagonist present (100%) and vehicle (0%). Gaddum/Schild EC.sub.50 Shift model (Prism), N=3 independent experiments. PACAP.sub.6-38 shifts the curve only at the highest concentration (1 M). FIG. 9C: Fixed concentration mode experiments with PACAP.sub.6-27, performed as in B. N=3 independent experiments. PACAP.sub.6-27 showed no detectable shifts in the agonist curves.

[0031] FIG. 10 shows PC12 cell morphology after vehicle vs PACAP treatment (100 nM). Upper Left: diluent only. Upper Right: PACAP.sub.1-27, Lower Left: PACAP.sub.1-27-S-G, Lower Right: PACAP.sub.1-27-S-L. The cell body volumes all showed increases when treated with each of the PACAP derivatives in Table 1. In all cases the process outgrowths on the treated cells were greater than 2X the cell body width.

DESCRIPTION OF PREFERRED EMBODIMENTS

[0032] As used herein, the term pseudoproline is defined as a proline-mimicking residue. The pseudoproline comprises a transient 5-membered ring having a secondary amine.

[0033] As used herein, the term glycoside is defined a molecule formed by a carbohydrate or a saccharide bound to another reactive functional group via a glycosidic bond, which is a covalent bond formed between the hemiacetal group of the carbohydrate and the reactive functional group, such as the hydroxyl group, of another compound.

[0034] As used herein, the term serine encompasses serine in either its L- or D-configuration, as well as racemate or in various mixtures of its isomers or extended alkyl derivatives. As used herein, the term threonine encompasses threonine in either its L- or D-configuration, as well as racemate or in various mixtures of its isomers or extended alkyl derivatives. As used herein, the term cysteine encompasses cysteine in either its L- or D-configuration, as well as racemate or in various mixtures of its isomers or extended alkyl derivatives.

Glycopeptides with Pseudoproline

[0035] Referring now to FIG. 14, the present invention features a glycopeptide having at least one pseudoproline residue. The glycopeptide may be according to formula 1:

##STR00001##

[0036] In some embodiments, A is an anomeric oxygen or a sulfur, R.sub.1 is a first peptide chain, R.sub.2 is a second peptide chain or (CO)NH.sub.2, R.sub.3 is a hydrogen or methyl, and R.sub.4 is a glycoside moiety. Surprisingly, a pseudoproline residue is formed by hydrogen bonding of an amino functionality to the A being glycosidically bonded to the glycoside moiety. In some preferred embodiments, the glycopeptide has an increased ability to cross a blood brain barrier (BBB) as compared to a version of the glycopeptide lacking the glycosylation.

[0037] In some embodiments, the glycoside moiety can be a saccharide, such as a mono-, di-, tri- or poly-saccharide. Non-limiting examples of which include glucose, maltose, lactose, cellobiose, or any other saccharide as known to one of ordinary skill in the art. Moreover, the glycoside moiety may further include and forms of the saccharides. In other embodiments, the side peptide chain can be an enkephalin, an endorphin, a dynorphin, a vasoactive intestinal peptide (VIP), a pituitary adenylate cyclase-activating polypeptide (PACAP), an endogenous neuropeptide, a secretin family peptide, an angiotensin-(1-7) peptide and derivatives thereof, or any other peptide. For example, the glycopeptide may be a hormone, an agonist, or an antagonist that can bind to a target receptor.

[0038] Another embodiment of the present invention may feature a method for synthesizing a glycopeptide having at least one pseudoproline residue. In one embodiment, the glycopeptide may be according to formula 1:

##STR00002##

wherein A is an anomeric oxygen or a sulfur, R.sub.1 is a first peptide chain, R.sub.2 is a second peptide chain or (CO)NH.sub.2, R.sub.3 is a hydrogen or methyl, and R.sub.4 is a glycoside moiety.

[0039] In some embodiments, the method may comprise synthesizing a glycosidic bond between a saccharide and a reactive functional group of a carrier amino acid to form a glycosylated amino acid, incorporating the glycosylated amino acid into an amino acid residue of R.sub.1, and effecting ring closure by spontaneously forming a hydrogen bond between an amino group of the amino acid residue and the reactive functional group to form a pseudoproline, thereby forming the glycosylated peptide. In some embodiments, the carrier amino acid may comprise R.sub.2, R.sub.3, and the reactive functional group comprising A. For example, the reactive functional group may be an OH or SH. In other embodiments, the saccharide may comprise R.sub.4.

[0040] In some embodiments, the carrier amino acid is serine, threonine, cysteine, or extended alkyl derivatives thereof. Examples of the carrier amino acid include, but are not limited to, Serine, D-Serine, Threonine, D-Threonine, allo-L-Threonine, allo-D-Threonine, Cysteine, D-Cysteine. In other embodiments, the first peptide chain or second peptide chain is an enkephalin, an endorphin, a dynorphin, a vasoactive intestinal peptide (VIP), a pituitary adenylate cyclase-activating polypeptide (PACAP), an endogenous neuropeptide, a secretin family peptide, an angiotensin-(1-7) peptide and derivatives thereof, or any other peptide. In further embodiments, the saccharide may be a mono-, di-, tri- or poly-saccharide. Non-limiting examples of which include glucose, maltose, lactose, cellobiose, or any other saccharide as known to one of ordinary skill in the art. Moreover, the glycoside moiety may further include and forms of the saccharides.

[0041] Embodiments of the glycopeptides described herein can have diverse biological functions. For example, the glycopeptide may be used for binding to a target receptor or for drug delivery. In some embodiments, the glycopeptide is amphipathic such that the glycopeptides can be water-soluble or membrane-bound. As used herein, the term amphipathic encompasses a molecule that has charged or highly polarized atoms bonds in the polar head, which is hydrophilic, and non-polar atoms and bonds in the tail, which are hydrophobic. As an example, a molecule can have a negatively or positively charged group or another water-soluble moiety, such as a sugar, in the polar head, which is hydrophilic, and non-polar atoms and bonds in the tail, which are hydrophobic. In one embodiment, the glycopeptide is folded along the side peptide chain such that charged groups of the glycopeptide are disposed on one end of the glycopeptide. In another embodiment, the glycopeptide is helical such that one surface of the helical glycopeptide has hydrophilic amino acids and the opposite face has hydrophobic (or lipophilic) amino acids. Preferably, the glycoside moiety, or saccharide, can effectuate crossing of the glycopeptide by adsorption and desorption from a biological membrane.

[0042] In some embodiments, the glycopeptides described herein may have various lengths. For example, in some embodiments, the glycopeptide is at least 5 amino acids in length. In other embodiments, the glycopeptide is at least 7 amino acids in length, at least 10 amino acids in length, at least 20 amino acids in length, or greater that 20 amino acids in length. In still further embodiments, the glycopeptide is not limited to the aforementioned lengths.

Glycosylated Peptides

[0043] Referring now to FIG. 5-10, the present invention features glycosylated peptides with glycosylation at or near the C-terminus. The glycosylated peptides have enhanced ability to cross the blood brain barrier (BBB) and/or enhanced half-lives. These glycosylated peptides may be used as drugs. For example, Pituitary Adenylate Cyclase Activating Peptide (PACAP) is a neuronal survival factor that acts as an agonist at the receptors PAC.sub.1, VPAC.sub.1, and/or VPAC.sub.2. A PACAP peptide with a C-terminal glycosylation, e.g., in lieu of the terminal leucine, functions as a PAC.sub.1 agonist with enhanced ability to cross the blood brain barrier and with enhanced half-life (without affecting receptor activation) (see Example 1). PACAP, as well as Vasoactive Intestinal Peptide (VIP) are closely related peptides from the Secretin Family, and are also survival factors that may be glycosylated in a similar fashion. The present invention also features PACAP and VIP antagonists.

[0044] As previously discussed, the present invention features glycosylated peptides, wherein at least one amino acid is glycosylated with a glycan. The peptides may be of various lengths. For example, in some embodiments, the peptide is at least 4 amino acids in length; or at least 5 amino acids in length; or at least 8 amino acids in length; or at least 10 amino acids in length; or at least 15 amino acids in length.

[0045] In some embodiments, the peptide is at least 20 amino acids in length; or at least 30 amino acids in length; or at least 40 amino acids in length; or at least 50 amino acids in length, or at least 60 amino acids in length. In other embodiments, the peptide is from 15 to 100 amino acids in length; or from 25 to 100 amino acids in length; or from 25 to 150 amino acids in length; or from 50 to 100 amino acids in length; or from 50 to 150 amino acids in length. The present invention is not limited to the aforementioned lengths.

[0046] In some embodiments, two or more amino acids are glycosylated with a glycan. In some embodiments, three or more amino acids are glycosylated with a glycan.

[0047] The C-terminal domain of the peptide may be defined by a group of amino acids at the C-terminus of the peptide. The C-terminal domain (e.g., the number of amino acids at the C-terminus of the peptide) may vary depending on the peptide. For example, in some embodiments, the C-terminal domain comprises the last amino acid at the C-terminus of the peptide. In some embodiments, the C-terminal domain comprises the last 2 amino acids at the C-terminus of the peptide. In some embodiments, the C-terminal domain comprises the last 3 amino acids at the C-terminus of the peptide. In some embodiments, the C-terminal domain comprises the last 4 amino acids at the C-terminus of the peptide. In some embodiments, the C-terminal domain comprises the last 5 amino acids at the C-terminus of the peptide. In some embodiments, the C-terminal domain comprises the last 6 amino acids at the C-terminus of the peptide. In some embodiments, the C-terminal domain comprises the last 7 amino acids at the C-terminus of the peptide. In some embodiments, the C-terminal domain comprises the last 8 amino acids at the C-terminus of the peptide. In some embodiments, the C-terminal domain comprises the last 9 amino acids at the C-terminus of the peptide. In some embodiments, the C-terminal domain comprises the last 10 amino acids at the C-terminus of the peptide. In some embodiments, the C-terminal domain comprises the last 15 amino acids at the C-terminus of the peptide. The C-terminal domain is not limited to the aforementioned number of amino acids at the C-terminus; in some embodiments, the C-terminal domain comprises the last 11, 12, 13, 14, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, etc. amino acids at the C-terminus of the peptide.

[0048] Glycosylation processes and glycans are well known to one of ordinary skill in the art. In some embodiments, the glycan is branched. In some embodiments, the glycan is unbranched. In some embodiments, the glycan is an N-linked glycan. In some embodiments, the glycan is an O-linked glycan. In some embodiments, the glycan is a C-linked glycan. In some embodiments, the glycan is an S-linked glycan. In some embodiments, the glycan is a phospho-serine glycan.

[0049] In some embodiments, the glycan comprises glucose. In some embodiments, the glycan comprises lactose. In some embodiments, the glycan comprises maltose. In some embodiments, the glycan comprises cellobiose. In some embodiments, the glycan comprises melibiose. In some embodiments, the glycan comprises linear or branched trisaccharides of glucose. In some embodiments, the glycan comprises N-acetylglucosamine. In some embodiments, the glycan comprises galactose. In some embodiments, the glycan comprises N-acetylgalactosamine. In some embodiments, the glycan comprises galactosamine. In some embodiments, the glycan comprises glucosamine. In some embodiments, the glycan comprises mannose. In some embodiments, the glycan comprises N-acetylmannosamine. In some embodiments, the glycan comprises mannosamine. In some embodiments, the glycan comprises xylose. In some embodiments, the glycan comprises N-acetylneuraminic acid. In some embodiments, the glycan comprises N-glycolylneuraminic acid. In some embodiments, the glycan comprises 2-keto-3-deoxynononic acid. In some embodiments, the glycan comprises fucose. In some embodiments, the glycan comprises iduronic acid. In some embodiments, the glycan comprises glucuronic acid. In some embodiments, the glycan comprises cellobiose. In some embodimetns, the glycan comprises melibiose. In some embodiments, the glycan comprises rhamnose. The present invention is not limited to the aforementioned glycans. For example, the glycan of the present invention may be selected from all mono-, di-, tri- and poly-saccharides.

[0050] In some embodiments, the glycosylated peptide has an increased ability to cross a blood brain barrier (BBB) as compared to a version of the glycosylated peptide lacking the glycosylation. In some embodiments, the glycosylated peptide has an increased half-life as compared to a version of the glycosylated peptide lacking the glycosylation.

[0051] In some embodiments, an amino acid in the C-terminal domain (of the peptide) is substituted with a different amino acid, the different amino acid is adapted to be glycosylated, the different amino acid being the amino acid glycosylated with the glycan. In some embodiments, a different amino acid (e.g., a glycosylated amino acid) is appended to the C-terminus of a peptide. The different amino acid may be selected from the group consisting of: serine, threonine, cysteine, tyrosine, hydroxylysine, hydroxyproline, asparagine, arginine, or tryptophan, and any reasonable alkyl derivative thereof.

[0052] Referring to Example 1 below, in some embodiments, the glycosylated peptide is a PAC.sub.1 agonist. The PAC.sub.1 agonist may comprise a PACAP peptide, e.g., a PACAP peptide wherein an amino acid in the C-terminal domain is glycosylated with a glycan. In some embodiments, the terminal leucine residue is substituted with a serine (the serine being the amino acid glycosylated with the glycan). As such, the present invention features a PAC.sub.1 agonist comprising a PACAP peptide, wherein an amino acid in a C-terminal domain of the PACAP peptide is glycosylated with a glycan. Again referring to Example 1, in some embodiments, the glycosylated peptide (e.g., the PAC, agonist) is selected from the group consisting of:

TABLE-US-00001 (SEQIDNO:1) HSDGIFTDSY.sub.10SRYRKQMAVK.sub.20KYLAAVL; (SEQIDNO:2) HSDGIFTDSY.sub.10SRYRKQMAVK.sub.20KYLAAVS-O--D-Glc; (SEQIDNO:3) FTDSY.sub.10SRYRKQMAVK.sub.20KYLAAVL; (SEQIDNO:4) FTDSY.sub.10SRYRKQMAVK.sub.20KYLAAVLGKR.sub.30YKQRVKNY; (SEQIDNO:5) FTDSY.sub.10SRYRKQMAVK.sub.20KYLAAVS-O--D-Glc; (SEQIDNO:6) FTDSY.sub.10SRYRKQMAVK.sub.20KYLAAVS-O--D-Glc--D-Gal; (SEQIDNO:7) FTDSY.sub.10SRYRRQLAVR.sub.20RYLAAVL; (SEQIDNO:8) FTDSY.sub.10SRYRRQLAVR.sub.20RYLAAVS-O--D-Glc; and (SEQIDNO:9) FTDSY.sub.10SRYRRQLAVR.sub.20RYLAAVS-O--D-Glc--D-Gal;

[0053] In some embodiments, the PAC.sub.1 agonist (e.g., the PAC.sub.1 agonist comprising a PACAP peptide wherein an amino acid in a C-terminal domain of the PACAP peptide is glycosylated with a glycan) has an increased ability to cross a blood brain barrier (BBB) as compared to a version of the glycosylated peptide lacking the glycosylation. In some embodiments, the PAC.sub.1 agonist (e.g., the PAC; agonist comprising a PACAP peptide wherein an amino acid in a C-terminal domain of the PACAP peptide is glycosylated with a glycan) has an increased half-life as compared to a version of the glycosylated peptide lacking the glycosylation.

[0054] Another embodiment of the present invention may feature a composition of a glycopeptide having at least one pseudoproline residue. The composition may comprise a structure as follows:

##STR00003##

wherein HSDGIFTDSY.sub.10SRYRKQMAVK.sub.20KYLAAV=pituitary adenylate cyclase-activating peptide (PACAP.sub.1-26) (SEQ ID NO: 10).

[0055] In another embodiment, the present invention may feature a composition of a glycopeptide having at least one pseudoproline residue. The composition may comprise a structure as follows:

##STR00004##

wherein FTDSY.sub.10SRYRKQMAVK.sub.20KYLAAV=pituitary adenylate cyclase-activating peptide (PACAP.sub.6-26) (SEQ ID NO: 11).

[0056] A further embodiment of the present invention may feature a composition of a glycopeptide having at least one pseudoproline residue. The composition may comprise a structure as follows:

##STR00005##

wherein FTDSY.sub.10SRYRKQMAVK.sub.20KYLAAV=pituitary adenylate cyclase-activating peptide (PACAP.sub.6-26) (SEQ ID NO: 11), and lactose=(--D-Glc--D-Gal).

[0057] In another embodiment, the present invention may feature a composition of a glycopeptide having at least one pseudoproline residue. The composition may comprise a structure as follows:

##STR00006##

wherein DRVYIH=angiotensin peptide (Ang-(1-6)) (SEQ ID NO: 12).

[0058] A composition of a glycopeptide having at least one pseudoproline residue. The composition may comprise a structure as follows:

##STR00007##

wherein DRVYIH=angiotensin peptide (Ang-(1-6)) (SEQ ID NO: 12), and lactose=(--D-Glc--D-Gal).

[0059] The present invention also features methods of use of said glycosylated peptides as well as various other methods involving said glycosylated peptides. For example, the present invention features a method of enhancing a peptide's ability to cross a blood brain barrier (BBB). The method may comprise introducing glycosylation to an amino acid in a C-terminal domain of the peptide (e.g., as described herein). The present invention also features a method of enhancing a peptide's half-life. In some embodiments, the method comprises introducing glycosylation to an amino acid in a C-terminal domain of the peptide. For example, in some embodiments, the method comprises forming the glycan-amino acid conjugate and including that conjugate in standard peptide synthesis conditions. In some embodiments, the carbohydrate is introduced synthetically.

[0060] In some embodiments, the method of enhancing the peptide's ability to cross the BBB and/or the method of enhancing a peptide's half-life further comprises substituting an amino acid in the C-terminal domain with a different amino acid adapted to be glycosylated (the different amino acid being the amino acid that is glycosylated with the glycan). As previously discussed, in some embodiments, the different amino acid is selected from the group consisting of: serine, threonine, cysteine, tyrosine, hydroxylysine, hydroxyproline, asparagine, arginine, or tryptophan, or derivatives thereof.

[0061] In some preferred embodiments, any of the glycopeptide or glycosylated peptides described herein can be cyclized either in solution or while still on a resin using amide bonds (OCN), disulfide bonds (SS), or olefins (CC, stapling).

EXAMPLE 1

[0062] Example 1 describes the study of peptides and O-linked glycopeptides related to Pituitary Adenylate Cyclase Activating Peptide (PACAP) for their potential as agonist neuroprotective agents and antagonist anti-migraine agents. Briefly, activation of human PAC.sub.1 receptors expressed in Chinese Hamster Ovary (CHO) cells was observed for PACAP.sub.1-27, as well as its glucoside analogue (PACAP.sub.1-27-S-G) with similar potency and efficacy. The glucoside (PACAP.sub.1-27-S-G) showed enhanced stability in the presence of mouse serum, and its presence in mouse brain could be detected after i.p. administration by flow-injection tandem mass spectrometry (MS.sup.3). Both the glucoside and the lactoside derivatives promoted differentiation of pheochromocytoma cells (PC12 cells) grown in culture, suggesting endogenous PAC.sub.1 agonism. In addition glycosylated derivatives of PACAP.sub.6-27 were tested as potential PAC.sub.1 antagonists for the treatment of migraine. These compounds did not display antagonist activity, yet neither did the putative PAC.sub.1 antagonists PACAP.sub.6-27 and PACAP.sub.6-38, suggesting that another target or mechanism may be involved. The present invention is not limited to the compounds, configurations, methods, and systems described in Example 1.

[0063] Methods

[0064] 1. Peptide and Glycopeptide Synthesis

[0065] Unless otherwise noted, all solvents were obtained from EMD Chemicals (Gibbstown, N.J.), and used without further purification. Fmoc-protected amino acids Fmoc-Ala-OH, Fmoc-Lys(Boc)-OH, Fmoc-Leu-OH, Fmoc-Met-OH. Fmoc-Tyr(tBu)OH, and condensing agent N,N-diisopropylcarbodiimide (DIC) were acquired from Advanced ChemTech (Louisville, Ky., USA). Fmoc-Asp(tBu)OH was acquired from Oxchem Corporation (Los Angeles, Calif., USA). Fmoc-Phe-OH, Fmoc-Gly-OH, Fmoc-Ile-OH, Fmoc-Gln(Trt)OH, Fmoc-Ser(tBu)OH, and Fmoc-Val-OH were acquired from Chem-Impex (Wood Dale, Ill., USA). Fmoc-His(Trt)OH, Fmoc-Arg(Pbf)OH, Fmoc-Thr(tBu)OH, Fmoc-Nle-OH, and Hydroxybenzotriazole hydrate (HOBt.H.sub.2O) were obtained from AAPPTec (Louisville, Ky., USA). 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) from TCI America (Portland, Oreg., USA), piperidine from Sigma-Aldrich (St. Louis, Mo., USA), piperizine from Alfa Aesar (Ward Hill, Mass., USA), 0-(7-Azabenzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphate (HATU) from Accela ChemBlo Co., Ltd (Hong Kong, China), 2,4,6-trimethylpyridine (TMP) from Acros Organics (Geel, Belgium), and 1-Methyl-2-pyrrolidinone from Avantor (Center Valley, Pa., USA). PACAP.sub.6-38 was obtained from commercial sources (Tocris, Bristol, UK).

[0066] Peptide and glycopeptide assembly was accomplished using a combination of manual and automated peptide methods. A general procedure follows: The C-terminal amino acids were loaded onto Fmoc-Rink resin (Advanced ChemTech, Louisville, Ky., USA) at 0.1 mmol/g resin loading in 25 mL fritted syringes. Initially, the resin was swelled using dimethylformamide (DMF, 5 mL solvent per gram resin), agitating at RT for two minutes (2). A solution of 2% DBU and 3% piperidine in DMF (v:v) was introduced and agitated for 5 minutes, refreshed, and agitated for an additional 10 minutes. The resin was washed with DMF (5), and finally with N-methylpyrrolidine (NMP). In a separate vial, Fmoc--OGlc(OAc).sub.4-Ser-OH (0.12 mmol, 1.2 eq) was dissolved in 5 mL NMP, and HOBt.H.sub.2O (0.13 mmol, 1.3 eq) was added and allowed to mix for 5 minutes. Condensing agent DIC (0.26 mmol, 2.6 eq) was then added, and mixed for 5 minutes. This solution was added to the resin and agitated for 10 minutes. Next, the syringe was placed in a microwave (Emerson 900 W MicrowaveMW9338SB) set to power level 1 and irradiated for 10 minutes, stopping to shake the syringe every 90 seconds. The syringe was then agitated at RT for an additional 30 minutes. The resin was washed with NMP (1), DMF (5), and CH.sub.2Cl.sub.2 (5), and dried in vacuo overnight.

[0067] Peptides and glycopeptides also were assembled on a Prelude Peptide Synthesizer (Protein Technologies, Inc., Tucson, Ariz., USA) using the reaction scheme that follows: Rink resin (100 mg) was placed into the fritted reaction vessels (RVs). Amino acids were dissolved in DMF at 250 mM concentration, HATU at 375 mM, and TMP at 3M. The following steps were performed for coupling: DMF Top Wash (1.5 mL, 2 min mix and drain; 6), Deprotection (2% DBU/3% piperidine in DMF; 1.5 mL, 4 min mix and drain; 8 min mix and drain), DMF Top Wash (1.5 ml, 2 min mix and drain; 5), Amino Acid Building Block (0.950 mL, 30 sec mix), Activator 1 (HATU, 0.650 mL, 30 sec mix), Base (TMP, 0.300 mL, 35 min mix and drain), DMF Top Wash (1.5 mL, 2 min mix and drain; x2). After coupling aspartic acid D7, the deprotection solution was changed to 0.1 M HOBt.H.sub.2O/5% piperazine in DMF to minimize aspartimide formation.

[0068] Cleavage of the peptides and glycopeptides from the resin was accomplished with a TFA cocktail of F.sub.3CCOOH:Et.sub.3SiH:H.sub.2O:CH.sub.2Cl.sub.2:Ph-OCH; (by volume, 9:0.3:0.2:1:0.05), agitating at RT for 2 hours. The resulting solutions were expelled into 15 mL centrifuge tubes, evaporated under argon, precipitated in ice-cold Et.sub.2O, decanted, and rewashed with Et.sub.2O, then dissolved in H.sub.2O and lyophilized to afford the crude material as fluffy white solids.

[0069] Purification of the crude glycopeptides was accomplished by Reversed Phase HPLC (RP-HPLC) with a preparative RP (C-18) Phenomenex (25022 mm) column using a CH.sub.3CNH.sub.2O gradient solvent system containing 0.1% F.sub.3CCOOH. Homogeneity of the purified glycopeptides was confirmed by analytical RP-HPLC and high resolution mass spectrometry.

[0070] 2. Stability Studies in Mouse Serum by MS.sup.n

[0071] Flow-injection tandem mass spectrometry (FI-MS.sup.n) was used to observe the degradation of the peptides and glycopeptides with a Thermo LCQ with electrospray ionization (ESI). The technique involved injection of a sample bolus of material in mouse serum via a six port valve with fluid flow delivered via a syringe pump, and subsequent electrospray ionization (ESI) followed by mass spectral analysis. Samples were diluted to a concentration of 5 M of each PACAP analogue, and were incubated at 37 C. for times varying from 1 to 60 minutes. After samples had been incubated for the prescribed amount of time they were prepared for mass spectrometry analysis by withdrawing 10 microliters of solution and spiking with 1 microliter of a 10 M solution of peptide internal standard (angiotensin II) in 50% acetic acid and subjecting them to a standard C18 zip tip desalting. These solutions, once eluted from the zip tip were diluted to 100 L in 50:50 acetonitrile/water with 0.1% formic acid. Tandem mass spectrometry analysis (MS.sup.3) was conducted to yield specific, quantitative signals proportional to the amount of PACAP analogue at each time point. This technique was also used with microdiasylate samples from a mouse after i.p. administration of PACAP1-27-S-G.

[0072] 3. PAC.sub.1 Cell Line and Cell Culture

[0073] A custom DNA clone of the human PAC1 gene with 3 hemagglutinin (HA) tags inserted 3 to the signal peptide sequence (to avoid proteolytic loss) was obtained from Genecopoeia (Rockville, Md.). The construct was electroporated into Chinese Hamster Ovary (CHO) cells, and selected for with 500 g/mL of G418. The resulting population was screened for high expressing clones, and one such clone selected for further analysis. The clonal cell line (PAC .sub.1-CHO) displayed high receptor expression by immunocytochemistry and Western blot, and showed selective activation of signaling in response to PACAP.sub.1-27 (data not shown). This cell line was used for all molecular pharmacology experiments. The cells were maintained in DMEM/F12 with 10% heat-inactivated FBS, 1X penicillin/streptomycin, and 500 g/mL G418, at 37 C. and 5% CO.sub.2.

[0074] 4. Fluorescent Imaging Plate Reader (FLIPR) Experiments

[0075] All molecular pharmacology experiments were carried out using a FLIPR Tetra from Molecular Devices (Sunnyvale, Calif.), set to image calcium flux using the manufacturer's recommended settings and protocols. The day before an experiment, the PAC.sub.1-CHO cells were split into 384 well black walled, clear bottom microplates, 10,000 cells per well. The cells were recovered overnight in growth medium (as above). The next day, the growth medium was replaced with Calcium 6 dye (Molecular Devices) using the manufacturer recommended buffer with 2.5 mM probenecid. The cells were incubated for 2 hours in the culture incubator, and removed during the last 15 minutes to allow equilibration to room temperature. Compound as indicated below was added to the cells using a 384 tip block, with real time monitoring before, during, and 15 minutes after compound addition. The resulting calcium flux was recorded, and the maximum-minimum response over the entire observation time calculated and reported as the meanSEM (4 wells per point).

[0076] For agonist mode experiments, compound was added in an 11 point concentration curve, with a vehicle control (buffer). The resulting response was normalized to the stimulation caused by PACAP.sub.1-27 (100%) and vehicle (0%). The response was analyzed using a 3 variable non-linear curve fit, and the EC.sub.50 (nM) and E.sub.Max (%) calculated and reported (Prism, GraphPad, La Jolla, Calif.).

[0077] For antagonist mode experiments, a concentration curve (variable concentration mode) or fixed amount (fixed concentration mode) of antagonist was added to the cells, and allowed to equilibrate for 2 minutes. Then, either a 5 nM fixed concentration (variable concentration mode) or an 11 point concentration curve (fixed concentration mode) of PACAP.sub.1-27 was added to the cells, and the max-min response recorded as above. For variable concentration mode experiments, the data was normalized to the stimulation caused by 5 nM PACAP.sub.1-27 (100%) and vehicle (0%), and analyzed with a 3 variable non-linear curve fit, with the IC.sub.50 (nM) and I.sub.Max (%) calculated and reported (Prism). For the fixed concentration mode experiments, each curve was normalized to the maximum stimulation caused by PACAP.sub.1-27 with no antagonist present (100%) and vehicle (0%). The resulting data was analyzed using a Gaddum/Schild EC.sub.50 shift model, (Schild, 1957, Gaddum, 1957) designed to analyze competitive antagonism. The data output was the pA2 (nM) and the Schild Slope, a measure of how closely the experimental data fits the operational model of competitive antagonism (Prism). For all analyses, each independent experiment performed in quadruplicate is considered to be a sample size of 1. The pharmacology values are calculated separately from each experiment, then combined and reported as the meanSEM for the entire set of experiments.

[0078] 5. Effects on Differentiation of Cultured PC12 Cells

[0079] The PC12 cells were cultured in RPMI containing 5% heat inactivated fetal bovine serum and 10% horse serum in the presence of 100 units/mL penicillin and 100 microgram/mL streptomycin. The cells were plated on poly-D-Lysine coated 6-well tissue culture plates at a density of 150,000 cells per well in 2 mL media. After 48 hours at 37 C. in 5% CO.sub.2 atmosphere, media exchange was performed and plates were dosed, using the peptide diluent (water) for the control samples. PACAP.sub.1-27, PACAP.sub.1-27-S-G, and PACAP.sub.1-27-S-L were used to screen for PAC.sub.1 receptor activation. Four groups of cells were used; one control group (diluent treated) and three treatment groups, each treatment group was exposed to 100 nM concentrations of PACAP.sub.1-27, PACAP.sub.1-27-S-G, or PACAP.sub.1-27-S-L. All groups were run in triplicate. Cell images of each treatment group were captured and compared to the control cells to screen for differentiation and cell body volume increases. Cells having neurite-like process outgrowth were noted and photographed. The neurite-like outgrowth was deemed positive if its length was at least two times the width of the cell body.

[0080] Results

[0081] 1. Synthesis of PACAP Derivatives

[0082] PACAP derivatives and glycosides were synthesized on a small scale (5 mg) using solid-phase methods previously described (Poll, et al., General Methods for - or -O-Ser/Thr Glycosides and Glycopeptides. Solid-Phase Synthesis of O-Glycosyl Cyclic Enkephalin Analogues. J. Am. Chem. Soc. 114, 10249-10258 (1992); Mitchell, et al., Solid-Phase Synthesis of O-Linked Glycopeptide Analogues of Enkephalin. J. Org. Chem. 66, 2327-2342 (2001)), For example, see FIG. 5 and Table 1 (below). Recent advances in the synthesis of Fmoc-protected glycosides of Serine and Threonine using minimally competent Lewis acid promoters allows for production of required glycosides in high yield and purity, which in turn provides either linear or cyclic variants of O-linked glycopeptides.

[0083] Table 1 shows peptides and glycopeptides in the present invention. The truncated peptides and glycopeptides are missing five N-terminal amino acids responsible for binding to the transmembrane portion of the GPCR receptors and were considered to be antagonists. For the glycosides, Leucine 27 has been replaced (underlined residues) by a Serine glycoside bearing glucose (--D-Glc) or lactose (--D-Glc--D-Gal). The final 3 alternate compounds have been modified by replacing methionine 17 with Leucine, and Lysines 15, 20 and 21 with Arginines to enhance stability in vivo, and were expected to be antagonists at PAC.sub.1.

TABLE-US-00002 TABLE1 Peptides\ SEQ Glycopeptides AminoAcidSequence(C-TerminalAmides) IDNO: MW PACAP1-27 HSDGIFTDSY.sub.10SRYRKQMAVK.sub.20KYLAAVL 1 3,938 PACAP1-27-S-G HSDGIFTDSY.sub.10SRYRKQMAVK.sub.20KYLAAVS-O-- 2 4,242 D-Glc PACAP6-27 FTDSY.sub.10SRYRKQMAVK.sub.20KYLAAVL 3 3,316 PACAP6-38 FTDSY.sub.10SRYRKQMAVK.sub.20KYLAAVLGKR.sub.30YK 4 4,534 QRVKNY PACAP6-27-S-G FTDSY.sub.10SRYRKQMAVK.sub.20KYLAAVS-O--D- 5 3,452 Glc PACAP6-27-S-L FTDSY.sub.10SRYRKQMAVK.sub.20KYLAAVS-O--D- 6 3,614 Glc--D-Gal PACAP6-27-Alt FTDSY.sub.10SRYRRQLAVR.sub.20RYLAAVL 7 3,382 PACAP6-27-S-G- FTDSY.sub.10SRYRRQLAVR.sub.20RYLAAVS-O--D- 8 3,518 Alt Glc PACAP6-27-S-L- FTDSY.sub.10SRYRRQLAVR.sub.20RYLAAVS-O--D- 9 3,681 Alt Glc--D-Gal

[0084] 2. Stability in Mouse Serum

[0085] Chemical stability of the glycopeptides in vivo clearly plays an important role in the deliverability of the drugs to the site(s) of action within the brain. It is also important to know what the chemical or metabolic instabilities are in order to inform the drug design process. Tandem mass spectroscopy (MS.sup.n) was used to determine both the stability of the PACAP compounds in mouse serum, and to identify specific cleavage products, which can stem from inherent chemical instability, or from enzymatic hydrolysis, as shown in FIG. 6 and FIG. 7.

[0086] 3. PAC.sub.1 Agonist Activity of PACAP.sub.1-27, PACAP.sub.6-27 and derivatives, and PACAP.sub.1-27-S-G

[0087] Using CHO cells that express human PAC.sub.1 receptors, PACAP.sub.1-.sub.27, the glucoside PACAP.sub.27-.sub.s-.sub.G, and the truncated putative antagonist PACAP.sub.6-27 and its derivatives were tested as agonists using FLIPR. It was found that PACAP.sub.1-27 and PACAP.sub.1-27-S-G, the serine glucoside, activated PAC.sub.1 with high potency (0.950.4 nM and 5.682.3 nM respectively, see FIG. 8, Table 2). These values are both very similar to what has been reported for PACAP.sub.1-27. In addition, the normalized efficacy of the PACAP.sub.1-27-S-G glucoside was nearly identical to the native PACAP.sub.1-27 peptide, at 101.91.6%.. These findings suggest that glycosylation of PACAP.sub.1-27 does not significantly alter binding and activation of the PAC.sub.1 receptor, supporting the use of such a glycopeptide for therapeutic purposes. None of the PACAP.sub.6-27 derivatives showed agonist activity at concentrations up to 1 M (see FIG. 8, Table 2).

[0088] 4. PACs Antagonist Activity by PACAP Derivatives

[0089] In addition to the agonist studies above, the ability of glycosylated and non-glycosylated PACAP.sub.6-27 derivatives to block activation of the PAC.sub.1 receptor by PACAP.sub.1-27 was tested. A variable concentration mode antagonist assay was used versus 5 nM of PACAP.sub.1-27 in the PAC.sub.1-CHO cells using FLIPR. Surprisingly, no antagonist activity of PACAP.sub.6-27 or any derivative up to 1 M (see FIG. 9, Table 2) was not detected. In addition, the known antagonist PACAP.sub.6-38 was tested, and only low potency antagonism was be detected (>333 nM, FIG. 9A and Table 2). These findings are at odds with a molecular pharmacology study of PACAP.sub.6-38 and PACAP.sub.6-27 with reported K.sub.i values of 1.5 and 60 nM, respectively (Robberecht et al., Structural requirements for the occupancy of pituitary adenylate-cyclase-activating peptide (PACAP) receptors and adenylate cyclase activation in human neuroblastoma NB-OK-1 cell membranes. Discovery of PACAP6-38 as a potent antagonist. Eur. J. Biochem. 1992, 207, 239-246).

[0090] However, one further difference with Robberecht el al. remained, which was the use of fixed concentration antagonist mode experiments. This mode is the most sensitive means of detecting antagonist activity, so experiments were performed using this method with the peptides PACAP.sub.6-27 and PACAP.sub.6-38. It was found that PACAP.sub.6-27 caused no shift in the agonist curves, while PACAP.sub.6-38 induced a shift only at 1 M (see FIG. 9B. FIG. 9C, Table 2). This resulted in a pA2 value of 200.655.4 nM for PACAP.sub.6-38, again well above the 1.5 nM value reported by Robberecht. Notably, PACAP.sub.6-38 also showed a Schild Slope of 2.00.1. A Schild Slope of 1 fits the assumptions of the model, while a slope above 1 suggests that the compound is more effective than would be expected for competitive antagonism. This could be due to the short incubation times in the FLIPR assay, which might not be long enough to allow the system to reach equilibrium. The model is only valid at equilibrium. This would result in a high Schild Slope. Alternatively, PACAP.sub.6-38 could function by a different mechanism, e.g. binding to VPAC.sub.1/2.

[0091] Table 2 shows activity of PACAP derivatives at PAC.sub.1. Mean valuesSEM reported, derived from curves in FIGS. 8 and 9. N=3-4 independent experiments. One experiment for PACAP.sub.6-38 in the Fixed Concentration set could not be fitted to the curves, so N=2 for those values (N=3 for curves in FIG. 9). NC=not converged, no curves could be fit or activity detected. ( )=% inhibition at highest concentration since the curve bottom could not be reliably fit.

TABLE-US-00003 TABLE 2 Antagonist Data (Variable Concentration) Agonist Data IC.sub.50 I.sub.Max Compound EC.sub.50 (nM) E.sub.Max (%) Compound (nM) (%) PACAP1-27 0.95 0.4 100 PACAP6-38 >333 (78.8) PACAP1-27-S-G 5.68 2.3 101.9 1.6 PACAP6-27 NC PACAP6-27 NC PACAP6-27-S-G NC PACAP6-27-S-G NC PACAP6-27-S-L NC PACAP6-27-S-L NC PACAP6-27-Alt NC PACAP6-27-Alt NC PACAP6-27-S-G-Alt NC PACAP6-27-S-G-Alt NC PACAP6-27-S-L-Alt NC PACAP6-27-S-L-Alt NC Antagonist Data (Fixed Concentration) Compound pA2 (nM) Schild Slope PACAP6-38 200.6 55.4 2.0 0.1 PACAP6-27 NC

[0092] 5. Differentiation in PC12 Cell Culture

[0093] PC12 cells are non-adherent cells, and in spite of using the poly-D-Lysine coated plates, the majority of the cells remained suspended. During the media exchange many of the cells were removed with the spent media. The remaining cells could be visually evaluated for qualitative morphological changes at the end of the treatment period, but meaningful cell quantification could not be done reliably using this approach. It was found that glucoside and lactoside PACAP.sub.1-27 derivative treatment produced neurite outgrowth and arborization when compared to vehicle treated cells (see FIG. 10). Qualitatively, it appeared that the arborization caused by PACAP.sub.1-27 may be more extensive than that caused by the glucoside and lactoside derivatives, but again this could not be quantified. In any case, both PACAP.sub.1-27 and the derivatives induced neurite outgrowth, suggesting native PAC.sub.1 agonist activity.

[0094] Discussion

[0095] Endogenous PACAP peptides occur as C-terminal peptide amides that have either 27 (10%) or 38 (90%) amino acid residues, and are typically regarded as PAC.sub.1 agonists in assays using intact tissue or in cell culture. In the present invention, a separate CHO cell line was developed expressing the PAC.sub.1 receptor individually. Use of solid-phase peptide synthesis has allowed replacing of the terminal Leucine amide with glycosides of Serine amide bearing the simple sugars glucose or lactose. Notably, these O-linked glycopeptides not only retained their agonist activity on PC12 cell cultures (FIG. 10) and in the quantitative CHO cell assay (FIG. 8 and Table 2), but also showed extended lifetimes in mouse serum (FIG. 7), and provided evidence via microdialysis studies that the glycopeptides can cross the blood brain barrier in mice (FIG. 7).

[0096] Given the earlier work of Robberecht, et al. and the widespread acceptance of the truncated peptides PACAP.sub.6-38 and PACAP.sub.6-27 as PAC.sub.1 antagonists, antagonist activity was expected to be seen in the CHO cell assay with this compound and other truncated peptides. However, the Ca.sup.++ flux response from the PAC.sub.1-CHO cells only showed very weak antagonist activity at PAC.sub.1 receptors from PACAP.sub.6-38, and none by PACAP.sub.6-27 and derivatives (FIG. 9 and Table 2). It may be worth noting that Robberecht's study is the only molecular pharmacology study of these compounds could be found, with other studies using functional assays like cell growth with endogenous receptor expression or in vivo dosing. Furthermore, Robberecht's study used membranes of neuroblastoma NB-OK-1 cells, which may express other relevant targets besides PAC; (e.g. VPAC.sub.i and VPAC.sub.2). Since the present invention is the only one to inventor's knowledge using isolated PAC.sub.1 expression to study receptor function (one earlier Robberecht group study with PAC.sub.1 in CHO only studied binding), this raises the possibility that PACAP.sub.6-27 and PACAP.sub.6-38 are not potent antagonists at PAC.sub.1 as described, and may prefer other targets. This question has considerable bearing on the potential use of PACAP.sub.6-27 derivatives as PAC.sub.1 antagonists in the treatment of migraine.

EXAMPLE 2

[0097] Example 2 describes the study of glycosides and the Ang-(I-7) peptide. The present invention is not limited to the compounds, configurations, methods, and systems described in Example 2.

[0098] Glycosides are added to the aqueous face of the native Ang-(I-7) peptide at (sites on the aqueous surface and not the lipophilic surface). Inherent binding of the glycopeptide to the native receptor is minimally affected. Therefore, the glycosylated Ang-(I-7) derivatives can, at minimum, maintain Mas binding similar to that of the native Ang-(I-7) peptide. Non-limiting examples of Ang-(I-7) derivatives are shown in Table 3.

TABLE-US-00004 TABLE 3 SEQ ID NO: Ang Peptides 1 2 3 4 5 6 7 8 13 Native AT.sub.1-7 Asp Arg Val Tyr Ile His Pro OH 14 ATn-1 Asp Arg Val Tyr Ile His Pro NH.sub.2 15 ATn-2 Asp Arg Val Tyr Ile His Pro Ser NH.sub.2 16 ATn-3 Asp Arg Val Tyr Ile His Pro Ser* NH.sub.2 17 ATn-4 Asp Arg Val Tyr Ile His Pro Ser** NH.sub.2 18 ATn-5 Asp Arg Val Tyr Ile His Ser* ATn-5-ATN11 Ala scan Tyr Ile etc . . . Pro Ser/*/** NH.sub.2 19 ATn-12 . . . Asp Arg Xxx Tyr Yyy His Pro Ser/*/** NH.sub.2 20 ATn-XX . . . Asp Arg Xxx Zzz Yyy His Pro Ser/*/** NH.sub.2

[0099] The degree of glycosylation (unglycosylated Ser, glucosylated Ser** or lactosylated Ser**) for optimal BBB transport is determined using the best binding compounds from these using the in vivo mouse model. Besides the disaccharide -lactose, the more robust disaccharide -cellobiose is examined using these structures. An alanine scan will allow for focused attention on the residues most likely to result in increased interaction of the glycopeptides with the Mas receptor (e.g. hot spots), and with a glycosylation state most likely to result in penetration of the BBB. Non-peptidic D-amino acids and N-methylated amino acids, as well as un-natural amino acids are substituted at 1-2 of the hottest spots to optimize binding (ATn-12 . . . , Table 1). Valine.sup.3 and Isoleucine.sup.5 have been used here to illustrate the process with Xxx and Yyy to indicated amino acid selection. After optimal substitution is achieved for these positions, an additional round of substitution (Zzz) is done to further optimize binding.

[0100] As used herein, the term about refers to plus or minus 10% of the referenced number.

[0101] Various modifications of the invention, in addition to those described herein, will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. Each reference cited in the present application is incorporated herein by reference in its entirety.

[0102] Although there has been shown and described the preferred embodiment of the present invention, it will be readily apparent to those skilled in the art that modifications may be made thereto which do not exceed the scope of the appended claims. Therefore, the scope of the invention is only to be limited by the following claims. Reference numbers recited in the claims are exemplary and for ease of review by the patent office only, and are not limiting in any way. In some embodiments, the figures presented in this patent application are drawn to scale, including the angles, ratios of dimensions, etc. In some embodiments, the figures are representative only and the claims are not limited by the dimensions of the figures. In some embodiments, descriptions of the inventions described herein using the phrase comprising includes embodiments that could be described as consisting of, and as such the written description requirement for claiming one or more embodiments of the present invention using the phrase consisting of is met.