Synthesis, conformational and pharmacological studies of glycosylated chimeric peptides of Met-enkephalin and FMRFa

Brain Research Bulletin 68 (2006) 329–334

Synthesis, conformational and pharmacological studies of glycosylated chimeric peptides of Met-enkephalin and FMRFa Gita Masand a , Kashif Hanif a , Somdutta Sen a , Aarif Ahsan b , Souvik Maiti c , Santosh Pasha a,∗ a

Peptide Synthesis Laboratory, Institute of Genomics and Integrative Biology, Mall Road, Delhi 110007, India Functional Genomics Unit, Institute of Genomics and Integrative Biology, Mall Road, Delhi 110007, India c Structural Biology Unit, Institute of Genomics and Integrative Biology, Mall Road, Delhi 110007, India

b

Received 29 June 2005; accepted 12 September 2005 Available online 7 October 2005

Abstract Our previous study showed that a chimeric peptide of Met-enkephalin and FMRFamide, YFa (YGGFMKKKFMRFa) not only caused antinociception and potentiated morphine analgesia but also blocked the development of tolerance and physical dependence. In the continuation of that study three chimeric analogues of YFa, [Ser5 ]YFa, [O-Glu-Ser5 ]YFa and [O-Gal-Ser5 ]YFa, were synthesized. To increase the bioavailability and penetration of blood brain barrier (BBB), glycosylated analogues, [O-Glu-Ser5 ]YFa and [O-Gal-Ser5 ]YFa, have been synthesized by solid phase peptide synthesis by building block method using anomeric acetate activation method. Circular dichroism studies showed that all the three chimeric peptides are stable and have a propensity for adopting helical conformation in the presence of membrane mimicking solvent. In comparison of parent chimeric peptide YFa, helicity of [Ser5 ]YFa, [O-Glu-Ser5 ]YFa and [O-Gal-Ser5 ]YFa has decreased. Pharmacological studies using tail-flick latency in mice showed that [O-Glu-Ser5 ]YFa have increased analgesia and bioavailability in comparison of [O-Gal-Ser5 ]YFa and non-glycosylated analogue [Ser5 ]YFa. Exhibition of enhanced analgesia by [O-Glu-Ser5 ]YFa as compared to [O-Gal-Ser5 ]YFa seems to be due to preference of GLUT-1 transporter system for glucose. © 2005 Elsevier Inc. All rights reserved. Keywords: Chimeric peptides; Met-enkephalin; FMRFa; Bioavailability; Glycosylated peptides

1. Introduction Bioavailability of peptide-based drugs to the brain is limited due to poor metabolic stability, or inability to cross the blood brain barrier (BBB) [5]. Peptide-based drugs and neuromodulators, administered peripherally, fail significantly to affect their target cells within the brain [17] because BBB that is characterized by high electrical resistance and low paracellular diffusion [11], excludes most of the peptides from reaching the brain [16]. A number of strategies have been used to improve the uptake of peptides through the BBB [2,22] and one of them is conjugation of the peptide with glucose moiety that may function as transport vector [17,4]. Glycosylation has proven to be a useful methodology for enhancing biodistribution of peptides to the brain. Improved analgesia has been reported for glycosylated deltrophin [14] and

∗

Corresponding author. Tel.: +91 11 27666156; fax: +91 11 27667471. E-mail address: [email protected] (S. Pasha).

0361-9230/$ – see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.brainresbull.2005.09.009

morphine [15]. A number of different sugar moieties have been investigated including glucose, galactose and xylose [4]. The improved analgesia exhibited by glycosylated-opioids may be due to increased bioavailability [18], reduced clearance [6] or improved BBB transport [4]. The role of opioids and antiopioids in modulation of antinociception becomes intriguing by endogenously present naloxone sensitive opioid Methionine-enkephalin-Arg6 -Phe7 (MERF) [10]. MERF consists of sequence of opioid Metenkephalin (at N-terminal) and Arg6 -Phe7 , a dipeptide that is an integral part of FMRFa/NPFF family of antiopioid peptides (at C-terminal) [7] and it binds to multiple opioid binding sites [1,12]. Based on MERF, two chimeric peptides of Met-enkephalin and FMRFa, YGGFMKKKFMRFamide (YFa) and [d-Ala2 ]YAGFMKKKFMRFamide ([d-Ala2 ]YFa) were designed and synthesized. In our previous studies [8,9], it was demonstrated that intraperitoneal (i.p.) administration of YFa and intracerebroventicular (i.c.v.) administration of [d-Ala2 ]YFa induced a naloxone reversible dose-dependent increase in tail-flick latency

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in mice, showing an antinociceptive effect due to involvement of opioid receptors. The chimeric peptides also potentiated morphine-induced antinociception and attenuated the development of tolerance to the antinociceptive action of morphine. This suggested that, besides opioid receptors, YFa and [d-Ala2]YFa are probably behaving as putative antagonists and not letting endogenous antiopioids bind to their receptors or they are down regulating antiopioid receptors by activating Gi proteins. Polt et al. [17] showed that l-serinyl ␤-d-glucoside analogues of [Met5 ] enkaphalin are transported across the BBB to bind to the opioid receptors and show enhanced analgesia as compared to non-glycosylated peptides. So to enhance the biodistribution and to study the effect of glycosylation on chimeric peptides, three serine-based analogues of YFa were designed in which Met5 has been replaced by Ser5 since position five can tolerate change without loss of activity [13]. Three analogues of YFa are as follows: (i) YGGFSKKKFMRFamide ([Ser5 ]YFa), wherein the Met5 position has been changed with Ser5 that can act as a site for glycosylation. (ii) [O-Glu-Ser5 ]YFa in which ␤-d-glucose is attached to Ser5 so as to utilize GLUT-1 transport system for the transport of chimeric peptide across the BBB. (iii) [O-Gal-Ser5 ]YFa in which ␤-d-glucose is replaced by ␤-dgalactose at Ser5 so that a comparison can be made between the two glycosylated analogues of chimeric peptides on their peripheral administration. These analogues were characterized by MALDI-Tof and conformations of three analogues of YFa were compared by circular dichroism (CD) studies. Pharmacological studies were performed by tail-flick method via intra peritoneal (i.p.) administration of all the three analogues in mice. 2. Materials and methods 2.1. Synthesis of glycosylated chimeric peptides 2.1.1. Chemical synthesis of building block of N-α-(9-fluorenylmethylcarbonyl)-3-O-(2,3,4,6-tetra-O-acetyl-β-d-glucopyranosyl)-l-serine [or Nα-(9-fluorenylmethyl-carbonyl)-3-O-(2,3,4,6-tetra-O-acetyl-β-d-galactopyranosyl)-l-serine] by activation of anomeric acetate The Lewis acid [BF3 ·Et2 O, 7.2 mmol] was added to a solution of pentaacetate glucose [2.3 mmol] and Fmoc-Serine [3 mmol] in CH2 Cl2 . The mixture was

stirred for 1 h and the solution was then washed with aqueous HCl (1 M), dried and concentrated (Scheme 1). The residue was purified by HPLC. 2.1.2. Solid-phase synthesis of glycopeptides Peptides [Ser5 ]YFa, [O-Glu-Ser5 ]YFa and [O-Gal-Ser5 ]YFa were assembled on the Rink amide methylbenzylhydramine resin (0.56 mmol/g substitution). All the solid phase reactions were carried out manually in a sintered glass tube. Peptide condensation was facilitated by using excess Fmoc amino acid (4 eq.) activated with DIPCDI, HOBt in N,N -dimethylformamide. At the fifth position instead of Fmoc-Ser-OH, N-␣-(9-Fmoc)-3-O-(2,3,4,6-tetra-O-acetyl␤-d-glucopyranosyl)-l-serine [or N-␣-(9-Fmoc)-3-O-(2,3,4,6-tetra-O-acetyl␤-d-galactopyranosyl)-l-serine], synthesized in previous step, was introduced for the synthesis of glycopeptides. Stepwise removal of N-Fmoc protecting group from growing chain of peptides was achieved by 20% piperidine in DMF. Synthesized glycopeptides were released from resin by treating with TFA (95%) and the reaction was quenched with ethanedithiol and crystalline phenol. After cleavage glycosylated chimeric peptides were deacetylated by dissolving the peptides in dry methanol (10 ml) followed by addition of methanolic sodium methoxide (6 M) till the pH of the solution reached 10. After 30 min at room temperature, the peptide solution was concentrated under reduced pressure. Products were purified by HPLC on semi preparative reverse phase column using isocratic gradient of acetonitrile (0.05%TFA):water (0.05%TFA)::70:30. The correct peptide sequences were confirmed by automated peptide sequencing (490 Applied Biosystems).

2.2. Mass spectrometry The mass analysis of three chimeric peptides [Ser5 ]YFa, [O-Glu-Ser5 ]YFa and [O-Gal-Ser5 ]YFa was carried out using MALDI-Tof (Kratos) in the positive ion mode using ␣-cyano-4-hydroxycinnamic acid as the matrix.

2.3. Circular dichroism studies The conformational studies were done by CD (Jasco J-715). CD spectra of YFa, [Ser5 ]YFa, [O-Glu-Ser5 ]YFa and [O-Gal-Ser5 ]YFa (1 mmole) were recorded in 3.3 mM Sodium chloride-Sodium citrate buffer with varying concentrations of trifluoroethanol (TFE) by using spectropolarimeter calibrated with d-10-camphorsulfonic acid in a cell of 0.5 pathlength at room temperature. CD band intensities are represented as mean residue ellipticity.

2.4. Pharmacological studies For study of antinociception, a locally made analgesiometer was used. The inhibition of tail-flick response was expressed as percentage maximum possible effect (%MPE) that was calculated as [(T1 − T0 )/(T2 − T0 )] × 100 where T0 and T1 were the tail-flick latency before and after the injection of peptide and T2 was the cut-off time. The antinociceptive response was measured by the radiant tail-flick test as described previously [8,9]. At the beginning of the study, the intensity of heat stimulus in the tail-flick apparatus was adjusted so as to elicit a response in control or untreated animal within 3–5 s. To minimize tail skin tissue damage, the cut-off was set at 10 s. Seven mice were used for each treatment

Scheme 1. Synthesis of N-␣-(9-fluorenylmethylcarbonyl)-3-O-(2,3,4,6-tetra-O-acetyl-␤-d-glucopyranosyl)-l-serine by anomeric acetate activation method.

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group. Mice were given three baseline trials, each separated by 10 min, and they were then injected with either saline (0.1 ml) or the peptide and tested 5, 15, 30, 45 and 60 min later.

2.5. Statistical analysis SPSS statistical software for windows (release 10) was used to carry out the statistical analysis. The variables were analyzed for normal distribution by Kolmogrov–Smirnov test. Variables that did not follow normal distribution were considered as log normal values and analyzed by Kruskal–Wallis and Mann–Whitney tests. MPE% of chimeric peptides were expressed as mean ± S.E.M. Differences within the effects of chimeric peptides at different time points as compared to saline and between the chimeric peptides were analyzed by one-way analysis of variance and unpaired t test with one-tailed values. A P-value of <0.05 was considered statistically significant.

Fig. 2. CD spectrum of chimeric peptide [Ser5 ]YFa (1 mmol). The spectra were recorded in different concentrations of TFE.

3. Results 3.1. Synthesis of [Ser5 ]YFa, [O-Glu-Ser5 ]YFa and [O-Gal-Ser5 ]YFa The first step in the synthesis of [O-Glu-Ser5 ]YFa and [OGal-Ser5 ]YFa is the synthesis of building block, i.e. glycosylated serine residue (Scheme 1). Using N-␣-(9-Fmoc)-3-O-(2,3,4,6tetra-O-acetyl-␤-d-glucopyranosyl)-l-serine [or N-␣-(9-Fmoc) -3-O-(2,3,4,6-tetra-O-acetyl-␤-d-galactopyranosyl)-l-serine] instead of Fmoc-serine, at the fifth position, [O-Glu-Ser5 ]YFa (or [O-Gal-Ser5 ]YFa) were synthesized with a yield of 30%. All three chimeric peptides [Ser5 ]YFa, [O-Glu-Ser5 ]YFa and [OGal-Ser5 ]YFa were analyzed by mass spectrometry. [Ser5 ]YFa showed m/z [M + H]+ 1494.4 (MW = 1494). m/z [M + H]+ of [OGlu-Ser5 ]YFa was found 1656 (MW = 1656). [O-Gal-Ser5 ]YFa was having m/z [M + H]+ of 1656.3 (MW = 1656). 3.2. CD studies The conformational studies were done by CD with varying concentrations of trifluoroethanol (TFE), a membrane mimicking solvent. In buffer YFa (Fig. 1), [Ser5 ]YFa (Fig. 2), [O-GluSer5 ]YFa (Fig. 3), [O-Gal-Ser5 ]YFa (Fig. 4) showed the same type of spectroscopic profile. These peptides show an unordered

Fig. 1. CD spectrum of chimeric peptide YFa (1 mmol). The spectra were recorded in different concentrations of TFE.

Fig. 3. CD spectra of chimeric peptide [O-Glu-Ser5 ]YFa (1 mmol). The spectra were recorded in different concentrations of TFE.

random coil conformation. On addition of increasing concentration of TFE, chimeric peptides displayed propensity for adopting helical conformation with minima around 206 and 222 nm [23]. In comparison of YFa, [Ser5 ]YFa, [O-Glu-Ser5 ]YFa, [O-GalSer5 ]YFa show less helicity upon increasing the concentration of TFE (Fig. 5).

Fig. 4. CD spectra of chimeric peptide [O-Gal-Ser5 ]YFa (1 mmol). The spectra were recorded in different concentrations of TFE.

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Fig. 5. Comparison of CD profile of YFa, [Ser5 ]YFa, [O-Glu-Ser5 ]YFa, [OGal-Ser5 ]YFa at different concentrations of TFE.

Fig. 6. Effect of intraperitoneal administration of chimeric peptide [Ser5 ]YFa and saline in mice in the radiant-heat tail-flick test. Each point represents the mean ± S.E. of seven animals. * Significant difference from the saline treated group (p < 0.05). Abbreviation: MPE, maximum possible effect.

3.3. Pharmacological studies All the peptides were administered through peripheral route, i.e. intraperitoneally (i.p.). The onset of analgesic activity was quick at both 40 and 60 mg/kg. At 40 and 60 mg/kg, respectively, antinociceptive effect of [Ser5 ]YFa (21.52 ± 7.61% MPE and 33.94 ± 4.6% MPE, p < 0.05, respectively, Fig. 6), of [O-Glu-Ser5 ]YFa (34.82 ± 6.69% MPE, p < 0.05 and 25.89 ± 8.03% MPE, respectively, Fig. 7) and of [O-Gal-Ser5 ]YFa (22.09 ± 4.26% MPE, p < 0.05 and 27.90 ± 7.75% MPE, respectively, Fig. 8) appeared just 5 min after administration of chimeric peptides. At 15 min at 40 and

Fig. 7. Effect of intraperitoneal administration of chimeric peptide [O-GluSer5 ]YFa and saline in mice in the radiant-heat tail-flick test. Each point represents the mean ± S.E. of seven animals. * Significant difference from the saline treated group (p < 0.05). Abbreviation: MPE, maximum possible effect.

Fig. 8. Effect of intraperitoneal administration of chimeric peptide [O-GalSer5 ]YFa and saline in mice in the radiant-heat tail-flick test. Each point represents the mean ± S.E. of seven animals. * Significant difference from the saline treated group (p < 0.05). Abbreviation: MPE, maximum possible effect.

60 mg/kg, respectively, the maximum antinociceptive effect of both [O-Glu-Ser5 ]YFa (48.66 ± 7.5% MPE, p < 0.05 and 57.85 ± 8.03% MPE, p < 0.05, respectively, Fig. 7) and [O-GalSer5 ]YFa (44.57 ± 8.13% MPE, p < 0.05 and 50.38 ± 8.13% MPE, p < 0.05, respectively, Fig. 8) was more than nonglycosylated [Ser5 ]YFa (26.15 ± 7.41% MPE and 41.05 ± 3.31% MPE, p < 0.01, respectively, Fig. 6). The antinociceptive effect of both glycosylated analogues lasted longer as compared to [Ser5 ]YFa. At 60 min at 40 and 60 mg/kg, respectively, the antinociceptive effect of both [O-Glu-Ser5 ]YFa (19.64 ± 8.03% MPE and 36.60 ± 8.03% MPE, p < 0.05, respectively, Fig. 7) and [OGal-Ser5 ]YFa (20.54 ± 7.36% MPE and 26.35 ± 7.36% MPE, respectively, Fig. 8) was more than [Ser5 ]YFa (13.74 ± 5.29% MPE and 13.90 ± 6.9% MPE, respectively, Fig. 6). At 40 and 60 mg/kg, on comparison, [O-Glu-Ser5 ]YFa shows an edge over [O-Gal-Ser5 ]YFa and [Ser5 ]YFa. At 40 mg/kg, the antinociceptive effect of [O-Glu-Ser5 ]YFa is more than that of [Ser5 ]YFa and YFa [8] but not very much conspicious as compared to [OGal-Ser5 ]YFa (Fig. 9A). At 60 mg/kg, the antinociceptive effect of [O-Glu-Ser5 ]YFa is more and shows longer bioavailability than that of [O-Gal-Ser5 ]YFa and [Ser5 ]YFa but YFa exhibits more potency and longevity in antinociception (Fig. 9B). 4. Discussion This study was conducted to evaluate the effect of glycosylation on conformation and antinociceptive properties of chimeric peptides. At fifth position Met was replaced by Ser, which is used as a site for glycosylation. Glycosylation of chimeric peptides was achieved by anomeric acetate activation. Though the yield in anomeric acetate activation was only 30% but anomeric activation gave considerable ease as peracetylated sugars are available in one step and the carboxylic acid of the glycosylated amino acid is unprotected which is easy in coupling methods used to introduce the building blocks into a peptide. Further, the bye-products could be removed easily. To assess the effect of replacement of Met by Ser at fifth position and effect of glycosylation on conformation, CD studies were done. CD data showed even after the replacement of Met by Ser at fifth position native conformation of the chimeric peptides is retained and YFa (Fig. 1), [Ser5 ]YFa (Fig. 2), [O-GluSer5 ]YFa (Fig. 3) [O-Gal-Ser5 ]YFa (Fig. 4) showed a tendency

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Fig. 9. Comparison of effect of intraperitoneal administration of chimeric peptides YFa, [Ser5 ]YFa, [O-Gal-Ser5 ]YFa and [O-Gal-Ser5 ]YFa at 40 mg/kg (A) and 60 mg/kg (B). Each point represents the mean ± S.E. of seven animals.

to adopt ␣-helical conformation in presence of membrane mimicking solvent. The tendency to adopt helical conformation in presence of TFE could be attributed to the proton donating nature of TFE that facilitates intramolecular hydrogen bonding more than intermolecular hydrogen bonding. It is evident from CD studies that on increasing concentration of TFE, YFa exhibits highest tendency to gain helical conformation (Fig. 5) resulting in more ability to diffuse across the BBB as the hydrophobic helix makes the membrane permeable [3]. Comparing to chimeric peptides YFa and [Ser5 ]YFa, the glycosylated analogues show an extended random coil with less alpha helical propensity (Fig. 5) indicating that native structure is stable as compared to chimeric peptides YFa and [Ser5 ]YFa. Helicity of both [O-Glu-Ser 5 ]YFa and [O-Gal-Ser 5 ]YFa have come down in comparison of YFa and [Ser5 ]YFa upon increasing the concentration of TFE (Fig. 5) because bulky carbohydrate moiety possibly posing steric hindrance to the formation of TFEinduced secondary structure [20]. All the three chimeric peptides were administered intraperitoneally in doses of 40 and 60 mg/kg. The pharmacological data of [Ser5 ]YFa (Fig. 6), [O-Glu-Ser5 ]YFa (Fig. 7) and [O-GalSer5 ]YFa (Fig. 8) indicate that these peptides, at both 40 and 60 mg/kg, show dose-dependent antinociception when administered intraperitonially indicating the central effect, which implies penetration of the BBB by all the three chimeric peptides. The quick onset of antinociceptive effect (in just 5 min) seems to be due to immediate migration of peptides from the i.p. space (peripheral region) into the CNS through BBB. This migration is likely to be facilitated by rapid diffusion of glycosylated chimeric peptides because glycosylated peptides have

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been found to be more water soluble than their nonglycosylated peptide counterparts [17]. It seems that glycosylation has imparted more bioavailability to both glycosylated analogues in comparison of [Ser5 ]YFa and more ability to be transported by GLUT-1 transporter system resulting in more and prolonged antinociception. Due to increased bioavailability, the antinociceptive effect of both [O-Glu-Ser5 ]YFa (19.64 ± 8.03% MPE and 36.60 ± 8.03% MPE, p < 0.05, respectively, Fig. 7) and [OGal-Ser5 ]YFa (20.54 ± 7.36% MPE and 26.35 ± 7.36% MPE, respectively, Fig. 8) was more than [Ser5 ]YFa (13.74 ± 5.29% MPE and 13.90 ± 6.9% MPE, respectively, Fig. 6) at 60 min at both 40 and 60 mg/kg, respectively. The antinociceptive effect of [O-Glu-Ser5 ]YFa is more than [O-Gal-Ser5 ]YFa in terms of both potency and longevity at both doses. At 60 mg/kg this fact is clearly accentuated when [O-Glu-Ser5 ]YFa clearly surpasses both [O-Gal-Ser5 ]YFa and [Ser5 ]YFa in antinociception as well as bioavailability (Fig. 9A and B). We suggest that diffusion may be responsible for the different potencies, a fact strengthened by other studies also [19]. It could be that diffusion of ␤-d-glucose linked chimeric peptide is facilitated by the glucose transporter GLUT-1, which may reject the ␤-d-galactose as a preferable substrate inhibiting its entry into the brain to some extent. On comparing with YFa it is found that YFa, in terms of antinociception, is more potent than [Ser5 ]YFa (Fig. 9A and B) though both are non-glycosylated. Lower analgesia by [Ser5 ]YFa may be due to its less helicity after replacement of Met (in YFa) by Ser (Fig. 5). Less helical peptides exhibit reduced bilayer-disturbing activity, showing that hydrophobic helix is decisive for binding and inducing permeability in membrane [3]. Moreover, the hydroxyl group of Ser tends to promote hydrogen bonding with solvating water leading to concomitant decrease in the lipophilicity with a resultant decrease in membrane permeability [21] and consequently in lower antinociception by [Ser5 ]YFa. On the other hand, after glycosylation both [O-Glu-Ser5 ]YFa and [O-Gal-Ser5 ]YFa showed more analgesia than parent compound YFa and [Ser5 ]YFa at 40 mg/kg. This again confirms that glycosylation has improved antinociception as well bioavailability. At 60 mg/kg antinociception produced by [O-Glu-Ser5 ]YFa and [O-Gal-Ser5 ] was still higher than [Ser5 ]YFa but YFa seems to be more potent and has the highest antinociception (Fig. 9B). The more antinociception by YFa at 60 mg/kg, in comparison of glycosylated analogues, needs further study. Though it can be speculated that at 60 mg/kg the more diffusibility of YFa, due to higher helicity, across BBB seems to surpass GLUT-1 mediated transport of [O-Glu-Ser5 ]YFa and [O-Gal-Ser5 ]YFa and results in higher antinociception by YFa in comparison of [O-Glu-Ser5 ]YFa and [O-Gal-Ser5 ]YFa. In our previous study [8,9], we have shown that administration of chimeric peptides not only potentiated the analgesia but also attenuated the development of tolerance by morphine. The results of present study show that after glycosylation, helicity of both glycosylated analogues has decreased in comparison of nonglycosylated analogues upon increasing the concentration of TFE. Both glycosylated chimeric peptides [O-Glu-Ser5 ]YFa and [O-Gal-Ser5 ]YFa show more prolong and higher antinoci-

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