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Properties of myelin altered peptide ligand cyclo(87-99)(Ala91,Ala96)MBP87-99 render it a promising drug lead for immunotherapy of multiple sclerosis

European Journal of Medicinal Chemistry, August 2015, Pages 13 - 23

Abstract

Multiple sclerosis (MS) is an inflammatory, demyelinating disease of the central nervous system, and it has been established that autoreactive T helper (Th) cells play a crucial role in its pathogenesis. Myelin basic protein (MBP) epitopes are major autoantigens in MS, and the sequence MBP87-99 is an immunodominant epitope. We have previously reported that MBP87-99 peptides with modifications at principal T-cell receptor (TCR) contact sites suppressed the induction of EAE symptoms in rats and SJL/J mice, diverted the immune response from Th1 to Th2 and generated antibodies that did not cross react with the native MBP protein. In this study, the linear and cyclic analogs of the MBP87-99 epitope, namely linear (Ala91,Ala96)MBP87-99 (P2) and cyclo(87-99)(Ala91,Ala96)MBP87-99 (P3), were evaluated for their binding to HLA-DR4, stability to lysosomal enzymes, their effect on cytokine secretion by peripheral blood mononuclear cells (PBMC) derived from MS patients or healthy subjects (controls), and their effect in rat EAE. P1 peptide (wild-type, MBP87-99) was used as control. P2 and P3 did not alter significantly the cytokine secretion by control PBMC, in contrast to P1 that induced moderate IL-10 production. In MS PBMC, P2 and P3 induced the production of IL-2 and IFN-γ, with a simultaneous decrease of IL-10, whereas P1 caused a reduction of IL-10 secretion only. The cellular response to P3 indicated that cyclization did not affect the critical TCR contact sites in MS PBMC. Interestingly, the cyclic P3 analog was found to be a stronger binder to HLA-DR4 compared to linear P2. Moreover, cyclic P3 was more stable to proteolysis compared to linear P2. Finally, both P2 and P3 suppressed EAE induced by an encephalitogenic guinea pig MBP74-85 epitope in Lewis rats whereas P1 failed to do so. In conclusion, cyclization of myelin altered peptide ligand (Ala91,Ala96)MBP87-99 improved binding affinity to HLA-DR4, resistance to proteolysis and antigen-specific immunomodulation, rendering cyclo(87-99)(Ala91,Ala96)MBP87-99 an important candidate drug for MS immunotherapy.

Graphical abstract

 

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Highlights

 

  • Cyclo(87-99)(Ala91,Ala96)MBP87-99 binds well to HLA-DR4.
  • Is resistant to proteolysis.
  • Fully and specifically suppresses EAE in Lewis rats.
  • Exerts antigen-specific immunomodulation on PBMC from MS patients.

Keywords: Multiple sclerosis, Myelin basic protein peptides, Experimental autoimmune encephalomyelitis, Cyclic peptide, Lysosomal enzymes, HLA-DR4(DRB*0401), Cytokines.

Abbreviations: APL - altered peptide ligand, MBP - myelin basic protein, PLP - proteolipid protein, MOG - myelin oligodendrocyte glycoprotein, TCR - T-cell receptor, EAE - experimental autoimmune encephalomyelitis, MS - multiple sclerosis, HLA-DR4 - human leukocyte antigen DR4 (DRB*0401).

1. Introduction

MS is chronic inflammatory, demyelinating disease of the central nervous system (CNS) [1] . Auto-reactive Th cells are considered responsible for the initiation and maintenance of auto-reactivity to CNS myelin, with downstream involvement of auto-antibodies, cytotoxic T-cells, NK cells, macrophages and microglial cells [2] . Autoimmune targets in MS include the myelin basic protein (MBP), proteolipid protein (PLP) and myelin oligodendrocyte glycoprotein (MOG). MBP constitutes enchephalitogenic Th-cell epitopes, a major one being MBP87-99 [3], [4], and [5].

MS is extremely heterogeneous in its initial presentation, rate and severity of relapses, pattern of disease progression, underlying immunopathology and response to disease modifying treatments [6] and [7], and, for these reasons, new therapeutic approaches are constantly sought. Such an approach, involves the design and use of cyclic peptide analogs of identified disease-associated myelin epitopes to modify T-cell responses. Our group has been extensively involved in the rational design, synthesis and biological evaluation of major MBP, PLP and MOG epitopes with emphasis on the MBP83-99 and MBP87-99 sequences. These studies include conformational analysis, appropriate cyclizations and pharmacology in a number of assays towards the selection of suitable candidates for clinical trials [8] and [9].

The use of cyclic instead of linear peptides as therapeutic entities has many advantages, including (i) stability and resistance to enzymatic degradation, (ii) increased receptor selectivity, consequently resulting in an improved pharmacological profile, (iii) locked conformation allowing identification of active sites, (iv) design of useful templates for the development of a non-peptide (mimetic) drug for oral administration, with potential oral activity if appropriately constrained [4], [10], and [11]. We have previously described [4] and [12] the rational design and synthesis of cyclic peptides, based on a combination of conformational analysis studies and theoretical calculations carried out on the linear MBP epitope 87-99 using 2D ROESY NMR spectroscopy.

In this study, we report the biological evaluation of MBP87-99 peptides with modifications at TCR contact sites. The substitutions were at the Lysine residue at position 91 (primary TCR contact) and Proline residue at position 96 with Alanine. Alanine was the selection for substitutions, because positions 91 and 96 are critical for TCR contact leading to antagonistic effects, as demonstrated by in vitro [13] , and in vivo [14] studies.

The importance of these two positions was later confirmed by x-ray crystallographic studies that showed that loss of H-bond contacts between the peptide and T-cell receptor (TCR) resulted in the dissociation of the trimolecular complex comprised of MHC class I-superagonist peptide-TCR [15] and [16]. It is presumed that similar contacts between TCR and peptides bound to MHC class II could also result in antagonist activity [17] . These observations are further supported by our previous studies with SJL/J mice that showed that injection of cyclo(87-99)(Ala91,Ala96)MBP87-99 resulted in reduced Th1 cytokine production [18] and [19]. Furthermore, using an EAE chronic pain model it was shown that vaccination with this analog ameliorated EAE and selectively mediated an analgesic effect on the animals [20] .

In this work, the linear (Ala91,Ala96)MBP87-99 and the cyclo(87-99)(Ala91,Ala96)MBP87-99 APLs were further evaluated for their binding to HLA-DR4, stability to lysosomal enzymes, their effect on cytokine secretion by peripheral blood mononuclear cells (PBMC) derived from MS patients or healthy subjects (controls), and their ability to induce or suppress experimental allergic/autoimmune encephalomyelitis (EAE) in rats.

2. Results

2.1. Resistance of peptides to proteolysis

To determine the stability of peptides P1-P3 to lysosomal enzymes, we used a lysosomal fraction of the EBV-transformed B cell line BSM that contains >50 different lysosomal enzymes ( Fig. 1 A), the aspartic protease Cathepsin D ( Fig. 1 B) and AEP ( Fig. 1 C), a cysteine proteinase that specifically cleaves at the N terminus and is an essential enzyme for antigen processing. The linear peptides P1 and P2 were not stable when digested with the lysosomal fraction, whereas the cyclic peptide was clearly stable. P2 and P3 were more stable to digestion with AEP compared with P1. All 3 peptides were stable to digestion with Cathepspin D. Overall, P3 exhibited the highest resistance to enzymatic degradation ( Fig. 1 ).

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Fig. 1 Stability of P1-P3 MBP peptide analogs (linear and cyclic) in the presence of lysosomal enzymes. A, lysosomal fraction; B, Cathepsin D; C, AEP. Stability (%) of the peptides after digestion at different time points (0 min, dark grey; 30 min, grey; 90 min, light grey). The experiments were done in triplicate and the results are given as mean (±SEM).

2.2. Binding of peptides to soluble HLA-DR4

Soluble HLA-DR4 (DRB1*0401) was used to determine the binding of P1-P3 peptides through competition with a fluorescent AMCA-labeled allele-specific HA306-318 peptide (cf. M&M and Table 1 ). As shown in Fig. 2 , P1 bound to HLA-DR4 (DRB1*0401) with high affinity, i.e. at 5- and 10-fold excess it competed the binding of HA306-318 by 69% and at 50-fold excess by 85%. In contrast, P2 bound weakly to HLA-DR4 (DRB1*0401), i.e. at 5-fold excess it failed to compete the binding of HA306-318, at 10-fold excess it competed the binding of HA306-318 by 6% and at 50-fold excess by 16%. Interestingly, cyclic P3 bound to HLA-DR4 (DRB1*0401) with moderate affinity which was much higher compared with its linear counterpart P2, i.e. at 5-fold excess it competed the binding of HA306-318 by 32%, at 20-fold excess by 38% and at 50-fold excess by 47%.

Table 1 Peptide analogs used in the study.

ID Sequence Peptide analog
HA306-318 P306KYVKQNTLKLAT318 Control peptide for HLA-DR4 binding assays: The influenza virus haemagglutinin (HA) peptide residues 306-318
P1 V87HFFKNIVTPRTP99 MBP87-99
P2 V87HFFANIVTARTP99 MBP87-99[A91,96]
P3 cyclo(87-99) V87HFFANIVTARTP99 cyclo(87-99)MBP87-99[A91,96]
MBP74-85 Q74KSQRSQDENPV85 MBP74-85 guinea pig sequence
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Fig. 2 Binding of linear and cyclic MBP peptide analogs to HLA-DR4 (DRB1*0401) assayed by competition with the allele-specific HA306-318 peptide. Competition (%) binding at 5-fold (dark grey), 20-fold (grey) and 50-fold (light grey) excess of MBP peptides. The experiments were done in triplicate and the results are given as mean (±SEM).

2.3. Effect of peptides on PBMC isolated from MS patients and controls

Proliferation assays of PBMC isolated from 7 patients with remitting-relapsing MS and 7 controls were carried out. The cells were cultured with increasing concentrations of P1-P3. P2 and P3 did not affect control PBMC proliferation whereas P1 decreased it at a concentration >10 pg/ml. In contrast, P1 had no effect on MS PBMC proliferation, whereas cell proliferation rates decreased when MS PBMC were cultured with P2 or P3 at a concentration of 10 pg/ml ( Fig. 3 ).

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Fig. 3 In vitro proliferation of PBMC isolated from 7 MS patients and 7 controls in the presence of various concentrations of P1, P2 and P3 peptide analogs. Each PBMC sample was cultured in triplicate, at a concentration of 106 cells/ml, in the presence of each peptide (P1, P2, P3, all at 10 pg/ml) or without peptides (CM, culture medium) for 72 h. The results were plotted as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001. The T-test was employed for the pairwise comparisons of the groups. Differences between groups were considered significant if p was ≤0.05. Data were analyzed using the GraphPad Prism v. 5.03 (San Diego, CA, USA).

PBMC isolated from 7 MS patients and 7 controls were cultured for 72 h in the presence or absence of 10 pg/ml of peptides P1-P3 (effective concentration as indicated by the proliferation results, cf. Fig. 3 ). Culture supernatants were collected and the concentrations of the cytokines IL-2, IFN-γ, IL-4 and IL-10 were determined by ELISA. P2 and P3 had no effect on IL-4 secretion by MS and control PBMC. Both P2 and P3 peptides induced strongly IL-2 and IFN-γ secretion. P3 induced the secretion of higher levels of IL-2 compared with P2. In parallel, both P2 and P3 caused a reduction in IL-10 secretion by MS PBMC, thus enhancing a type-1 cytokine secretion pattern of the cells. P1 increased IL-10 secretion by control PBMC and decreased IL-10 secretion by MS PBMC ( Fig. 4 ).

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Fig. 4 Effect of peptide analogs on cytokine secretion by PBMC of controls (n = 7) and MS patients (n = 7). Each PBMC sample was cultured in triplicate, at a concentration of 106 cells/ml, in the presence of each peptide (P1, P2, P3, all at 10 pg/ml) or without peptides (CM) for 72 h. The data are expressed as the average net change in cytokine production over placebo values (CM). The results were plotted as mean ± SD. ***p < 0.001. The T-test was employed for the pairwise comparisons of the groups. Differences between groups were considered significant if p was ≤0.05. Data were analyzed using the GraphPad Prism v. 5.03 (San Diego, CA, USA).

2.4. Effect of P2 and P3 in a rat EAE animal model

The induction of EAE in female Lewis rats using the MBP74-85 peptide epitope was inhibited in the presence of P2 and P3 peptide analogs, after 8 days of co-injection ( Fig. 5 ). Linear analog P2 protected the rats when co-injected with linear MBP74-85 from 8 to 16 days of the EAE experiment, whereas cyclic P3 protected the rats completely from the disease (until the end of the observation period which lasts for 18–20 days in this acute EAE model; cf. Fig. 7 ).

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Fig. 5 EAE experiments using the linear and cyclic MBP analogues in Lewis rats. The linear epitope MBP74-85 when injected alone induced EAE at clinical score level 2. Suppression of EAE was demonstrated after 8 days of P2 and P3 co-injection. Three rats were used per immunization protocol. The results were plotted as mean ± SEM. Statistical significance after comparisons between groups of rats, using the Kruskal–Wallis test, is shown (*p < 0.05). Data were analyzed using the GraphPad Prism v. 5.03 (San Diego, CA, USA).

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Fig. 6 Paraffin sections of the spinal cord of Lewis rats, stained with hematoxylin and eosin. A & B: Representative sample of spinal cord of Lewis rat injected with linear MBP74-85 peptide (A:×200 magnification; B: x400 magnification). C. Representative sample of spinal cord of Lewis rat injected with linear MBP74-85 and cyclo(87-99)(Ala91,Ala96)MBP87-99 peptides (×400 magnification). D. Representative sample of spinal cord of Lewis rat injected with linear MBP74-85 and linear (Ala91,Ala96)MBP87-99 peptides (×200 magnification). E. Representative sample of spinal cord of a healthy Lewis rat (×200 magnification). F. The results of the analysis of the whole experimental series (3 rats/immunization protocol). The number of infiltrating lymphocytes was counted from 2 consecutive sections/point/rat, at ×400 magnification. The results were plotted as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001. The T-test was employed for the pairwise comparisons of the groups. Differences between groups were considered significant if p was ≤0.05. Data were analyzed using the GraphPad Prism v. 5.03 (San Diego, CA, USA).

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Fig. 7 Control EAE experiments to test the effect of the linear MBP87-99 epitope in Lewis rats. The linear epitope MBP74-85, when injected alone, induced EAE at clinical score level 2. When injected together with the linear MBP87-99 epitope EAE was mildly suppressed to clinical score level 1.6, demonstrated after 16 days of MBP74-85 and MBP87-99 co-injection. Three rats were used per immunization protocol. The results were plotted as mean ± SEM. Comparisons between groups of rats, using the Kruskal–Wallis test, showed no statistical significance (p > 0.05). Data were analyzed using the GraphPad Prism v. 5.03 (San Diego, CA, USA).

Tissue samples of spinal cord of rats injected with MBP74-85, showed perivascular infiltrates of mononuclear cells around small vessels, particularly between the white and the gray matter. Scattered small lymphocytes were also observed in the adjacent parenchyma ( Fig. 6 A, B, F). Significantly lower cell infiltrates were present in rats co-injected with MBP74-85 and P3 ( Fig. 6 C and F), in contrast to rats co-injected with MBP74-85 and P2 in which higher cell infiltrates were observed ( Fig. 6 D and F).

To compare the effects of P2 and P3 with P1, EAE experiments were performed to test the effect of P1 (the linear MBP87-99 epitope) in Lewis rats ( Fig. 7 ). The linear epitope MBP74-85, when injected alone, induced EAE at clinical score level 2. Co-injection of MBP74-85 with the linear MBP87-99 epitope resulted in mild suppression of EAE (clinical score level 1.6). The difference between the results of the 2 injection protocols did not reach statistical significance ( Fig. 7 ).

3. Discussion

In the present study we have evaluated the linear APL (Ala91,Ala96)MBP87-99 (P2) and its counterpart, the head-to-tail cyclic APL cyclo(87-99)(Ala91,Ala96)MBP87-99 (P3). These peptides were assayed for their stability to different proteases, their ability to bind to HLA-DR4, their in vivo effect on an EAE model, and their in vitro effect on human PBMC derived from MS patients or control subjects.

We have chosen to study the MBP87-99 sequence, because MBP peptides 83-99 and 87-99 are encephalitogenic in rodent strains susceptible to acute (Lewis rat) and chronic (SJL/J mouse) EAE [19] and [21]. In addition, MBP epitopes are major autoantigens in MS, and the MBP87-99 sequence is an immunodominant epitope [3], [4], [5], [12], and [16].

Our previous studies have shown that the APLs cyclo(91-99)(Ala96)MBP87-99 and cyclo(87-99)(Arg91,Ala96)MBP87-99 (i) could suppress the proliferation of an MBP87-99-specific CD4+ T-cell clone created in vitro from T-cells isolated from an MS patient, (ii) scored the best Th2/Th1 cytokine ratio in PBMC cultures derived from 13 MS patients, by selectively inducing the secretion of IL-10, (iii) could bind to HLA-DR4, first to be reported for cyclic MBP peptides, and (iv) were more stable to lysosomal enzymes Cathepsin B, D, and H, compared to their linear counterparts [4]. These findings demonstrated that the design and synthesis of linear and cyclic APLs could help delineate the amino acid sequences and molecular conformations that can affect antigen presentation to TCR and influence the ensuing immune response.

The development of cyclic molecules that mimic the immunomodulatory activity of MBP linear epitopes, while maintaining an advantage over regular peptides in term of stability and HLA binding, is a necessary step before these molecules can be proposed as drug leads.

There are two major approaches in the development of such molecules. One is the design and synthesis of non-peptide or constrained peptide mimetics with antagonist activity and improved pharmacological profile (stability, binding, desired biological activity) compared with the wild-type MBP87-99 linear epitope peptide. Cyclic analogs of MBP87-99 offer advantages compared with their linear counterparts that include resistance to metabolic degradation and conformational restriction, that meet the demands of improved pharmacokinetics. In this approach, mutation of diverse-associated epitopes can actively inhibit disease through the loss of H-bond contacts between the MHC-peptide and TCR trimolecular complex, which trigger the proliferation of autoreactive CD4+ T-cells implicated in MS pathogenesis [14], [15], and [16].

The other approach is the regulation of the immune response with the use of immunomodulatory epitopes alone or conjugated with reduced mannan through KLH or Lys–Gly bridge. In recent studies by our group, mannosylation of mutated human MBP83-99 peptides gave promising results because they diverted the immune responses from Th1 to Th2 in SJL/J mice and generated antibodies which did not cross react with the native MBP protein. In addition, conjugated peptides MBP83-99[Y91] and MBP83-99[F91] gave the best cytokine and antibody profile constituting promising candidate peptides for immunotherapy of MS [20] and [22].

In the EAE model that is widely used to evaluate the therapeutic potential of peptides, the actual mechanism through which the peptides prevent the development of EAE is yet unclear. The peptides can act as antagonists at the same receptor with the encephalitogenic epitope or they can bind to different receptors causing activation of Th2 or Treg cells that, in turn, suppress the activation of Th1 cells by the encephalitogenic epitope. From our results, we assume that the mechanism of inhibition could involve the competition between peptides P2 or P3 and the encephalitogenic epitope MBP74-85 for binding to the TCR and/or the same sites of MHC molecules, although previous work with altered peptide ligands suggests that the interaction is more likely due to competition at TCR sites. Previous studies have shown that APL antagonistic effects may be mediated through reduced production of pro-inflammatory cytokines that are critical in the EAE pathogenesis, up-regulation of anti-inflammatory cytokines, and bystander suppression through regulatory T-cell amplification [14], [23], [24], [25], [26], and [27].

Our previous studies in EAE models using different MBP APLs, have demonstrated that the antagonistic effects of the peptides were due to their effect on different T-cell populations with opposite functions [12], [23], and [28], raising the possibility that the mechanism of inhibition by APLs is mediated through activation of antigen-specific regulatory T-cells. Thus, T-cells specific for one epitope may down regulate T-cells specific for another epitope of the same protein, creating a dominant subpopulation of T-cells for a given antigen. Factors that determine this “motif dominance” may include the antigenicity of the ligand and/or the binding affinity of the APL to the TCR.

The results from our in vitro experiments that investigated the effect of the peptides on MS or control PBMC, showed that all 3 peptides inhibited the secretion of IL-10. In contrast to the wild-type P1, the altered peptides increased IL-2 and IFN-γ secretion by MS PBMC, with P3 inducing higher levels of IL-2 secretion. We can therefore suggest that replacement of the 91 (Lys) and 96 (Pro) contact residues with Ala resulted in a stronger agonist effect compared to the native peptide. In addition, cyclization did not affect conformationally the critical binding sites 91, 96 of the peptide as TCR contact sites (responsible for triggering agonist activity), i.e. the cyclic peptide maintained a similar spatial orientation towards the MHC. This is assumed because linear P2 and cyclic P3 peptides, with identical sequences but different conformations, have the critical amino acids at the correct orientation towards the TCR.

Smith et al. reported that in the MBP83-99 immunodominant epitope the primary anchor residues i.e. Val 87 and Phe 90, occupy specific pockets of HLA-DR2 [29] .

The binding motif for DRB*0401 is well characterized (SYFPEITHI Databank) [30] . Typically, DRB*0401 needs a big hydrophobic amino acid (preferentially aromatic amino acids like F, Y, W) for pocket P1. This pocket does not accept alanine. In our peptide (Ala91,Ala96)MBP87-99, the pockets for DR4 can be predicted as 90F for P1; 93I for P4; 95T for P6 and 98T for P9. As expected, the linear MBP peptide competes efficiently the hemagglutinin peptide HA(306-318) which uses tyrosine for P1. Residue Ala91 cannot use a MHC binding site and therefore the peptide is not able to shift with another motif. Residues Ala91 and Ala96 therefore must point to the TCR site. Indeed, cyclization reduces the optimal MHC-peptide binding as demonstrated by the binding experiments but the peptide must use the same amino acids for MHC binding. Therefore we conclude that Ala91 and Ala96 are still TCR sites in the cyclised molecule also.

To note, the increased levels of IL-2 secretion that resulted when MS PBMC were cultured with P3, taken together with the improved binding ability of P3 to HLA-DR4, and the higher effectiveness of P3 to inhibit the development of the disease in the EAE rat model, indicate that cyclization per se altered favorably the function of the peptide.

Since linear MBP sequences, one example being MBP82-98 [31] , have been the subject of clinical trials, evaluation of cyclo(87-99)(Ala91,Ala96)MBP87-99 as a potential drug lead for MS immunotherapy is warranted.

4. Experimental section

4.1. Peptides

The peptides used in this work are described in Table 1 .

4.2. Solid-phase peptide synthesis of linear and cyclic MBP87-99 analogs

Peptides MBP87-99 (VHFFKNIVTPRTP)(P1) and (Ala91,Ala96)MBP87-99 (VHFFANIVTARTP) (P2) were prepared on 2-chlorotrityl chloride resin (CTLR-Cl) using Fmoc/tBu methodology [4], [12], and [23] ( Scheme 1 ). All amino acids were purchased from the Chemical and Biopharmaceutical Laboratories of Patras (Patras, Greece). The use of the 2-chlorotrityl resin under mild conditions (DCM/TFE, 7/3) for cleaving the peptide-resin bond, allowed protected peptide release from the resin and the subsequent cyclization of the desired peptide. Cyclization of the protected (Ala91,Ala96)MBP87-99 peptide (P3) was achieved using O-benzotriazol-1-yl-N,N,N′,N’-tetramethyluronium tetrafluoroborate (TBTU) and 1-hydroxy-7-azabenzotriazole (HOAt), 2,4,6 collidine in DMF solution, allowing fast reactions and high yield cyclization products (Scheme 1 and Scheme 2A,B). The peptides were >98% pure as analyzed by HPLC and electron spray ionization mass spectrometry (cf. Supplementary Fig. S1 and S2 ). The cyclization reaction was monitored using the ninhydrin test, and the reaction mixture was resolved by thin-layer chromatography, nbutanol/acetic acid/water (4/1/1) solvent system and analytical HPLC in C4 Nucleosil RP column 5 nm packing material. The protected cyclic analog was then released from side chain protected groups with 90% trifluoroacetic acid (TFA) in DCM solution containing 10% Dithiothreitol/water/Anisole as scavengers. Preparative HPLC for the purification of peptide analogs were performed using a Nucleosil RP-18 reversed phase semi-preparative column with 7 μm packing material. The identification of the molecular weight of the linear and cyclic peptide was achieved by ESI-Mass Spectrometry.

sc1

Scheme 1 An outline of the synthetic pathway of linear (Ala91,Ala96)MBP87-99 and cyclo(87-99)(Ala91,Ala96)MBP87-99 peptides.

sc2

Scheme 2 Chemical structure of (A) linear (Ala91,Ala96)MBP87-99 and (B) cyclo(87-99)(Ala91,Ala96)MBP87-99. Ala mutations at positions 91 and 96 are highlighted in blue. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

4.3. Binding of peptides to HLA-DR4 (DRB1*0401)

Binding of peptides to HLA-DR4 was performed as previously demonstrated [4] . Briefly, the EBV transformed homozygous human B cell line BSM (DRB1*0401) was used for isolating MHC class II. BSM cell pellets were lyzed by nonidet P-40 and HLA-DR was isolated from homogenates by affinity chromatography using the monoclonal antibody L243. The purity of the preparation was checked by SDS-PAGE, HPSEC and western blotting (not shown). Competition binding assays for MBP87-99 peptide analogs were carried out using Fluorescent AMCA-labeled allele-specific HA306-318 peptide immune affinity-purified HLA-DR4. Briefly, solubilized HLA-DR4 (0.13 mM) was incubated for 48 h at 37 °C with the N-terminally 7-amino-4-methylcoumarin-3-acetic acid (AMCA)-labeled influenza matrix protein (306-318) peptide (AMCA-peptide) dissolved in 150 mM sodium phosphate, pH 5.5, containing 15% acetonitrile, 0.1% Zwittergent-12 (Calbiochem) and a cocktail of protease inhibitors (0.2 mM PMSF, 5 μM leupeptin, 10 μM pepstatin, and 1 μM chymostatin). Competition assays were performed in a 1.5 μM solution of AMCA-peptide. As competitors, different MBP peptides (P1, P2 and P3) were added in 5-, 20- and 50- fold excess of the AMCA-peptide. All samples were analyzed on a Pharmacia Superdex 75 HR 5/20 high performance gel filtration column. The column was operated at a flow rate of 0.4 ml/min using the HPSEC buffer, pH 6.0. The eluent passed through a Shimadzu fluorescence spectrometer (350/450 nm) and a Merck ultraviolet detector (214 nm) set in series. Fluorescence and UV signals eluting with the HLA-DR dimers were recorded by a model D 2500 integrator (Merck-Hitachi). The eluent passed through a fluorescence spectrometer (350/450 nm) and an ultraviolet detector (214 nm) set in series.

4.4. Stability of peptides to proteolysis

The peptides were digested with different lysosomal enzymes as described previously [4] . Lysosomal fractions were isolated from BSM cells by differential centrifugation and hypotonic lysis. Cathepsin D and AEP were obtained from Sigma–Aldrich. Peptides 2.5 μg were dissolved in 50 μl of the different substrate buffers, and incubated with enzymes at 37 °C. Between 0 and 90 min, 15 μl of solution were taken and quenched with 5 μl 20% TFA at 4 °C. Samples were analyzed by analytical HPLC using a Vydac C18 5 μm column with a linear gradient of buffer A (0.05%TFA/H2O) and buffer B (80%Acetonitril/0.05%TFA/H2O) from 5 to 80% B in 40 min at 214 nm. The relative concentration of each compound was estimated from the area under the sample peak.

4.5. Effect of peptides on peripheral blood mononuclear cells isolated from MS patients and controls

4.5.1. Study subjects

Whole blood samples (10 ml) were collected from 7 patients with definite remitting-relapsing MS, at the acute phase of the disease (3 males, 4 females, mean age 35.4 years, range 17–65 years, mean duration of disease 7.6 years, range 0–18 years) and 7 healthy volunteers (controls, 4 males, 3 females, mean age 28.5 years, range 20–35 years).

All subjects gave written informed consent before enrollment in the study. The study protocol was approved by the Patras University Hospital Ethics (Re: 296/23.9.08) and Scientific (Re: 451/17.10.08) Committees as part of a general application to collect biological samples from patients attending the Neurology Clinic to study in vitro the role of T helper cell populations and cytokines in the pathogenesis, prognosis and natural course of multiple sclerosis. The Hospital abides by the Helsinki declaration on ethical principles for medical research involving human subjects.

4.6. Effect of peptides on cell proliferation

Peripheral blood mononuclear cells (PBMC) were isolated from MS patients (n = 7) and controls (n = 7) by density gradient centrifugation using Biocoll separating solution (BIOCHROM, Plymouth, UK). The cells were cultured in RPMI1640 medium (GIBCO BRL, Gaithersburg, MD, USA), containing 10% fetal bovine serum and 1% penicillin/streptomycin (CM) at a concentration of 106 cells/ml, in the presence or absence of various concentrations of peptide analogs P1–P3. For the quantification of cell proliferation, a BrdU colorimetric immunoassay was used (Roche Diagnostics GmbH, Mannheim, Germany). Briefly, PBMC were cultured for 48 h; subsequently, BrdU was added and the cells were re-incubated for an additional 24 h. BrdU incorporation was determined following the assay procedures described by the manufacturers.

4.7. Cytokine secretion

PBMC were isolated from MS patients (n = 7) and controls (n = 7) by density gradient centrifugation using Biocoll separating solution (BIOCHROM). The cells were cultured in CM at a concentration of 106 cells/ml, in the presence or absence of peptide analogs P1–P3 at a concentration of 10 pg peptide/ml, for 72 h. At the end of the culture period, cell culture supernatants were collected and analyzed by ELISA, as described by the manufacturers. The following ELISA kits were used: human (h) IL-2 and h–IFN–γ from Endogen Inc (Woburn, MA, USA) and h-IL-4 and h-IL-10 from R&D Systems QuantikineTM (Mineapolis, USA).

4.8. Induction and suppression of EAE using MBP analogs

Inbred female Lewis rats, 6–8 weeks old, were immunized with MBP74-85 epitope (30 μg) as a positive control (i.e. a peptide that induces EAE in Lewis rats), MBP74-85 (30 μg) plus linear peptide analog P1 (500 μg), MBP74-85 (30 μg) plus linear peptide analog P2 (500 μg) and MBP74-85 (30 μg) plus cyclic peptide analog P3 (500 μg) in 200 μl of an emulsion composed of equal volumes of Freund's complete adjuvant (CFA) containing 4 mg/ml of heat-killed M. tuberculosis (H37Ra) in PBS. Immunization was performed subcutaneously in the two hint foot pads. Rats were examined daily for clinical signs of EAE and scored as follows: 0, no clinical disease; 0.5, weight loss; 1, tail weakness; 2, paraparesis of hind limbs; 3, paraplegia of hind limbs; 4, paraplegia with forelimb weakness moribund; 5, death. The experiments were conducted in the ELPEN laboratories and were approved by the veterinary authorities of East Attica Region in accordance with the Greek law No 160/91 in accordance to the European Community regulations (directive 86/609).

4.9. Histological examination

Immunized rats were sacrificed at the peak of clinical disease (day 15). Healthy littermates were sacrificed on the same day and served as controls. Spinal cord (the whole cervical and thoracic vertebral column containing the intact spinal cord) was removed and fixed in 4% buffered paraformaldehyde overnight at 4 °C. After fixation, the whole spinal cord was dissected and serially sectioned at about 2 mm intervals and embedded in paraffin. Paraffin sections, 4 μm thick, were stained with hematoxylin and eosin.

Conflicts of interest

The authors declare that they have no conflict of interest.

Acknowledgments

This work was supported by the Greek General Secretariat of Research and Technology “Cooperation” grant 09SYN-21-609 (O. P. Competitiveness & Entrepreneurship, EPAN ΙΙ) to AM and JM, by a Du Pre' grant from Multiple Sclerosis International Federation (MSIF) to GD and an unconditional grant by Genesis Pharma S.A. to ND.

Appendix A. Supplementary data

The following are the supplementary data related to this article:

figs1

Fig. S1 Electrospray ionization-mass spectrometry (ESI-MS) of linear MBP linear (Ala91,Ala96)MBP87-99. M+: 1473.58, M+2H+/2: 736.84.

figs2

Fig. S2 Electrospray ionization-mass spectrometry (ESI-MS) of cyclo(87-99)(Ala91,Ala96)MBP87-99. M+: 1454.88, M+2H+/2: 728.33.

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Footnotes

a Department of Chemistry, University of Patras, Patras, Greece

b Division of Hematology, Department of Internal Medicine, Faculty of Medicine, University of Patras, Patras, Greece

c Interfaculty Institute of Biochemistry, University of Tubingen, Tubingen, Germany

d Experimental Research Unit of ELPEN, ELPEN Pharmaceutical Co. Inc., 19009, Pikermi, Greece

e Eldrug S.A., Pharmaceutical Company, 26504, Platani, Patras, Greece

f Department of Neurology, Patras University Hospital, Patras, Greece

g College of Health and Biomedicine, Centre for Chronic Disease Prevention and Management, Victoria University, Melbourne, VIC 3021, Australia

Corresponding author.

∗∗ Corresponding author. Eldrug S.A., Pharmaceutical Company, 26504, Platani, Patras, Greece.

1 Both senior authors.


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