PEPTIDE STABILITY IN DRUG DEVELOPMENT .2. EFFECT OF SINGLE AMINO-ACIDSUBSTITUTION AND GLYCOSYLATION ON PEPTIDE REACTIVITY IN HUMAN SERUM

The determination of peptide stability in human serum (HS) or plasma c onstitutes a powerful screening assay for eliminating unstable peptide s from further development. Herein we report on the stability in HS of several major histocompatibility complex (MHC)-binding peptides. Some of these peptides are in development for the novel treatment of selec ted autoimmune disorders such as rheumatoid arthritis and insulin-depe ndent diabetes. For most of the 1-amino acid peptides studied, the pre dominant degradation mechanism is exopeptidase-catalyzed cleavage. Pep tides that were protected by d-amino acids at both termini were found to be more stable than predicted, based on additivity of single substi tutions. In addition, N-acetylglucosamine glycopeptides were significa ntly stabilized, even when the glycosylation site was several amino ac ids from the predominant site(s) of cleavage. This indicates that long -range stabilization is possible, and likely due to altered peptide co nformation. Finally, the effect of single amino acid substitutions on peptide stability in HS was determined using a model set of poly-Ala p eptides which were protected from exopeptidase cleavage, allowing the study of endopeptidase cleavage pathways.