Next Article in Journal
Benzothiadiazole vs. iso-Benzothiadiazole: Synthesis, Electrochemical and Optical Properties of D–A–D Conjugated Molecules Based on Them
Next Article in Special Issue
Harnessing the Anti-Nociceptive Potential of NK2 and NK3 Ligands in the Design of New Multifunctional μ/δ-Opioid Agonist–Neurokinin Antagonist Peptidomimetics
Previous Article in Journal
Expanding the Myxochelin Natural Product Family by Nicotinic Acid Containing Congeners
Previous Article in Special Issue
Facile Synthesis of 2H-Benzo[h]Chromenes via an Arylamine-Catalyzed Mannich Cyclization Cascade Reaction
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Molecules
Volume 26
Issue 16
10.3390/molecules26164932
molecules-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Synthesis and In Vitro Characterization of Glycopeptide Drug Candidates Related to PACAP1–23

by
Christopher R. Apostol
1,
Parthasaradhireddy Tanguturi
2,
Lajos Z. Szabò
1,
Daniel Varela
3,
Thiago Gilmartin
3,
John M. Streicher
2 and
Robin Polt
1,*
1
Department of Chemistry and Biochemistry, BIO5, The University of Arizona, 1306 E. University Blvd, Tucson, AZ 85721, USA
2
Department of Pharmacology, College of Medicine, The University of Arizona, 1501 N. Campbell Ave, Tucson, AZ 85724, USA
3
Facultat de Quìmica Tarragona, Universitat Rovera I Virgili, 43007 Barcelona, Spain
*
Author to whom correspondence should be addressed.
Molecules 2021, 26(16), 4932; https://doi.org/10.3390/molecules26164932
Submission received: 22 July 2021 / Revised: 10 August 2021 / Accepted: 11 August 2021 / Published: 14 August 2021
(This article belongs to the Special Issue A Themed Issue Dedicated to Professor Victor Hruby)
Browse Figures
Versions Notes

Abstract

:
The search for efficacious treatment of neurodegenerative and progressive neuroinflammatory diseases continues, as current therapies are unable to halt or reverse disease progression. PACAP represents one potential therapeutic that provides neuroprotection effects on neurons, and also modulates inflammatory responses and circulation within the brain. However, PACAP is a relatively long peptide hormone that is not trivial to synthesize. Based on previous observations that the shortened isoform PACAP1–23 is capable of inducing neuroprotection in vitro, we were inspired to synthesize shortened glycopeptide analogues of PACAP1–23. Herein, we report the synthesis and in vitro characterization of glycosylated PACAP1–23 analogues that interact strongly with the PAC1 and VPAC1 receptors, while showing reduced activity at the VPAC2 receptor.

1. Introduction

Neurodegenerative disorders continue to negatively impact the health and quality of life of millions of people worldwide [1,2,3,4]. Many current therapeutic strategies involve only symptomatic alleviation, as there is a severe lack of treatments with the potential to halt or reverse disease progression [5,6]. A plethora of studies have elucidated the molecular pathophysiology of various neurodegenerative diseases, revealing a complex interplay between neuronal apoptosis (oxidative stress, mitochondrial dysfunction, imbalances in ion homeostasis) and the inflammatory response in the brain [7,8,9,10,11]. Hyperactivation of microglia, the resident macrophages of the brain, results in the continual release of pro-inflammatory cytokines, which induces oxidative stress, mitochondrial dysfunction, imbalances in ion homeostasis, and eventual cell death. These apoptotic events will induce further stimulation of hyperactive microglia, establishing a continual cycle of neuronal cell death and neuroinflammation that promotes disease progression [7,12,13]. Thus, one of the biggest challenges associated with treating neurodegenerative diseases is effectively addressing both the apoptotic and neuroinflammatory aspects simultaneously. Endogenous pleiotropic peptide hormones are a representative class of compounds that may be able to address this critical problem. The pituitary adenylate cyclase activating peptide (PACAP) is one such endogenous peptide that has been shown to elicit neuroprotection and anti-inflammatory activity in animal models of Parkinson’s disease (PD), ischemic stroke, Alzheimer’s disease (AD), traumatic brain injury (TBI), and ethanol toxicity [14,15,16,17,18,19,20,21]. Interestingly, PACAP’s primary sequence has been conserved for millions of years across different species, which implies that it regulates critical biological functions [22]. The wide distribution of PACAP and its cognate receptors in different organ systems throughout the body further indicates their important regulatory roles [23]. PACAP exists as two different isoforms containing either 27 or 38 amino acid residues (Table 1). PACAP’s biological activities are elicited through three class B G-protein coupled receptors (GPCRs) known as PAC1, VPAC1, and VPAC2. Of note, class B GPCRs contain a large extracellular domain thought to be an affinity trap to initially bind their relatively large cognate peptide ligands, making them structurally distinct from other members of the GPCR family tree [24,25]. PACAP’s neuroprotective effects are mediated through PAC1, while immunomodulatory effects are modulated through VPAC1 and VPAC2 [23]. More specifically, VPAC1 activation elicits the production of anti-inflammatory mediators, while activation of VPAC2 can induce a pro-inflammatory response. Thus, relatively lower affinity for VPAC2 is desirable. PACAP binds and activates PAC1, VPAC1, and VPAC2 with equally high affinity whereas the vasoactive intestinal peptide (VIP), a structurally homologous relative of PACAP (Table 1), exhibits high affinity for VPAC1 and VPAC2, but is not selective for PAC1. Due to the promiscuous binding profile of PACAP, several research groups have attempted to prepare PAC1-selective PACAP analogues with potent neuroprotective activity. Some success has been attained, but there is yet to be a synthetic purely PAC1-selective agonist with no activity at the VPAC1/VPAC2 receptors [26]. This difference in affinity is intriguing considering there is striking structural similarity between VIP and PACAP1–27 (Table 1). However, positions 4 and 5 in PACAP’s and VIP’s primary sequences may be the key to fine-tuning receptor selectivity [27,28]. Positions 4 and 5 in PACAP are occupied by Gly and Ile, respectively, whereas the corresponding residues in VIP are Ala and Val. Gly is a known β-turn inducer while Ala is known to promote α-helical conformations, and Ile and Val have subtly different steric profiles. Thus, it is hypothesized that amino acid substitutions in the “hinge region” may lead to analogues with more diverse selectivity profiles.

1.1. Previous SAR Work and Evidence of PACAP1–23 Neuroprotection In Vitro

Several structure–activity relationship (SAR) studies have been carried out on PACAP to identify important pharmacophoric elements and sites prone to proteolytic cleavage [28,29,30,31]. C-terminal truncation studies revealed that the minimum sequence required to maintain adequate receptor binding is PACAP1–23, whereas studies investigating N-terminal deletions demonstrated that the 1st six residues are required to maintain agonist activity [30]. Due to the weak receptor binding profile of PACAP1–23, it was rarely investigated further for its neuroprotective potential [32]. However, in 2019, Chatenet and coworkers explored the in vitro functional activity and neuroprotective potential of PACAP1–23 in neuroblastoma cells [33]. They demonstrated that PACAP1–23 is capable of attenuating MPP+-induced apoptosis, mitochondrial dysfunction, and glutamate-induced excitotoxicity despite drastically reduced binding affinity at PAC1 [33]. In addition, they observed comparable potency between PACAP1–23 and PACAP1–38 in activating specific downstream signaling pathways. These data demonstrate that PACAP1–23 represents a potential candidate for the treatment of neurodegenerative diseases.

1.2. PACAP1-23 Glycopeptide Design Considerations

Despite the neuroprotective potential of PACAP1–23, the native peptide itself is a poor drug candidate due to its rapid in vivo metabolism and limited blood–brain barrier (BBB) permeability. Furthermore, activation of the VPAC2 receptor may be undesirable in some contexts, making it important to pursue the discovery of selective agonists of the PAC1 receptor [34]. PACAP1–38 enters the brain through a selective transporter at the BBB endothelium known as peptide transporter system 6 (PTS-6), whereas PACAP1–27 appears to enter the brain via passive diffusion [35,36]. However, it is unknown if PACAP1–23 can penetrate the BBB like the native PACAP isoforms. Many strategies have been implemented to improve the pharmacokinetics and membrane permeability of peptides, including N-methylation, cyclization, lipidation, and PEGylation [37,38,39]. One strategy that is highly effective yet often overlooked is glycosylation. Several laboratories have demonstrated that glycosylation of peptides dramatically increases stability in vivo and enhances permeability of the BBB, which is evident through observing central effects and microdialysis in the striatum followed by mass spectrometry analysis following peripheral administration (i.v., i.p.) [40,41,42,43,44,45]. Our laboratory has extensively investigated glycosylation as a means to address the concerns of BBB permeability and stability for many endogenous peptides including opioid peptides, angiotensin1–7, and PACAP [46,47,48,49,50]. In fact, we have shown that glycosylated PACAP analogues not only successfully penetrate the BBB, but that they also elicit potent neuroprotection in animal models of TBI, stroke, and PD [51,52]. Carbohydrates provide the necessary steric bulk to protect peptides from proteolytic degradation and modulate the amphipathicity of peptides such that it influences how they interact with biological membranes [40,43]. We hypothesized that converting relatively lipophilic peptides to amphipathic glycopeptides allows them to “hop” along the surface of cell membranes and, therefore, increases the probability that the glycopeptide will find and interact with the target receptor of interest. In the context of BBB permeability, we have hypothesized that glycopeptides can pass through the BBB via adsorptive transcytosis similarly to peptides with a high degree of positive charge [53]. However, the exact mechanism by which glycopeptides penetrate the BBB is still unknown. Considering our past successes in enhancing the in vivo stability and BBB penetrance of endogenous peptide hormones by glycosylation, particularly PACAP1–27 (unpublished results), we envisioned that applying this strategy to PACAP1–23 would provide analogues with enhanced stability and BBB penetrance. We incorporated various carbohydrate motifs (glucose, di-glucose, lactose, Figure 1) to determine which carbohydrate will provide the optimal balance between stability and in vitro functional activity in ongoing studies. The carbohydrates investigated in this study were chosen based on previously successful studies on glycosylated endogenous peptide scaffolds including angiotensin1–7, the enkephalins, and PACAP1–27 [40,43,48,51,52]. In addition to glycosylation of PACAP1–23, we set out to examine structural modifications predicted to increase selectivity for PAC1 and provide additional stability. We opted to replace Met17, which is susceptible to oxidation, with norvaline (Nva), a non-natural amino acid with an all-carbon side chain that cannot undergo oxidation. To fine-tune receptor selectivity, we chose to modify PACAP’s N-terminal region. The N-terminal region of PACAP exhibits high similarity to VIP (Table 1), but those similarities diverge at position 4. In PACAP, position 4 is occupied by Gly, which is a known β-turn inducer. In VIP, position 4 is occupied by Ala, which is well known as an α-helix stabilizer. Thus, it has been hypothesized that incorporation of amino acids capable of biasing the conformation of the N-terminus towards β-turns and α-helices would lead to selectivity for PAC1 or VPAC1/VPAC2, respectively. To this end, we prepared analogues containing either Ala, a known α-helix promoter, and N-methylglycine (Sar) (Figure 1), which has been shown to induce β-turn-like conformations. Herein, we present the synthesis and in vitro characterization of glycosylated analogues of PACAP1–23.

2. Results

2.1. Synthesis

The PACAP1–23 glycopeptide analogues were synthesized on a Rink amide MBHA resin (0.25 mmol scale, d.s. ~0.5 mmol/g) using the Prelude® automated peptide synthesizer from Gyros Protein Technologies. The carbohydrate motifs were introduced as pre-assembled Fmoc protected serine glycoside buildings blocks, which were prepared via established methods in our laboratory utilizing minimally competent InBr3 catalysis [54]. Several different coupling protocols were utilized in this synthesis due to the fact that PACAP is considered a “difficult” peptide sequence [55]. PACAP’s relatively long length necessitates the use of stronger coupling reagents later on in the synthesis, and there are two motifs present that are highly prone to aspartimide formation (Asp3-Gly4 and Asp8-Ser9). Aspartimide formation involves the base-promoted cyclization of an aspartic acid side chain with the α-amino nitrogen of the preceding residue, with Gly, Ser, Thr, and Cys being the most problematic [56,57,58]. To circumvent this issue, we used dipeptide building blocks that suppress aspartimide formation. In the case of the Asp8-Ser9 motif, we utilized a pseudoproline dipeptide building block (Fmoc-DS, Figure 2), and for the Asp3-Gly4, we utilized a dimethoxybenzyl (DMB)-containing dipeptide (Fmoc-DG, Figure 2) [57]. The Fmoc-DS and Fmoc-DG dipeptide building blocks are unable to undergo aspartimide formation due to the alkyl protection of the amide nitrogens of the Ser and Gly motifs. In the case of Fmoc-DS, the serine amide nitrogen is locked into a pseudoproline ring that is cleaved upon treatment with TFA during resin cleavage. In the case of Fmoc-DG, the amide nitrogen of the Gly moiety is protected with a 2,4-dimethoxybenzyl group, which can be removed under the acidic of resin cleavage and global side chain deprotection. In addition to suppressing aspartimide formation, these specially protected dipeptide building blocks are also beneficial in that they minimize peptide–peptide aggregation on the resin, ultimately enhancing the efficiency of subsequent amino acid couplings.
With this plan in hand, we then set out to complete the glycopeptide assembly. First, the desired Fmoc protected glycosyl amino acid or Fmoc-Ser(OtBu)-OH was loaded onto the resin using equimolar amounts of Cl-HOBt and DIC in NMP (Scheme 1, Figure 3). The resin was then treated with a mixture of DIPEA/Ac2O in DCM (10%/10% v/v) to cap any unreacted sites on the resin. The next 14 residues (Tyr10-Leu23) were coupled utilizing a standard HBTU/N-methylmorpholine coupling protocol (Scheme 1). The Fmoc-DS dipeptide was coupled using the more reactive Cl-HOBt/DIC to introduce Asp8 and Ser9 due to increased steric hinderance. The relatively lipophilic tripeptide motif Ile5-Phe6-Thr7 was introduced using a modified HBTU/N-methylmorpholine coupling protocol where greater excesses of the coupling reagent and base were used to achieve complete coupling. The Fmoc-DG dipeptide was utilized in cases where the Asp3-Gly4 motif was present. In the cases of 5 and 6, the Cl-HOBt/DIC coupling protocol was utilized to couple Ala4 or Sar4. As in the case of Fmoc-DS, the Cl-HOBt/DIC protocol was employed due to additional steric hinderance. The remaining amino acids were coupled using the Cl-HOBt/DIC in NMP protocol. After cleavage of the final Fmoc group on His1, the resin was treated with 50% NH2NH2•H2O in NMP to remove the carbohydrate acetate protecting groups. The crude peptides were precipitated in cold diethyl ether and purified by preparative HPLC. To our delight, the final purity of the shorter PACAP1–23 glycopeptides (≥98%) was superior to PACAP1–27 glycopeptides previously prepared in our laboratory [52]. The retention time data of the pure glycopeptides are reported in Table 2. The molecular masses of the glycopeptide analogues were determined by ESI-MS, resulting in agreement with the theoretical masses calculated from the predicted primary structures. The most predominant peaks in the mass spectra were in the +4 or +5 charge state, which was expected due to the presence of a significant number of basic residues in PACAP’s primary sequence (Table 2) (Supplementary Materials).

2.2. In Vitro Characterization

Following synthesis, purification, and characterization, the glycopeptides were assessed for their ability to stimulate cAMP in CHO cells individually expressing the PAC1, VPAC1, and VPAC2 receptors (Table 3). Introduction of a serine with no carbohydrate (1) yielded a compound with low nanomolar activity at PAC1 and VPAC1, but activity at VPAC2 was greatly diminished compared to the control. The incorporation of a serine glucoside moiety at the C-terminus (2) did not alter activity at VPAC2 significantly, but potency at PAC1 was greatly enhanced. When a second serine glucoside was introduced to give the di-glucoside-containing compound 3, activity at VPAC2 dropped sharply, while potency and efficacy were maintained at PAC1 and VPAC1, albeit with reduced potency at PAC1 compared to 2. The compounds containing serine lactoside motifs at the C-terminus (4, 5, 6) were PAC1/VPAC1 selective, with slightly greater potency at PAC1 over VPAC1. Additionally, these compounds exhibited extremely weak activity at the VPAC2 receptor, similarly to the serine–glucoside and serine–di-glucoside compounds.

3. Discussion

Our group has previously investigated the glycosylation of native PACAP1–27 as a strategy to improve its PK properties while maintaining its intrinsic potency and efficacy in a rodent model of PD [51,52]. Even shorter versions of this hormone (e.g., PACAP1–23) demonstrate neuroprotective activity in vitro [33], showing potency similar to native PACAP1–38 in the attenuation of glutamate-induced cytotoxicity and MPP+-induced cell death, suggesting that shorter PACAP analogues could be viable alternatives to the longer hormones. This is advantageous for several reasons. First, PACAP1–27 and PACAP1–38 are quite long sequences and difficult to synthesize, and the shorter PACAP glycosides are marginally easier to prepare in their pure state. Six glycopeptide analogues were prepared in satisfactory yields and higher purity than our previously prepared PACAP glycopeptide analogues (Supplementary Materials, [52]). The modifications we introduced include various glycoside motifs at the C-terminus (glucose, di-glucose, lactose) to enhance stability and BBB transport and N-terminal substitutions in position 4 (Ala, N-methyl glycine) to fine-tune receptor selectivity. To our delight, the introduction of carbohydrate-bearing amino acids into the C-termini of our compounds did not negatively impact functional activity. PAC1/VPAC1 selectivity was maintained, and activity at VPAC2 was drastically reduced. Interestingly, functional activity at VPAC2 was drastically reduced upon introduction of a di-glucoside motif (3), or a lactose residue (4, 6). However, 5 seems to be an exception to this trend, but this was expected considering the Ala4 substitution, which is known to enhance selectivity for the VPAC1/VPAC2 receptors. This decrease in activity at VPAC2 may be attributed to disfavored binding interactions between the carbohydrate moiety and the extracellular domain (ECD) of the receptor, but this has not been confirmed. Thus, further exploration into the origin of carbohydrate-mediated decreases in VPAC2 selectivity are warranted. Although the Sar4 substitution yielded a compound that was PAC1 selective, potency was decreased by a considerable amount compared to the other compounds. The best compound in the series was determined to be the mono-glucoside 2, with ~58-fold selectivity for PAC1 over VPAC1 and ~886-fold selectivity for PAC1 over VPAC2. However, 3 (serine di-glucoside) and 4 (serine lactoside) also exhibited ideal functional activity and receptor selectivity profiles and are scaffolds worth pursuing in future PACAP glycopeptide drug discovery efforts. Ongoing studies will elucidate the effects of the substitutions investigated in this study on the in vitro and in vivo stability of our PACAP1–23 glycopeptide analogues.

4. Materials and Methods

4.1. Glycopeptide Synthesis and Purification

Fmoc-based solid phase peptide synthesis (SPPS) was used to synthesize the 6 drug candidates on Rink resin to produce the C-terminal amides. Acetate removal from the glycosides was accomplished “on resin” with hydrazine hydrate (H2N–NH2•H2O) per previously published methods [47]. Three distinct coupling methods were used to assemble the glycopeptide backbone that contains two “difficult sequences” that can form aspartimides, leading to α to β amide migration. This complication was avoided by the use of dipeptides with pre-formed amide linkages to each aspartic acid residue [57,59,60].

4.1.1. General

Peptide synthesis was performed on a Prelude® automated peptide synthesizer (Gyros Protein Technologies, Tucson, AZ, USA). Synthesis was performed either in an automated fashion or semi-manually where reagents were loaded into the reaction vessels using a syringe. The resin was agitated (mixed) using a steady flow of argon. The washing steps with DMF and DCM were performed for 2 min each.

4.1.2. Rink Amide Resin Preparation

A sample of 0.25 mmol of Rink amide-MBHA resin (0.6 g) resin was placed in a 45 mL reaction vessel and swelled in DMF for 1 h. Fmoc removal was achieved by addition of a solution containing 2%DBU-3%piperidine in DMF (6 mL) and mixing for 4 min. The mixture was then drained, and the resin was washed once with 6mL of DMF. Fmoc removal was then repeated for an additional 8 min followed by 6 DMF washes (6 mL, 2 min).

4.1.3. Serine or Glycosyl Amino Acid Loading

A sample of 0.20 mmol (0.8 eq.) of the desired first amino acid Fmoc-Ser(tBu)-OH, Fmoc-Ser(Glc(OAc)4)-OH, or Fmoc-Ser(Lac(OAc)7)-OH) and 0.2 mmol (0.8 eq.) 6-Cl-HOBt were placed into a vial and dissolved in 4 mL NMP. A sample of 0.2 mmol (0.8 eq.) of DIC was then added into the solution. The mixture was vortexed and/or sonicated for 1 min and then added to the resin. The reaction mixture was mixed overnight for 16 h. The mixture was diluted with DMF (10 mL) and drained immediately. Then, the resin was washed 6 times with DMF (6 mL) and then 6 times with DCM (6 mL). The unreacted NH2 sites on the resin were then capped with a solution of 10% N,N-diisopropylethylamine and 10% Ac2O in 8mL DCM. This reaction was allowed to proceed for 1 h. The resin was then washed 6 times with DCM (6 mL) and then washed 4 times with DMF (6 mL) to prepare the resin for the next automated steps.

4.1.4. Loading of Additional Fmoc-Ser(Glc(OAc)4)-OH (3 Only)

Samples of 0.5 mmol (2.0 eq.) of Fmoc-Ser(Glc(OAc)4)-OH and 0.5 mmol (2.0 eq.) 6-Cl-HOBt were placed into a vial and dissolved in 4 mL NMP. A sample of 0.5 mmol (2.0 eq.) of DIC was then added into the solution. The mixture was vortexed and/or sonicated for 1 min and then added to the resin. The reaction mixture was mixed overnight for 16 h. The mixture was diluted with DMF (10 mL) and drained immediately. Then, the resin was washed 6 times with DMF (6 mL).

4.1.5. Prelude® Automated Synthesis

The Leu23-Tyr10 amino acid series was prepared using the automated SPPS feature on the Prelude® automated peptide synthesizer. The Fmoc group was removed as described above and a solution containing the desired Fmoc amino acid (2 equivalents), HBTU (2 equivalents) and N-methylmorpholine (10 equivalents) was loaded to the resin. The reaction mixture was mixed for 30 min followed by a single DMF wash (6 mL). The coupling reaction was repeated a second time for 30 min, and the resin was then washed 6 times with DMF (6 mL). Subsequent deprotection and coupling cycles were then performed up to tyrosine10.

4.1.6. Manual Loading of DS Dipeptide

The Fmoc group was initially removed as described above. Then, 0.3 mmol of Fmoc-DS-OH or Fmoc-DG-OH (1.2 equiv.) and 0.3 mmol of 6-Cl-HOBt (1.2 equiv.) were added to a vial and dissolved in 4 mL of NMP. A sample of 0.3 mmol of DIC (1.2 equiv.) was then added to the solution. The mixture was vortexed and/or sonicated for 1 min and then added to the resin. The reaction mixture was mixed for 16 h. The mixture was diluted with DMF (10 mL) and drained immediately. Then, the resin was washed 6 times with DMF (6 mL).

4.1.7. Automated Addition of IFT

The Ile5-Thr7 amino acid series was prepared using the automated SPPS feature on the Prelude® automated peptide synthesizer. The Fmoc group was removed as described above and a solution containing the desired Fmoc amino acid (4 equivalents), HBTU (4 equivalents), and N-methylmorpholine (16 equivalents) in 10 mL of DMF was loaded into the resin. The reaction mixture was mixed for 30 min followed by a single DMF wash (10 mL). The coupling reaction was repeated a second time for 30 min, and the resin was then washed 6 times with DMF. Subsequent deprotection and coupling cycles were then performed up to isoleucine5.

4.1.8. Manual Loading of DG Dipeptide

The Fmoc group was initially removed as described above. Then, 0.5 mmol Fmoc-DG-OH (2.0 equiv.) and 0.5 mmol of 6-Cl-HOBt (2.0 equiv.) were added to a vial and dissolved in 4 mL of NMP. A sample of 0.5 mmol of DIC (2.0 equiv.) was then added to the solution. The mixture was vortexed and/or sonicated for 1 min and then added to the resin. The reaction mixture was then mixed for 6 h. The mixture was diluted with DMF (10 mL) and drained immediately. Then, the resin was washed 6 times with DMF (6 mL).

4.1.9. Manual Loading of Fmoc-Ala-OH (5) or Fmoc-Sar-OH (6)

The Fmoc group was initially removed as described above. Then, 1.0 mmol of amino acid (4 equiv.) and 1.0 mmol of 6-Cl-HOBt (4 equiv.) were added to a vial and dissolved in 4 mL of NMP. A sample of 1.0 mmol of DIC (4 equiv.) was then added to the solution. The mixture was vortexed for 1 min and then added to the resin. The reaction mixture was mixed for 2 h. The mixture was diluted with DMF (10 mL) and drained immediately. Then, the resin was washed 6 times with DMF (6 mL).

4.1.10. Manual Loading of Remaining Amino Acids Fmoc-Asp(tBu)-OH (5 and 6 Only), Fmoc-Ser(tBu)-OH, and Fmoc-His(Trt)-OH

The Fmoc group was initially removed as described above. Then, 1.0 mmol amino acid (4 equiv.) and 1.0 mmol of 6-Cl-HOBt (4 equiv.) were added to a vial and dissolved in 4 mL of NMP. A sample of 1.0 mmol of DIC (4 equiv.) was then added to the solution. The mixture was vortexed for 1 min and then added to the resin. The reaction mixture was mixed for 60 min. The mixture was diluted with DMF (10 mL) and drained immediately. Then, the resin was washed 6 times with DMF (6 mL). After addition of the final amino acid, the Fmoc group was removed as described above.

4.1.11. Acetyl Cleavage

One hundred and twenty milliliters of a 50% solution containing NH2NH2 × H2O in NMP (10 mL per reaction vessel) was prepared and added to the resin. The solution was mixed overnight for 16 h. The solution was then drained, and a second 10 mL portion of 50% NH2NH2 x H2O was added to each reaction vessel. This solution was mixed for an additional 2 h. The 50% NH2NH2 x H2O was then drained, and the resin was washed 8 times with DMF (10 mL), 8 times with DCM (10 mL), and dried under vacuum for 3 h.

4.1.12. Cleavage from the Resin and Global Side Chain Deprotection

The dried resin was treated with an acidic cleavage cocktail containing TFA, DCM, H2O, triethylsilane, and anisole (90:10:2:3:0.5). The resin was mixed for 1 h, and the solution was collected in a 45 mL centrifuge tube. The cleavage step was repeated 2 more times for 10 min periods. The combined fractions were slowly evaporated over a stream of argon until the peptide began to crash out. Cold ether (~40 mL) was then added to precipitate the peptide and the mixture was centrifuged for 10 min at 5 G. The ether layer was decanted off and ether (~40 mL) was added to the crude peptide and centrifuged once more. This process was repeated for a third time. After decanting the ether layer, the crude peptide was dried under vacuum overnight.

4.1.13. HPLC Purification and Characterization of Peptides

These crude samples were then purified on a Gilson system with a UV detector (at 280 nm) using a Vydak C18 preparative reversed-phase column (250 mm × 50 mm) using a gradient of 5–80% CH3CN vs. 0.1% CF3COOH in H2O over 60 min to give the glycopeptides in pure form, which were assessed for purity by analytical HPLC (Inspire C18 5 μm 250 mm × 4.6 mm column) on a Varian LC with a diode array detector system (at 280 nm) employing the same gradient over a period of 25 min.
The pure fractions obtained from preparative HPLC purification were frozen at −80 °C and then lyophilized to afford the pure peptides as white and fluffy solids. The pure peptides were then characterized using mass spectrometry (ESI-MS) (University of Arizona Analytical and Biological Mass Spectrometry Core Facility, Tucson, AZ, USA).

4.2. Cell Culture

CHO cells stably expressing cloned PAC1, VPAC1, and VPAC2 were produced by electroporation with human PAC1/VPAC1/VPAC2 N-3xHA tag cDNA constructs (GeneCopoeia). Cells were grown on 10 cm dishes in DMEM/F-12 50/50 mix w/L-glutamine and 15 mM HEPES (Corning) containing 10% heat inactivated fetal bovine serum, 100 units/mL penicillin, 100 μg/mL streptomycin, and 500 μg/mL G418 under 5% CO2 at 37 °C. The cells were enriched into high expressing populations using flow cytometry, selecting the top ~2% of expressing cells.

4.3. cAMP Accumulation Assay

At ~80% confluence, cells were plated into 96-well plates (20,000 cells/well) and grown in the same medium and conditions as described above for 24 h. The cells were then serum starved for 4 h. After a 20 min incubation at 37 °C with 500 μM 3-Isobutyl-1-methylxanthine (IBMX), serum-free medium containing 500 μM IBMX and the appropriate agonists was added and then incubated for 10 min at 37 °C. The reaction was terminated by removing the medium and adding 60 μL of ice-cold assay buffer (50 mM Tris-HCl pH 7.4, 100 mM NaCl, 5 mM ethylenediaminetetraacetic acid (EDTA)). Plates were sealed with boiling mats and then boiled at 95 °C for 10 min. Plates were then centrifuged at 4000 rpm, 4 °C, for 10 min to remove debris. Fifty microliters of lysate was transferred to a 96-well plate. Lysate was incubated with ~1 pmol 3H-cAMP (PerkinElmer), and 7 μg protein kinase A (Sigma-Aldrich, St. Louis, MI, USA) with 0.05% bovine serum albumin (BSA). The assay was incubated at room temperature for 1 h. The reactions were then harvested onto GF/B filter plates (PerkinElmer) via rapid filtration by a 96-well plate Cell Harvester (Brandel) and washed 3 times with ice-cold water. Filter plates were dried, 40μL of Microscint-PS scintillation cocktail was added to each well, and then counted in a TopCount or Microbeta2 (PerkinElmer) microplate scintillation counter.

5. Conclusions

The shorter glycosidic PACAP compounds display unique receptor profiles with greatly reduced activity for the VPAC2 receptor while retaining PAC1 and VPAC1 agonist activity. These compounds may be useful in the treatment of traumatic brain injury, stroke, Parkinsonism, or other progressive disorders.

6. Patents

The novel PACAP glycosides have been patented by Tech Launch Arizona, the University of Arizona, University Services Annex West, 4th Floor, 220 West 6th Street, Tucson, AZ 85721.

Supplementary Materials

The following are available online. Table S1: Glycopeptide yields. The original analytical HPLC chromatograms and ESI-MS spectra are available in the supplementary material. The binding curves corresponding to the in vitro cAMP accumulation data are also available in the supplementary material.

Author Contributions

Conceptualization, C.R.A., L.Z.S., P.T., J.M.S. and R.P.; synthesis of glycopeptides, C.R.A., T.G. and D.V.; in vitro cAMP accumulation experiments, P.T. and J.M.S.; HPLC and ESI-MS characterization, C.R.A., T.G. and D.V.; writing—original draft preparation, C.R.A. and R.P.; writing—review and editing, C.R.A. and R.P.; project administration, J.M.S. and R.P.; funding acquisition, R.P. and J.M.S. All authors have read and agreed to the published version of the manuscript.

Funding

We thank the University Rovira i Virgili, Tarragona for facilitating the studies of T.G. and D.V. at UArizona. This research was supported by the National Institute of Neurological Disorders and Stroke (NINDS) of the National Institutes of Health (NIH) under Award Number R01NS091238. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data of the compounds are available from the authors.

Acknowledgments

We would like to express our gratitude for the enormous contributions that Victor J. Hruby has made to the field of peptide drugs, as well as his invaluable help and scientific advice over the years, the training of numerous students at all levels, from undergraduate research (C.R.A.) to assistant professors (J.M.S.) and even tenured principal investigators (R.P.), to say nothing of the warm friendship that has made the University of Arizona a great place to work and do research.

Conflicts of Interest

R.P. and J.M.S. have equity in Teleport Pharmaceuticals, LLC, a UArizona biotech startup. This interest played no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Sample Availability

Not available.

References

  1. Marras, C.; Beck, J.C.; Bower, J.H.; Roberts, E.; Ritz, B.; Ross, G.W.; Abbott, R.D.; Savica, R.; Eeden, S.K.V.D.; Willis, A.W.; et al. Prevalence of Parkinson’s disease across North America. npj Park. Dis. 2018, 4, 1–7. [Google Scholar] [CrossRef] [Green Version]
  2. Alzheimer’s Association; 2017 Alzheimer’s disease facts and figures. Alzheimer’s Dement. 2017, 13, 325–373. [CrossRef]
  3. Dewan, M.C.; Rattani, A.; Gupta, S.; Baticulon, R.; Hung, Y.-C.; Punchak, M.; Agrawal, A.; Adeleye, A.O.; Shrime, M.G.; Rubiano, A.M.; et al. Estimating the global incidence of traumatic brain injury. J. Neurosurg. 2019, 130, 1080–1097. [Google Scholar] [CrossRef] [Green Version]
  4. Feigin, V.L.; Norrving, B.; Mensah, G.A. Global Burden of Stroke. Circ. Res. 2017, 120, 439–448. [Google Scholar] [CrossRef] [PubMed]
  5. Smith, Y.; Wichmann, T.; Factor, S.A.; DeLong, M.R. Parkinson’s Disease Therapeutics: New Developments and Challenges Since the Introduction of Levodopa. Neuropsychopharmacology 2011, 37, 213–246. [Google Scholar] [CrossRef]
  6. Tsang, K.K.-T.; Whitfield, P.C. Traumatic brain injury: Review of current management strategies. Br. J. Oral Maxillofac. Surg. 2012, 50, 298–308. [Google Scholar] [CrossRef]
  7. Streit, W.J.; Mrak, R.E.; Griffin, W.S.T. Microglia and neuroinflammation: A pathological perspective. J. Neuroinflammation 2004, 1, 14. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  8. Hirsch, E.C.; Hunot, S. Neuroinflammation in Parkinson’s disease: A target for neuroprotection? Lancet Neurol. 2009, 8, 382–397. [Google Scholar] [CrossRef]
  9. Becher, B.; Spath, S.; Goverman, J. Cytokine networks in neuroinflammation. Nat. Rev. Immunol. 2016, 17, 49–59. [Google Scholar] [CrossRef]
  10. Ransohoff, R.M. How neuroinflammation contributes to neurodegeneration. Science 2016, 353, 777–783. [Google Scholar] [CrossRef]
  11. Peixoto, C.A.; de Oliveira, W.H.; Araújo, S.M.D.R.; Nunes, A.K.S. AMPK activation: Role in the signaling pathways of neuroinflammation and neurodegeneration. Exp. Neurol. 2017, 298, 31–41. [Google Scholar] [CrossRef] [PubMed]
  12. Perry, V.H.; Holmes, C. Microglial priming in neurodegenerative disease. Nat. Rev. Neurol. 2014, 10, 217–224. [Google Scholar] [CrossRef] [PubMed]
  13. Block, M.L.; Zecca, L.; Hong, J.-S. Microglia-mediated neurotoxicity: Uncovering the molecular mechanisms. Nat. Rev. Neurosci. 2007, 8, 57–69. [Google Scholar] [CrossRef] [PubMed]
  14. Vaudry, D.; Rousselle, C.; Basille, M.; Falluel-Morel, A.; Pamantung, T.F.; Fontaine, M.; Fournier, A.; Vaudry, H.; Gonzalez, B.J. Pituitary adenylate cyclase-activating polypeptide protects rat cerebellar granule neurons against ethanol-induced apoptotic cell death. Proc. Natl. Acad. Sci. USA 2002, 99, 6398–6403. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  15. Botia, B.; Jolivel, V.; Burel, D.; Le Joncour, V.; Roy, V.; Naassila, M.; Bénard, M.; Fournier, A.; Vaudry, H.; Vaudry, D. Neuroprotective Effects of PACAP Against Ethanol-Induced Toxicity in the Developing Rat Cerebellum. Neurotox. Res. 2010, 19, 423–434. [Google Scholar] [CrossRef]
  16. Farkas, O.; Tamás, A.; Zsombok, A.; Reglodi, R.; Pal, J.; Buki, A.; Lengvári, I.; Povlishock, J.T.; Doczi, T. Effects of pituitary adenylate cyclase activating polypeptide in a rat model of traumatic brain injury. Regul. Pept. 2004, 123, 69–75. [Google Scholar] [CrossRef]
  17. Reglodi, D.; Tamás, A.; Lubics, A.; Szalontay, L.; Lengvári, I. Morphological and functional effects of PACAP in 6-hydroxydopamine-induced lesion of the substantia nigra in rats. Regul. Pept. 2004, 123, 85–94. [Google Scholar] [CrossRef]
  18. Dejda, A.; Seaborn, T.; Bourgault, S.; Touzani, O.; Fournier, A.; Vaudry, H.; Vaudry, D. PACAP and a novel stable analog protect rat brain from ischemia: Insight into the mechanisms of action. Peptides 2011, 32, 1207–1216. [Google Scholar] [CrossRef] [PubMed]
  19. Chen, Y.; Samal, B.; Hamelink, C.R.; Xiang, C.C.; Chen, Y.; Chen, M.; Vaudry, D.; Brownstein, M.J.; Hallenbeck, J.M.; Eiden, L.E. Neuroprotection by endogenous and exogenous PACAP following stroke. Regul. Pept. 2006, 137, 4–19. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  20. Kojro, E.; Postina, R.; Buro, C.; Meiringer, C.; Gehrig-Burger, K.; Fahrenholz, F. The neuropeptide PACAP promotes ?-secretase pathway for processing Alzheimer amyloid precursor protein. FASEB J. 2006, 20, 512–514. [Google Scholar] [CrossRef]
  21. Liao, C.; De Molliens, M.P.; Schneebeli, S.T.; Brewer, M.; Song, G.; Chatenet, D.; Braas, K.M.; May, V.; Li, J. Targeting the PAC1 Receptor for Neurological and Metabolic Disorders. Curr. Top. Med. Chem. 2019, 19, 1399–1417. [Google Scholar] [CrossRef] [PubMed]
  22. Dejda, A.; Sokołowska, P.; Nowak, J.Z. Neuroprotective potential of three neuropeptides PACAP, VIP and PHI. Pharmacol. Rep. 2005, 57, 307–320. [Google Scholar]
  23. Vaudry, D.; Falluel-Morel, A.; Bourgault, S.; Basille, M.; Burel, D.; Wurtz, O.; Fournier, A.; Chow, B.K.C.; Hashimoto, H.; Galas, L.; et al. Pituitary Adenylate Cyclase-Activating Polypeptide and Its Receptors: 20 Years after the Discovery. Pharmacol. Rev. 2009, 61, 283–357. [Google Scholar] [CrossRef]
  24. Pal, K.; Melcher, K.; Xu, H.E. Structure and mechanism for recognition of peptide hormones by Class B G-protein-coupled receptors. Acta Pharmacol. Sin. 2012, 33, 300–311. [Google Scholar] [CrossRef]
  25. Hollenstein, K.; de Graaf, C.; Bortolato, A.; Wang, M.-W.; Marshall, F.H.; Stevens, R.C. Insights into the structure of class B GPCRs. Trends Pharmacol. Sci. 2013, 35, 12–22. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  26. Ramos-Álvarez, I.; Mantey, S.A.; Nakamura, T.; Nuche-Berenguer, B.; Moreno, P.; Moody, T.W.; Maderdrut, J.L.; Coy, D.H.; Jensen, R.T. A structure–function study of PACAP using conformationally restricted analogs: Identification of PAC1 receptor-selective PACAP agonists. Peptides 2015, 66, 26–42. [Google Scholar] [CrossRef] [Green Version]
  27. Doan, N.-D.; Bourgault, S.; Dejda, A.; Létourneau, M.; Detheux, M.; Vaudry, D.; Vaudry, H.; Chatenet, D.; Fournier, A. Design and in vitro characterization of PAC1/VPAC1-selective agonists with potent neuroprotective effects. Biochem. Pharmacol. 2011, 81, 552–561. [Google Scholar] [CrossRef] [PubMed]
  28. Bourgault, S.; Chatenet, D.; Wurtz, O.; Doan, N.D.; Leprince, J.; Vaudry, H.; Fournier, A.; Vaudry, D. Strategies to Convert PACAP from a Hypophysiotropic Neurohormone Into a Neuroprotective Drug. Curr. Pharm. Des. 2011, 17, 1002–1024. [Google Scholar] [CrossRef]
  29. Bourgault, S.; Vaudry, D.; Ségalas-Milazzo, I.; Guilhaudis, L.; Couvineau, A.; Laburthe, M.; Vaudry, H.; Fournier, A. Molecular and Conformational Determinants of Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) for Activation of the PAC1 Receptor. J. Med. Chem. 2009, 52, 3308–3316. [Google Scholar] [CrossRef]
  30. Robberecht, P.; Gourlet, P.; Neef, P.; Woussen-Colle, M.-C.; Vandermeers-Piret, M.-C.; Vandermeers, A.; Christophe, J. 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 PACAP(6-38) as a potent antagonist. JBIC J. Biol. Inorg. Chem. 1992, 207, 239–246. [Google Scholar] [CrossRef]
  31. Robberecht, P.; Gourlet, P.; De Neef, P.; Woussen-Colle, M.C.; Vandermeers-Piret, M.C.; Vandermeers, A.; Christophe, J. Receptor occupancy and adenylate cyclase activation in AR 4-2J rat pancreatic acinar cell membranes by analogs of pituitary adenylate cyclase-activating peptides amino-terminally shortened or modified at position 1, 2, 3, 20, or 21. Mol. Pharmacol. 1992, 42, 347–355. [Google Scholar]
  32. Gourlet, P.; Vandermeers, A.; Vandermeers-Piret, M.-C.; Rathé, J.; De Neef, P.; Robberecht, P. C-Terminally shortened pituitary adenylate cyclase-activating peptides (PACAP) discriminate PACAP I, PACAP II-VIP1 and PACAP II-VIP2 recombinant receptors. Regul. Pept. 1996, 62, 125–130. [Google Scholar] [CrossRef]
  33. Lamine, A.; De Molliens, M.P.; Létourneau, M.; Hébert, T.; Vaudry, D.; Fournier, A.; Chatenet, D. The amidated PACAP1–23 fragment is a potent reduced-size neuroprotective agent. Biochim. et Biophys. Acta (BBA)-Gen. Subj. 2019, 1863, 129410. [Google Scholar] [CrossRef]
  34. Fizanne, L.; Sigaudo-Roussel, D.; Saumet, J.L.; Fromy, B. Evidence for the involvement of VPAC1 and VPAC2 receptors in pressure-induced vasodilatation in rodents. J. Physiol. 2004, 554, 519–528. [Google Scholar] [CrossRef] [Green Version]
  35. Banks, W.A.; Kastin, A.; Komaki, G.; Arimura, A. Passage of pituary adenylate cyclase activating polypeptide1-27 and pituary adenylate cyclase activating polypeptide1-38 across the blood-brain barrier. J. Pharmacol. Exp. Ther. 1993, 267, 690–696. [Google Scholar]
  36. Banks, W.A. Peptides and the blood–brain barrier. Peptides 2015, 72, 16–19. [Google Scholar] [CrossRef] [Green Version]
  37. Muttenthaler, M.; King, G.F.; Adams, D.J.; Alewood, P.F. Trends in peptide drug discovery. Nat. Rev. Drug Discov. 2021, 20, 309–325. [Google Scholar] [CrossRef]
  38. Lau, J.L.; Dunn, M.K. Therapeutic peptides: Historical perspectives, current development trends, and future directions. Bioorganic Med. Chem. 2018, 26, 2700–2707. [Google Scholar] [CrossRef]
  39. Kaspar, A.A.; Reichert, J.M. Future directions for peptide therapeutics development. Drug Discov. Today 2013, 18, 807–817. [Google Scholar] [CrossRef]
  40. Apostol, C.R.; Hay, M.; Polt, R. Glycopeptide drugs: A pharmacological dimension between “Small Molecules” and “Bio-logics”. Peptides 2020, 131, 170369. [Google Scholar] [CrossRef]
  41. Varamini, P.; Toth, I. Lipid- and sugar-modified endomorphins: Novel targets for the treatment of neuropathic pain. Front. Pharmacol. 2013, 4, 155. [Google Scholar] [CrossRef] [Green Version]
  42. Moradi, S.V.; Hussein, W.M.; Varamini, P.; Simerska, P.; Toth, I. Glycosylation, an effective synthetic strategy to improve the bioavailability of therapeutic peptides. Chem. Sci. 2016, 7, 2492–2500. [Google Scholar] [CrossRef] [Green Version]
  43. Jones, E.M.; Polt, R. CNS active O-linked glycopeptides. Front. Chem. 2015, 3, 40. [Google Scholar] [CrossRef] [Green Version]
  44. Mabrouk, O.S.; Falk, T.; Sherman, S.J.; Kennedy, R.T.; Polt, R. CNS penetration of the opioid glycopeptide MMP-2200: A microdialysis study. Neurosci. Lett. 2012, 531, 99–103. [Google Scholar] [CrossRef] [Green Version]
  45. Bartlett, M.J.; Mabrouk, O.S.; Szabò, L.; Flores, A.J.; Parent, K.L.; Bidlack, J.M.; Heien, M.L.; Kennedy, R.T.; Polt, R.; Sherman, S.J.; et al. The Delta-Specific Opioid Glycopeptide BBI-11008: CNS Penetration and Behavioral Analysis in a Preclinical Model of Levodopa-Induced Dyskinesia. Int. J. Mol. Sci. 2020, 22, 20. [Google Scholar] [CrossRef] [PubMed]
  46. Carmo, G.P.D.; Polt, R.; Bilsky, E.J.; Rice, K.C.; Negus, S.S. Behavioral Pharmacology of the μ/δ Opioid Glycopeptide MMP2200 in Rhesus Monkeys. J. Pharmacol. Exp. Ther. 2008, 326, 939–948. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  47. Li, Y.; Louis, L.S.; Knapp, B.I.; Muthu, D.; Anglin, B.; Giuvelis, D.; Bidlack, J.M.; Bilsky, E.J.; Polt, R. Can Amphipathic Helices Influence the CNS Antinociceptive Activity of Glycopeptides Related to β-Endorphin? J. Med. Chem. 2014, 57, 2237–2246. [Google Scholar] [CrossRef] [PubMed]
  48. Hay, M.; Polt, R.; Heien, M.L.; Vanderah, T.W.; Largent-Milnes, T.M.; Rodgers, K.; Falk, T.; Bartlett, M.J.; Doyle, K.P.; Konhilas, J. A Novel Angiotensin-(1-7) Glycosylated Mas Receptor Agonist for Treating Vascular Cognitive Impairment and Inflammation-Related Memory Dysfunction. J. Pharmacol. Exp. Ther. 2019, 369, 9–25. [Google Scholar] [CrossRef] [Green Version]
  49. Lowery, J.J.; Yeomans, L.; Keyari, C.M.; Davis, P.; Porreca, F.; Knapp, B.I.; Bidlack, J.M.; Bilsky, E.J.; Polt, R. Glycosylation Improves the Central Effects of DAMGO. Chem. Biol. Drug Des. 2007, 69, 41–47. [Google Scholar] [CrossRef]
  50. Li, Y.; Lefever, M.R.; Muthu, D.; Bidlack, J.M.; Bilsky, E.; Polt, R. Opioid glycopeptide analgesics derived from endogenous enkephalins and endorphins. Futur. Med. Chem. 2012, 4, 205–226. [Google Scholar] [CrossRef] [Green Version]
  51. Bernard, K.; Bartlett, M.J.; Liu, C.; Molnar, G.; Apostol, C.R.; Szabò, L.Z.; Sherman, S.J.; Madhavan, L.; Streicher, J.M.; Polt, R.; et al. Evaluation of a neuroprotective PACAP glycopeptide as systemically delivered CNS active drug to treat Parkinson’s disease. In Proceedings of the Society for Neuroscience, Chicago, IL, USA, 11–13 January 2021; Program No. 117.08. 2021 Neuroscience Meeting Planner. Available online: https://www.sfn.org/meetings/virtual-events/sfn-global-connectome-a-virtual-event/abstracts (accessed on 10 August 2021).
  52. Apostol, C.R.; Bernard, K.; Molnar, G.; Bartlett, M.J.; Szabò, L.; Liu, C.; Ortiz, J.B.; Saber, M.; Giordano, K.R.; Green, T.F.R.; et al. Design and Synthesis of Brain Penetrant Glycopeptide Analogues of PACAP with Neuroprotective Potential for Stroke, Traumatic Brain Injury, and Parkinsonism. Unpublished work.
  53. Lalatsa, A.; Butt, A.M. Physiology of the Blood–Brain Barrier and Mechanisms of Transport Across the BBB. In Nanotechnology-Based Targeted Drug Delivery Systems for Brain Tumors; Kesharwani, P., Gupta, U., Eds.; Elsevier Academic Press: Amsterdam, The Netherlands, 2018; pp. 49–74. [Google Scholar] [CrossRef]
  54. Lefever, M.R.; Szabò, L.Z.; Anglin, B.; Ferracane, M.; Hogan, J.; Cooney, L.; Polt, R. Glycosylation of α-amino acids by sugar acetate donors with InBr3. Minimally competent Lewis acids. Carbohydr. Res. 2012, 351, 121–125. [Google Scholar] [CrossRef] [Green Version]
  55. Paradís-Bas, M.; Puche, J.T.; Albericio, F. The road to the synthesis of “difficult peptides”. Chem. Soc. Rev. 2015, 45, 631–654. [Google Scholar] [CrossRef] [PubMed]
  56. Dölling, R.; Beyermann, M.; Haenel, J.; Kernchen, F.; Krause, E.; Francke, P.; Brudel, M.; Bienert, M. Piperidine–mediated Side Product Formation for Asp(OBut)–containing Peptides. J. Chem. Soc. Chem. Commun. 1994, 7, 853–854. [Google Scholar] [CrossRef]
  57. Subirós-Funosas, R.; El-Faham, A.; Albericio, F. Aspartimide formation in peptide chemistry: Occurrence, prevention strategies and the role of N-hydroxylamines. Tetrahedron 2011, 67, 8595–8606. [Google Scholar] [CrossRef]
  58. Yang, Y.; Sweeney, W.V.; Schneider, K.; Thörnqvist, S.; Chait, B.T.; Tam, J.P. Aspartimide formation in base-driven 9-fluorenylmethoxycarbonyl chemistry. Tetrahedron Lett. 1994, 35, 9689–9692. [Google Scholar] [CrossRef]
  59. Cardona, V.; Eberle, I.; Barthelemy, S.; Beythien, J.; Doerner, B.; Schneeberger, P.; Keyte, J.; White, P.D. Application of Dmb-Dipeptides in the Fmoc SPPS of Difficult and Aspartimide-Prone Sequences. Int. J. Pept. Res. Ther. 2008, 14, 285–292. [Google Scholar] [CrossRef]
  60. Sampson, W.R.; Patsiouras, H.; Ede, N.J. The synthesis of “difficult” peptides using 2-hydroxy-4- methoxybenzyl or pseu-doproline amino acid building blocks: A comparative study. J. Pept. Sci. 1999, 5, 403–409. [Google Scholar] [CrossRef]
Molecules 26 04932 g001 550
Figure 1. Strategic amino acid substitutions to fine-tune receptor selectivity and optimize stability and functional activity: (a) the bioactive conformation of our analogues can be fine-tuned by introducing amino acids into the 4th position that are biased towards either α-helical or β-turn-like conformations; (b) Met17 was replaced with norvaline (Nva) to enhance the stability of our glycopeptides. Methionine is easily oxidized and likely contributes to the low in vivo stability of native PACAP isoforms. (c) Various carbohydrate motifs were investigated in these studies to examine the effects on in vitro efficacy and potency of our analogues. Specifically, we examined serine, glucose, lactose, and two sequential serine glucoside residues as a lactose mimic.
Figure 1. Strategic amino acid substitutions to fine-tune receptor selectivity and optimize stability and functional activity: (a) the bioactive conformation of our analogues can be fine-tuned by introducing amino acids into the 4th position that are biased towards either α-helical or β-turn-like conformations; (b) Met17 was replaced with norvaline (Nva) to enhance the stability of our glycopeptides. Methionine is easily oxidized and likely contributes to the low in vivo stability of native PACAP isoforms. (c) Various carbohydrate motifs were investigated in these studies to examine the effects on in vitro efficacy and potency of our analogues. Specifically, we examined serine, glucose, lactose, and two sequential serine glucoside residues as a lactose mimic.
Molecules 26 04932 g001
Molecules 26 04932 g002 550
Figure 2. Amino acid building blocks utilized to suppress aspartimide formation.
Figure 2. Amino acid building blocks utilized to suppress aspartimide formation.
Molecules 26 04932 g002
Molecules 26 04932 g003 550
Figure 3. Resin and coupling protocols utilized in the synthesis.
Figure 3. Resin and coupling protocols utilized in the synthesis.
Molecules 26 04932 g003
Molecules 26 04932 sch001 550
Scheme 1. General synthetic scheme for glycopeptide preparation. The glycopeptides were constructed on a Rink amide HMBA resin. Coupling protocols using DIC/6-Cl-HOBt or HBTU/NMM were utilized depending on the identity of the amino acids. The acetate protecting groups in the glycoside moieties were cleanly removed using 50% H2N–NH2•H2O in NMP. Global side chain deprotection and resin cleavage were carried out using a cocktail consisting of TFA/Et3SiH/anisole/DCM/H2O.
Scheme 1. General synthetic scheme for glycopeptide preparation. The glycopeptides were constructed on a Rink amide HMBA resin. Coupling protocols using DIC/6-Cl-HOBt or HBTU/NMM were utilized depending on the identity of the amino acids. The acetate protecting groups in the glycoside moieties were cleanly removed using 50% H2N–NH2•H2O in NMP. Global side chain deprotection and resin cleavage were carried out using a cocktail consisting of TFA/Et3SiH/anisole/DCM/H2O.
Molecules 26 04932 sch001
Table
Table 1. Structures of relevant peptides/glycopeptides for this study.
Table 1. Structures of relevant peptides/glycopeptides for this study.
CompoundStructure 1
PACAP1–38HSDGIFTDSY10SRYRKQMAVK20KYLAAVLGKRYKQRVKNK-CONH2
PACAP1–27HSDGIFTDSY10SRYRKQMAVK20KYLAAVL-CONH2
PACAP1–23HSDGIFTDSY10SRYRKQMAVK20KYL-CONH2
VIPHSDAVFTDNY10TRLRKQMAVK20KYLNSILN-CONH2
1HSDGIFTDSY10SRYRKQÑAVK20KYL-Ser(OH)-CONH2
2HSDGIFTDSY10SRYRKQÑAVK20KYL-Ser(Glc)-CONH2
3HSDGIFTDSY10SRYRKQÑAVK20KYL-Ser(Glc)-Ser(Glc)-CONH2
4HSDGIFTDSY10SRYRKQÑAVK20KYL-Ser(Lac)-CONH2
5HSDAIFTDSY10SRYRKQÑAVK20KYL-Ser(Lac)-CONH2
6HSD∑IFTDSY10SRYRKQÑAVK20KYL-Ser(Lac)-CONH2
1 Ñ = Νorvaline, Σ = Sarcosine.
Table
Table 2. Characterization of synthetic PACAP1–23 glycopeptides.
Table 2. Characterization of synthetic PACAP1–23 glycopeptides.
CompoundMolecular
Formula		Calculated Mass1 Experimental
Mass →		2 HPLC Ret. Time (min)
1 C128H199N37O372846.48570.71 (M+5H)5+2848.5513.98
2 C134H209N37O423008.54603.09 (M+5H)5+3010.4513.60
3 C143H224N38O493257.62652.76 (M+5H)5+3258.8013.24
4 C140H219N37O473170.59793.93 (M+4H)4+3171.7213.59
5 C141H221N37O473184.60638.09 (M+5H)5+3185.4513.48
6 C141H221N37O473184.60638.01 (M+5H)5+3185.4513.42
1 m/z values evaluated by ESI-MS. 2 HPLC conditions: 5–80% CH3CN vs. 0.1% CF3COOH in H2O over 60 min.
Table
Table 3. In vitro cAMP stimulation on PAC1-CHO, VPAC1-CHO, and VPAC2-CHO cells in the presence of PACAP1–23. Glycopeptide analogues.
Table 3. In vitro cAMP stimulation on PAC1-CHO, VPAC1-CHO, and VPAC2-CHO cells in the presence of PACAP1–23. Glycopeptide analogues.
Drug CandidatePAC1VPAC1VPAC2
EC50 (nM)EMAX (%)EC50 (nM)EMAX (%)EC50 (nM)EMAX (%)
PACAP1–2720, 9.0100, 10030, 6.5100, 10071, 29100, 100
12.21985.242571111
20.6418137104567110
31.9152261477369128
41.8174211181943169
57.018138124321139
667134195107NCNC
NC = No convergence.
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Share and Cite

MDPI and ACS Style

Apostol, C.R.; Tanguturi, P.; Szabò, L.Z.; Varela, D.; Gilmartin, T.; Streicher, J.M.; Polt, R. Synthesis and In Vitro Characterization of Glycopeptide Drug Candidates Related to PACAP1–23. Molecules 2021, 26, 4932. https://doi.org/10.3390/molecules26164932

AMA Style

Apostol CR, Tanguturi P, Szabò LZ, Varela D, Gilmartin T, Streicher JM, Polt R. Synthesis and In Vitro Characterization of Glycopeptide Drug Candidates Related to PACAP1–23. Molecules. 2021; 26(16):4932. https://doi.org/10.3390/molecules26164932

Chicago/Turabian Style

Apostol, Christopher R., Parthasaradhireddy Tanguturi, Lajos Z. Szabò, Daniel Varela, Thiago Gilmartin, John M. Streicher, and Robin Polt. 2021. "Synthesis and In Vitro Characterization of Glycopeptide Drug Candidates Related to PACAP1–23" Molecules 26, no. 16: 4932. https://doi.org/10.3390/molecules26164932

APA Style

Apostol, C. R., Tanguturi, P., Szabò, L. Z., Varela, D., Gilmartin, T., Streicher, J. M., & Polt, R. (2021). Synthesis and In Vitro Characterization of Glycopeptide Drug Candidates Related to PACAP1–23. Molecules, 26(16), 4932. https://doi.org/10.3390/molecules26164932

Article Metrics

Back to TopTop