Crystal structures of antigenic peptides bound to class I MHC proteins suggest that chemical modifications of the central part of the bound peptide should not alter binding affinity to the MHC restriction protein but could perturb the T-cell response to the parent epitope. In our effort in designing nonpeptidic high-affinity ligands for class I MHC proteins, oligomers of (R)-3-hydroxybutanoate and(or) beta-homoalanine have been substituted for the central part of a HLA-B27-restricted T-cell epitope of viral origin. The affinity of six modified peptides to the B2705 allele was determined by an in vitro stabilization assay. Four out of the six designed analogues presented an affinity similar to that of the parent peptide. Two compounds, sharing the same stereochemistry (R,R,S,S) at the four stereogenic centers of the nonpeptidic spacer, bound to B2705 with a 5-6-fold decreased affinity. Although the chiral spacers do not strongly interact with the protein active site, there are configurations which are not accepted by the MHC binding groove, probably because of improper orientation of some lateral substituents in the bound state and different conformational behavior in the free state. However we demonstrate that beta-amino acids can be incorporated in the sequence of viral T-cell epitopes without impairing MHC binding. The presented structure-activity relationships open the door to the rational design of peptide-based vaccines and of nonnatural T-cell receptor antagonists aimed at blocking peptide-specific T-cell responses in MHC-associated autoimmune diseases.

Class I MHC 1 molecules are highly polymorphic proteins that play a key role in immune surveillance by presenting foreign peptides to cytotoxic T lymphocytes (1). The molecular mechanisms of peptide selection have been characterized by x-ray diffraction studies of several MHC proteins in complex with either a peptide pool or single ligands (2). Peptides, generally nonamers, tightly bind to conserved MHC residues in a sequence-independent manner at their N and C termini (3), whereas the central part of the bound peptide bulges out of the binding groove (4). Peptide specificity is governed by the position and chemical nature of some anchoring side chains (often P2, P3, and P9) that bind to MHC polymorphic pockets (5, 6). Complementary to x-ray structure determinations, sequencing self-peptides naturally bound to MHC proteins allows the determination of peptide binding motifs (7,8) and thus the identification of conserved amino acids responsible for MHC binding (named dominant anchors, generally at positions P2 and P9) and more variable residues hypothesized to account for TcR recognition (usually in the central part of the peptide sequence, from P4 to P8). Peptide mutation (9, 10) as well as recently determined x-ray structures of ␣␤ TcRs in complex with a MHC-peptide (11, 12) unambiguously support this assumption. Since some class I MHC alleles are associated with either susceptibility or resistance to human diseases (13-15), altering TcR contact residues of T cell epitopes has been proposed for designing altered peptide ligands with TcR antagonist properties (16), leading to in vivo T cell anergy (17). However, natural peptides cannot be easily used as immunosuppressors because of poor enzymatic stability and pharmacokinetic properties (18). Herewith, we describe the substitution of nonpeptidic moieties for the TcR contact amino acids of several T cell epitopes naturally presented by the class I MHC protein B*2705, which is strongly linked to severe inflammatory diseases like ankylosing spondylitis (13) or reactive arthritis (19). Some reports in which a similar strategy has been followed (20 -22) show that the altered peptide ligands still form stable complexes with their host MHC protein but often present a reduced affinity relative to the parent peptide. The present study describes a novel oligomeric spacer able not only to link two MHC anchoring positions (P3 and P9) but also to significantly improve binding to the restricting class I MHC protein. EXPERIMENTAL PROCEDURESComputer-assisted Ligand Design-Molecular mechanics and dynamics calculations were carried out using the AMBER 4.1 package (23), using the parm94 parameter set (24) and an all-atom force field representation. Force field parameters for the ester group were taken from the literature (25). Atomic charges for the Aua and HB monomers were calculated using the GAUSSIAN 94 package (26) and the HF/6 -31G* basis set by fitting atom-centered charges to an ab initio electrostatic potential, using the RESP method (27) according to a previously describe...

In the center of the immune system, there are major hisrocompafibilify (MHC) proteininonapeptide complexes which are recognized by T cells. The nonapeptides consist of three regions, an N-terminal one containing three amino-acid residues with a mandatory arginine in position 2, a C-terminal one with a lysine or arginine in position 9, and a central, variable one of five residues (cJ Fig. 1). We have now synthesized the first conjugates (1-4) of oligopeptides with oligo[(R)-3-hydroxybutanoates] (OHB) as analogs of MHC-binding peptides. Of the approaches chosen (Scheme I), a fragment coupling of a hydroxy-butanoyl-amido ester (17 and 19) with an [(aminoalkanoyl)oxy]butanoyl chloride (27; Scheme 3), followed by two peptide-coupling steps (Scheme 4), turned out to be most efficient. The conjugates H-Gln-Arg-Leu-(HB),,,-Lys-OH (1 and 2) and H-Ala-Arg-Leu-(HB),.,-Lys-OH (3 and 4) were thus obtained in pure form. The conjugates I and 2 with N-terminal glutamine have a tendency to undergo cyclization with formation of a pyroglutamate residue (cf. Fig. 2). CD Measurements at different temperatures and so-called epitope-stabilization assays show that the complexes of the conjugates 2 and 4, containing four HB units, with the HLA-B27 class-I-MHC protein are more stable than those of a model nonapeptide (C50 values of 2.25 and 1.60 V M vs. 10 VM), while the conjugates 1 and 3 with three HB units incorporated form less stablecomplexes (C'50 values of 30 and 21 p~) . The tetra(hydroxybutanoate)-peptide conjugates 2 and 4 are the first nonapeptide analogs for which the modification of the central part leads to increased aflinities for a class-I-MHC protein, as compared to a model nonapeptide.