Application of N-Dodecyl l-Peptide to Enhance Serum Stability while Maintaining Inhibitory Effects on Myometrial Contractions Ex Vivo.

N-Alkylation and N-acylation of the prostaglandin-F2α allosteric modulator l-PDC31 were performed to install various alkyl, PEG and isoprenoid groups onto the l-enantiomer of the peptide. Among the different bio-conjugates studied, the N-dodecyl analog reduced prostaglandin-F2α-induced mouse myometrium contractions ex vivo. Furthermore, N-dodecyl-l-PDC31 exhibited improved stability in a mouse serum assay, likely due to protection from protease degradation by the lipid chain.

A Cyclic Altered Peptide Analogue Based on Myelin Basic Protein 87-99 Provides Lasting Prophylactic and Therapeutic Protection Against Acute Experimental Autoimmune Encephalomyelitis.

In this report, amide-linked cyclic peptide analogues of the 87-99 myelin basic protein (MBP) epitope, a candidate autoantigen in multiple sclerosis (MS), are tested for therapeutic efficacy in experimental autoimmune encephalomyelitis (EAE). Cyclic altered peptide analogues of MBP87-99 with substitutions at positions 91 and/or 96 were tested for protective effects when administered using prophylactic or early therapeutic protocols in MBP72-85-induced EAE in Lewis rats. The Lys91 and Pro96 of MBP87-99 are crucial T-cell receptor (TCR) anchors and participate in the formation of trimolecular complex between the TCR-antigen (peptide)-MHC (major histocompability complex) for the stimulation of encephalitogenic T cells that are necessary for EAE induction and are implicated in MS. The cyclic peptides were synthesized using Solid Phase Peptide Synthesis (SPPS) applied on the 9-fluorenylmethyloxycarboxyl/tert-butyl Fmoc/tBu methodology and combined with the 2-chlorotrityl chloride resin (CLTR-Cl). Cyclo(91-99)[Ala96]MBP87-99, cyclo(87-99)[Ala91,96]MBP87-99 and cyclo(87-99)[Arg91, Ala96]MBP87-99, but not wild-type linear MBP87-99, strongly inhibited MBP72-85-induced EAE in Lewis rats when administered using prophylactic and early therapeutic vaccination protocols. In particular, cyclo(87-99)[Arg91, Ala96]MBP87-99 was highly effective in preventing the onset and development of clinical symptoms and spinal cord pathology and providing lasting protection against EAE induction.

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

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.

Molecular evolution of myelin basic protein, an abundant structural myelin component.

Rapid nerve conduction in jawed vertebrates is facilitated by the myelination of axons, which evolved in ancient cartilaginous fish. We aim to understand the coevolution of myelin and the major myelin proteins. We found that myelin basic protein (MBP) derived from living cartilaginous fish (sharks and rays) associated with the plasma membrane of glial cells similar to the phosphatidylinositol (4,5)-bisphosphate (PIP2)-binding marker PH-PLCδ1, and that ionomycin-induced PIP2-hydrolysis led to its cellular redistribution. We identified two paralogous mbp genes in multiple teleost species, consistent with a genome duplication at the root of the teleost clade. Zebrafish mbpb is organized in a complex transcription unit together with the unrelated gene-of-the-oligodendrocyte-lineage (golli) while mbpa does not encode GOLLI. Moreover, the embryonic expression of mbpa and mbpb differed, indicating functional specialization after duplication. However, both mbpa and mbpb-mRNAs were detected in mature oligodendrocytes and Schwann cells, MBPa and MBPb were mass spectrometrically identified in zebrafish myelin, both associated with the plasma membrane via PIP2, and the ratio of nonsynonymous to synonymous nucleotide-substitution rates (Ka/Ks) was low. Together, this indicates selective pressure to conserve many aspects of the cellular expression and function of MBP across vertebrate species. We propose that the PIP2-binding function of MBP is evolutionarily old and that its emergence in ancient gnathostomata provided glial cells with the competence to myelinate.

Divergent and convergent synthesis of polymannosylated dibranched antigenic peptide of the immunodominant epitope MBP(83-99).

Multiple antigenic peptide (MAP) systems are dendrimeric structures bearing multiple copies of identical or different peptide epitopes, and they have been demonstrated to show enhanced immunogenicity. Herein, we report the direct (divergent) and indirect (convergent) synthesis, using contemporary synthetic approaches, of a di-branched antigenic peptide (di-BAP) containing the immunodominant epitope MBP(83-99), which is implicated in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). The direct synthesis (di-BAP 1) was performed using microwave irradiation. The indirect synthesis (di-BAP 2) was carried out performing an efficient chemoselective coupling reaction through the formation of a thioether bond. Both di-BAPs were conjugated to polysaccharide mannan since mannosylation is a promising technique to achieve modulation in immune response. The conjugation was achieved through free amino groups of both di-BAPs via the formation of Schiff bases. The mannan-conjugated di-BAPs were further evaluated in vivo in a prophylactic vaccination protocol, prior to EAE induction in Lewis rats.

NMR structural elucidation of myelin basic protein epitope 83-99 implicated in multiple sclerosis.

Myelin basic protein peptide 83-99 (MBP83-99) is the most immunodominant epitope playing a significant role in the multiple sclerosis (MS), an autoimmune disease of the central nervous system. Many peptide analogues, linear or cyclic have been designed and synthesized based on this segment in order to inhibit the experimental autoimmune encephalomyelitis, the best well-known animal model of MS. In this study, the solution structural motif of MBP(83-99) has been performed using 2D (1)H-NMR spectroscopy in dimethyl sulfoxide. A rather extended conformation, along with the formation of a well defined alpha-helix spanning residues Val(87)-Phe(90) is proposed, as no long-range NOE are presented. Moreover, the residues of MBP peptide that are important for T-cell receptor recognition are solvent exposed. The spatial arrangement of the side chain all over the sequence of our NMR based model exhibits great similarity with the solid state model, while both TCR contacts occupy the same region in space.

Design and synthesis of a cyclic double mutant peptide (cyclo(87-99)[A91,A96]MBP87-99) induces altered responses in mice after conjugation to mannan: implications in the immunotherapy of multiple sclerosis.

Altered peptide ligands that alter immune responses are a promising approach to the immunotherapy of multiple sclerosis. Cyclic peptides are of interest because the limited stability of linear peptides restricts their use in vivo. We designed and synthesized a cyclic double mutant peptide from MBP(87-99)-[cyclo(87-99)[A(91),A(96)]MBP(87-99)]. Immunization of mice, in CFA reduced Th1 responses. However, when conjugated to reduced mannan, a significant further reduction of Th1 responses and moderate Th2 responses were induced.

MBP-8298, a synthetic peptide analog of myelin basic protein for the treatment of multiple sclerosis.

BioMS Medical Corp, under license from the University of Alberta, is developing MBP-8298, a synthetic peptide analog of myelin basic protein, for the potential treatment of multiple sclerosis. Phase II and III clinical trials of MBP-8298 are underway.

Synthesis and study of the electrophoretic behavior of mannan conjugates with cyclic peptide analogue of myelin basic protein using lysine-glycine linker.

New approaches for the treatment of multiple sclerosis involve the design and synthesis of peptide or nonpeptide analogues of myelin sheath, which could alter the immune response of patients. For this purpose, the cyclo(75-82) myelin basic protein (MBP)(74-85) analogue was conjugated to mannan (a polymannose) via (Lys-Gly)(5) linker. Monitoring of synthesis of the (Lys-Gly)(5)-containing cyclic analogue of MBP, mannan oxidation, and the conjugation reaction of this analogue to oxidized mannan was performed with capillary electrophoresis (CE) in operating buffers of different pH values. The (Lys-Gly)(5)cyclo(75-82)MBP(74-85) was efficiently synthesized by solid-phase synthesis and purified to a high degree, as confirmed by CE analysis in a low-pH (3.0) phosphate buffer and normal polarity. Oxidation of mannan was monitored using a high-pH (9.3) borate buffer, and the generation of heterogeneous products and even UV-absorbing peaks was shown by CE. CE analysis in a pH 5.1 phosphate buffer offers high resolution of oxidized mannan and the conjugation product and can be used for screening of the reaction products. Mannan-peptide conjugates of varying degrees of substitution and unreacted mannan were observed. The developed CE analysis presents distinct advantages over sodium dodecyl sulfate-polyacrylamide gel electrophoresis such as high versatility, high separation efficiency, short analysis time, low cost, and low solvent consumption.

Design and synthesis of a novel potent myelin basic protein epitope 87-99 cyclic analogue: enhanced stability and biological properties of mimics render them a potentially new class of immunomodulators.

A cyclic analogue, [cyclo(87-99)MBP(87)(-)(99)], of the human immunodominant MBP(87)(-)(99) epitope, was designed based on ROESY/NMR distance information and modeling data for linear epitope 87-99, taking into account T-cell (Phe(89), Lys(91), Pro(96)) and HLA (His(88), Phe(90), Ile(93)) contact side-chain information. The cyclic analogue was found to induce experimental allergic encephalomyelitis (EAE), to bind HLA-DR4, and to increase CD4 T-cell line proliferation, like that of the conformationally related linear MBP(87)(-)(99) epitope peptide. The mutant cyclic peptides, the cyclo(91-99)[Ala(96)]MBP(87)(-)(99) and the cyclo(87-99)[Arg(91)Ala(96)]MBP(87)(-)(99), reported previously for suppressing, to a varying degree, autoimmune encephalomyelitis in a rat animal model, were found in this study to possess the following immunomodulatory properties: (i) they suppressed the proliferation of a CD4 T-cell line raised from a multiple sclerosis patient, (ii) they scored the best in vitro TH2/TH1 cytokine ratio in peripheral blood mononuclear cell cultures derived from 13 multiple sclerosis patients, inducing IL-10 selectively, and (iii) they bound to HLA-DR4, first to be reported for cyclic MBP peptides. In addition, cyclic peptides were found to be more stable to lysosomal enzymes and Cathepsin B, D, and H, compared to their linear counterparts. Taken together, these data render cyclic mimics as putative drugs for treating multiple sclerosis and potentially other Th1-mediated autoimmune diseases.

Novel synthetic amino acid copolymers that inhibit autoantigen-specific T cell responses and suppress experimental autoimmune encephalomyelitis.

Copolymer 1 (Cop 1, Copaxone [Teva Marion Partners, Kansas City, Missouri, USA]), a random amino acid copolymer of tyrosine (Y), glutamic acid (E), alanine (A), and lysine (K), reduces the frequency of relapses by 30% in relapsing-remitting multiple sclerosis (MS) patients. In the present study, novel random four-amino acid copolymers, whose design was based on the nature of the anchor residues of the immunodominant epitope of myelin basic protein (MBP) 85-99 and of the binding pockets of MS-associated HLA-DR2 (DRB1*1501), have been synthesized by solid-phase chemistry. Poly (Y, F, A, K) (YFAK) inhibited binding of the biotinylated MBP 86-100 epitope to HLA-DR2 molecules more efficiently than did either unlabeled MBP 85-99 or any other copolymer including Cop 1. Moreover, YFAK and poly (F, A, K) (FAK) were much more effective than Cop 1 in inhibition of MBP 85-99-specific HLA-DR2-restricted T cell clones. Most importantly, these novel copolymers suppressed experimental autoimmune encephalomyelitis, induced in the susceptible SJL/J (H-2(s)) strain of mice with the encephalitogenic epitope PLP 139-151, more efficiently than did Cop 1. Thus, random synthetic copolymers designed according to the binding motif of the human immunodominant epitope MBP 85-99 and the binding pockets of HLA-DR2 might be more beneficial than Cop 1 in treatment of MS.

Antagonistic effects of human cyclic MBP(87-99) altered peptide ligands in experimental allergic encephalomyelitis and human T-cell proliferation.

The immunodominant myelin basic protein (MBP) peptide comprising residues 87-99 is a self-antigen in multiple sclerosis (MS). In Lewis rats this epitope induces experimental allergic encephalomyelitis (EAE), a demyelinating disease of the central nervous system, and is a model of MS. Structure-activity studies have shown that Lys(91) and Pro(96) residues are important for encephalitogenicity. Replacement of Lys and/or Pro residues with Arg and/or Ala, respectively, results in suppression of EAE. A potent linear altered peptide ligand of the immunodominant sequence MBP(83-99) has been selected for clinical trial (Nat. Med. 2000, 6, 1167, 1176). In the present report, two cyclic analogues, cyclo(91-99)[Ala(96)]MBP(87-99) and cyclo(87-99)[Arg(91), Ala(96)]MBP(87-99) were designed by NMR and molecular modeling data on human MBP(87-99) epitope (Val(87)-His-Phe-Phe-Lys-Asn-Ile-Val-Thr-Pro-Arg-Thr-Pro(99)) and its linear antagonist peptide analogue [Arg(91), Ala(96)]MBP(87-99). These analogues (altered peptide ligands) inhibited EAE in Lewis rats and decreased inflammation in the spinal cord. In addition, the analogue cyclo(87-99)[Arg(91), Ala(96)]MBP(87-99) induced proliferation of human peripheral blood T-cells. These cyclic MBP(87-99) peptide analogues may lead to the design of potent antagonist mimetics for treating MS.

Treatment of experimental allergic encephalomyelitis (EAE) by a rationally designed cyclic analogue of myelin basic protein (MBP) epitope 72-85.

In this report the rational design, synthesis and pharmacological properties of an amide-linked cyclic antagonist analogue of the guinea pig myelin basic protein epitope MBP(72-85) are described. Design of the potent cyclic analogue was based on 2D NOESY nuclear magnetic resonance and molecular dynamics studies carried out in the linear antagonist Ala81MBP(72-85). The cyclic antagonist completely prevented the induction of experimental allergic/autoimmune encephalomyelitis when coinjected with linear and cyclic agonist analogues MBP(72-85) and cyclo(2-9)MBP(72-85).

Design and synthesis of a potent cyclic analogue of the myelin basic protein epitope MBP72-85: importance of the Ala81 carboxyl group and of a cyclic conformation for induction of experimental allergic encephalomyelitis.

Experimental allergic encephalomyelitis (EAE) is induced in susceptible animals by immunodominant determinants of myelin basic protein (MBP), such as guinea pig sequence MBP72-85. Two linear and one cyclic analogues based on MBP72-85 have been synthesized and evaluated for EAE induction in Lewis rats. The linear peptide Gln1-Lys2-Ser3-Gln4-Arg5-Ser6-Gln7-+ ++Asp8-Glu9-Asn10-Pro11-Val12 (1) was found to induce EAE, while substitution of the Asp residue at position 8 with Ala resulted in an analogue (2) which suppressed the induction of EAE by its parent peptide. Nuclear magnetic resonance studies of analogue 1 in dimethyl sulfoxide (DMSO) using TOCSY/ROESY techniques revealed a head-to-tail intramolecular interaction (ROE connectivity between betaVal12-gammaGln1), indicating a pseudocyclic conformation for the immunogenic peptide 1. A conformational model was developed using NMR constraints and molecular dynamics. Based on this model, a novel amide-linked cyclic analogue has been designed and synthesized by connecting the side-chain amino and carboxyl groups of Lys and Glu at positions 2 and 9, respectively, of linear analogue 1. The cyclic analogue (3) had similar activity to the linear peptide 1, and the EAE effects induced by cyclic analogue 3 were completely suppressed by co-injection with the Ala81-substituted analogue 2 in Lewis rats. The similar potencies of analogues 1 and 3 support the proposed cyclic comformation suggested for analogue 1 from NMR studies and computer modeling and provides the basis for designing more potent molecules with improved properties such as increased resistance to degradation.15 The present findings suggest that a cyclic conformation for the MBP72-85 epitope positions the carboxyl group of Asp81 correctly and presumably other side groups of the peptide such as Arg78 in a manner which enables functional binding of the trimolecular complex MHC-peptide-T cell receptor resulting in EAE.

The effect of intrathecal MBP synthetic peptides containing epitope P85 VVHFFKNIVTP96 on free anti-MBP levels in acute relapsing multiple sclerosis.

Acute relapses of multiple sclerosis (MS) are characterized by elevated Free (F)/Bound (B) anti-MBP ratios during the initial phase, followed by a steady decline of F antibody as the recovery/remission phase develops. The (human) MBP epitope for MS anti-MBP is: Pro85-Val-Val-His-Phe-Phe-Lys-Asn-Ile-Val-Thr-Pro96. In phase one clinical research, synthetic peptides (p) containing this epitope, namely pMBP86-95 and/or pMBP82-98, were intrathecally administered to MS patients with monosymptomatic or polysymptomatic relapses to determine the dosage, frequency and duration of administration which will immediately neutralize F circulating CSF anti-MBP. Patients with monosymptomatic relapses required 50 mg of peptide administered daily for 4-5 days. In patients with polysymptomatic relapses, F anti-MBP can be neutralized with dosages between 50 mg peptide daily for 4 days up to 100 mg twice a day for 2 days; however due to the prolonged nature of polysymptomatic relapses, antibody neutralization could not be maintained by these short courses of intrathecal peptide administration. Intravenous administration of these same peptides did not prevent occurrence of future relapses.

Suppression of experimental allergic encephalomyelitis in the Lewis rat, by administration of an acylated synthetic peptide of myelin basic protein.

A Myelin Basic Protein (MBP) epitope encephalitogenic for the Lewis rat (amino acid residues 68-86) was synthesized and acylated by the attachment of a palmitoyl residue. Lewis rats treated intravenously (i.v.) with the palmitoylated peptide alone were better protected against clinical manifestations of experimental allergic encephalomyelitis (EAE) than rats treated with the peptide inserted into liposomes or with the native peptide at similar doses. The administration of the acylated peptide (PAL68 86) conferred excellent protection against a challenge with the encephalitogenic peptide (p68-86) or with the intact MBP molecule, both before and after induction of active disease, and also when administered to recipients after the transfer of lymphocytes from MBP-challenged donors. Histological manifestations were also reduced to a statistically significant degree. Treatment with a palmitoylated peptide from a non-encephalitogenic region of the MBP molecule (PAL44-62) or with a palmitoylated unrelated peptide were ineffective. In vitro Ag-specific proliferative responses as well as the ability to transfer disease to syngeneic recipients, by lymph node lymphocytes from PAL68-86-treated donors, were considerably reduced. Addition of IL-2 to these cultures failed to restore either Ag-specific responsiveness or the ability of the cells to transfer disease. The results suggest that the administration of acylated peptides induces a profound state of unresponsiveness, and thus may provide an effective means for treating T cell-mediated autoimmune inflammatory disorders.

Immunological effects of an arginine side chain contaminating synthetically prepared peptides.

The side chain, 4-methoxy-2,3,6-trimethylbenzenesulphonyl (Mtr), is a protective group coupled to arginine to mask the omega-nitrogen, in order to protect the guanidino function during peptide synthesis by the 9-fluorenylmethoxycarbonyl (Fmoc) procedure (Walker, 1994). This group is removed at the completion of peptide synthesis; however, the cleavage process can be incomplete. We have found that animals injected with a mixed population of pure, i.e. unmodified, and Mtr-containing MBP peptides have an immunodominant humoral response to the Mtr-bearing peptide. This response is dependent on the characteristics of the MBP peptide involved. For two MBP peptides, the Mtr-containing peptide had increased binding to antibody over pure peptide. For two other peptides, only the Mtr-containing peptide bound antibody while the unmodified peptide did not. In a separate system involving a polyclonal response to an unrelated peptide from beta2-microglobulin (beta2 m), the dominance of the Mtr group was also evident. These results provide further evidence that a small side chain on a single amino acid in a peptide can markedly alter the immunogenicity and antigenicity of that peptide for antibody reactivity. This evidence emphasizes the need for a critical awareness of each component of peptide synthesis and its potential to alter the immunoreactivity of the final product.

Administration of myelin basic protein synthetic peptides to multiple sclerosis patients.

A double blind Phase 1 clinical research project was conducted in vivo in multiple sclerosis (MS) patients to determine the effect of myelin basic protein (MBP) synthetic peptides on free (F) and bound (B) titers of anti-MBP in cerebrospinal fluid (CSF). Intrathecal administration of peptide MBP75-95, either as a single dose, or as repeated injections for periods up to 10 weeks, produced complete binding-neutralization of F anti-MBP with no change in B levels. A control peptide MBP35-58 had no effect on F and B anti-MBP levels. Intravenous administration of MBP75-95 resulted in significant decline of F and B CSF anti-MBP levels over a period of one month. Administration of MBP synthetic peptides to MS patients either intrathecally or intravenously did not have any adverse neurological effects and systemic complications did not occur. The MBP epitope for MS anti-MBP was further localized to an area between Pro85 and Pro96.

Stimulation of T cells by antigenic peptide complexed with isolated chains of major histocompatibility complex class II molecules.

Major histocompatibility complex (MHC) class II molecules are heterodimeric glycoproteins with one alpha and one beta polypeptide chain of similar molecular size. In this report, we describe the binding of an acetylated N-terminal peptide of myelin basic protein, [Ala4]MBP-(1-14), to purified individual alpha and beta chains of murine I-Ak molecules. Purified complexes of isolated single chains and antigenic peptide bind to cloned T cells restricted by I-Ak and [Ala4]MBP-(1-14) tetradecapeptide. The binding is blocked by alpha/beta anti-T-cell receptor (TCR) monoclonal antibody. Cell triggering as measured by an increase in extracellular acidification rate is observed when cloned T cells are exposed to purified complexes of isolated chains and antigenic peptide. This increase in the extracellular acidification rate is antigen specific and MHC-restricted, as chains alone or irrelevant chain-peptide complexes do not trigger an increase in the metabolic acidification rate. These results together demonstrate that in vitro cloned T cells are triggered by complexes of specific antigenic peptides and isolated individual chains of their cognate MHC proteins.