Stimulation of the leukocyte N-formylpeptide receptor (FPR) induces chemotaxis, cell adhesion, free radical release, and degranulation, responses associated with infection and inflammation. Under conditions where continuous activation of the receptor prevails, neutrophil-dependent tissue damage ensues. Antagonists of the FPR have potential for use as diagnostic and therapeutic agents. Hence, we have synthesized and evaluated a series of amino-terminal carbamate analogues of the peptide Met-Leu-Phe (MLF) in order to determine the structural requirements for imparting agonist or antagonist activity at the human neutrophil FPR. Peptides were evaluated in three in vitro assays: receptor binding, superoxide anion release, and cell adhesion. Unbranched carbamates (methoxycarbonyl, ethoxycarbonyl, and n-butyloxycarbonyl) resulted in agonist activity, whereas branched carbamates (iso-butyloxycarbonyl, tert-butyloxycarbonyl, and benzyloxycarbonyl) were antagonists. The peptide antagonists were more potent inhibitors of superoxide anion release than cell adhesion by 4-7-fold. When iso-butyloxycarbonyl-MLF (i-Boc-MLF) was further modified at the carboxy terminus with Lys, antagonist potency was retained but without functional selectivity. Further C-terminal modification with the radionuclide linker diethylenetriaminepentaacetic acid did not alter the potency of i-Boc-MLFK. These results indicate that the switch from agonist to antagonist activity can be achieved by modifying the overall size and shape of the amino-terminal group; that modifications at both the amino and carboxy termini can alter the functional selectivity of the peptide; and that modifications can be tolerated at the carboxy terminus to allow for development of an antagonist for diagnostic applications.
The potency of previously isolated peptides that are modest agonists of the EPO receptor was dramatically increased by PEG-induced dimerization. The EPO receptor is thought to be dimerized during activation, so our results are consistent with the proposed 2:2 receptor : peptide stoichiometry. The conversion of an inactive peptide into an agonist further supports the idea that dimerization can mediate receptor activation.
A peptide mapping procedure was developed to locate regions of a monoclonal antibody, OKT3, that undergo chemical modification as the molecule degrades upon storage. The structures of these peptide degradation products were investigated. Deamidation at specific asparagine residues and oxidation of a cysteine and several methionines were found to be major routes of OKT3 degradation. A unique chain cross-linked degradation product was also observed and characterized. Changing the storage conditions of the antibody affected the relative distribution of degradation products. These results were useful in the development of more stable formulations for OKT3, and the methods can be used in the characterization of other monoclonal antibodies intended for therapeutic use.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.