Design and Synthesis of a Potent Cyclic Analogue of the Myelin Basic Protein Epitope MBP_72-85:  Importance of the Ala⁸¹Carboxyl Group and of a Cyclic Conformation for Induction of Experimental Allergic Encephalomyelitis

Abstract

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.

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

“…15,20,[23][24][25][26][27][28] We had previously demonstrated that injection of Lewis rats with linear or cyclic guinea pig MBP 72-85 peptides induced EAE. 19 15 In our in vivo rat studies, EAE was induced by the agonist guinea pig epitope MBP [72][73][74][75][76][77][78][79][80][81][82][83][84][85] and not by the human MBP 83-99 agonist peptide. Injections with human agonist peptides caused anaphylactic shock and weak signs of EAE.…”

Conformational analysis of the ΜΒΡ83–99 (Phe91) and ΜΒΡ83–99 (Tyr91) peptide analogues and study of their interactions with the HLA-DR2 and human TCR receptors by using Molecular Dynamics

“…Effort must be made to understand: (a) the forces that determine the folding of the epitope; (b) the conservation of the linearity and extended structure of the altered epitope; (c) the molecular features of the altered epitope that maximize the productive interactions with HLA-DR2 and minimize the contacts with TCR. The linear peptides were prepared on 2-chlorotrityl chloride resin (CLTR-Cl) using the Fmoc/tBu solid-phase methodology [28][29][30][31]. The first N a Fmoc (9-fluorenylmethyloxycarboxyl)-protected amino acid (Fmoc-Pro-OH) was esterified to the resin in the presence of diisopropylethylamine (DIPEA) in dichloromethane (DCM) in 1 h at RT [28][29][30][31].…”

“…The linear peptides were prepared on 2-chlorotrityl chloride resin (CLTR-Cl) using the Fmoc/tBu solid-phase methodology [28][29][30][31]. The first N a Fmoc (9-fluorenylmethyloxycarboxyl)-protected amino acid (Fmoc-Pro-OH) was esterified to the resin in the presence of diisopropylethylamine (DIPEA) in dichloromethane (DCM) in 1 h at RT [28][29][30][31]. DCM/MeOH/DIPEA (85:10:5) was then added and the resulting mixture was stirred for another 10 min at RT.…”

“…DCM/MeOH/DIPEA (85:10:5) was then added and the resulting mixture was stirred for another 10 min at RT. The remaining protected peptide chains were assembled by sequential couplings of the appropriate Fmoc protected amino acids (2.5 equiv), in the presence of N,N'-diisopropylcarbodiimide (DIC) (2.75 equiv) and 1-hydroxybenzotriazole (HOBt) (3.75 equiv) in N,N-dimethylformamide (DMF) [28][29][30][31]. The completeness of each coupling was verified by the Kaiser test and TLC using a BAW, 4:1:1 (v/v/v) eluent system and the Fmoc protecting group was removed by treatment with piperidine solution (20% in DMF).…”

“…NMR spectroscopy is coupled with MD simulations to study the binding motif of the altered peptide epitopes of MBP 83-99 with HLA-DR2 [19][20][21][22][23][24][25][26][27][28][29][30][31].…”