AF12198, a Novel Low Molecular Weight Antagonist, Selectively Binds the Human Type I Interleukin (IL)-1 Receptor and Blocks in Vivo Responses to IL-1

Abstract

A wide range of immune and inflammatory responses are influenced by the cytokine IL-1 1 (1). The two agonists IL-1␣ and IL-1␤ can cause both local and systemic inflammatory responses. Prolonged expression of even low levels of IL-1 can have detrimental consequences in chronic inflammatory diseases such as rheumatoid arthritis (2) and inflammatory bowel disease (3). The naturally occurring antagonist IL-1ra plays an important role in physiological regulation of IL-1-induced activities. The three IL-1 ligands, IL-1␣, IL-1␤ and IL-1ra, bind to the two IL-1 receptors (IL-1R), type I and type II (1). Only the type I receptor induces signaling upon binding of IL-1␣ or -␤. The type II receptor appears to act as a negative regulator of activity by effectively competing for binding of ligand and thus regulating IL-1 concentrations (4). The type II receptor released from cells selectively binds IL-1␤ or -␣ over IL-1ra, therefore having little effect on the inhibitory action of the antagonist (5).IL-1 antagonists, including IL-1ra and soluble IL-1R, have been therapeutically effective in animal models of inflammatory disease and are currently in human trials. Recent studies have shown that IL-1ra can reduce some of the symptoms of established disease in model systems, bacterial cell wall-induced arthritis in rats (6), antigen-induced rheumatoid arthritis in rabbits (7), and lung fibrosis in mice (8). A small, active IL-1 antagonist would have several advantages over the recombinant IL-1ra protein including ease of synthesis, the ability to deliver locally in high concentrations, and possible oral delivery. We have screened large libraries of recombinant peptides in search of IL-1 receptor antagonists. The peptide described here, AF12198, is derived from a family of peptide antagonists we recently reported (9). AF12198, Ac-FEWTPGWYQJYALPL-NH 2 , binds specifically to the human type I IL-1R and blocks both in vitro and in vivo IL-1-induced activities. This peptide is the first low molecular weight IL-1 receptor antagonist to show in vivo activity. EXPERIMENTAL PROCEDURESPeptide Synthesis-Routine peptide synthesis was performed on an Advanced Chemtech model ACT350 multiple peptide synthesizer using Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry (10), 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate activation, and polystyrene resin derivatization with trifluoroacetic acid-labile Knorr linker. Peptides were cleaved from the resin using a solution of 95:5 trifluoroacetic acid/water and appropriate scavengers for 90 min. The cleavage solution was then filtered through a glass wool plug, concentrated to dryness, and the peptide purified to homogeneity by reverse-phase (C18) high performance liquid chromatography (HPLC) (water/acetonitrile gradient). Peptides prepared in this fashion afforded satisfactory results upon characterization by analytical HPLC, mass spectrometry, and high resolution mass spectrometry.Binding Analysis-Competition binding analysis was as described (9). The soluble extracellular dom...

Anti-inflammatory peptide-functionalized hydrogels for insulin-secreting cell encapsulation

“…The IL-1R inhibitory peptide sequence FEWTPGWYQPY-NH 2 (IL-1RIP) was designed based on a previously reported inhibitory peptide for IL-1R [50]. Maleimide-terminated IL-1RIP was attached to hydrogels using Michael-Addition reaction as described previously, with the peptide density in the hydrogel varying from 1% to 5% of total thiol groups (calculated values).…”

Development and in vitro assessment of enzymatically-responsive poly(ethylene glycol) hydrogels for the delivery of therapeutic peptides

“…The peptide drugs investigated here were restricted to pro-angiogenic peptides; however, controlled release of any therapeutic peptide is feasible, including anti-inflammatory [3, 4] and chemotherapeutic [5, 6] peptides. Additionally, while therapeutic peptides were the exclusive focus of this study, controlled release of any therapeutic agent whose chemical composition allows them to be flanked by MMP-degradable substrates is also theoretically possible [62, 63].…”

“…Peptide drugs have been identified for a variety of applications including to promote vascularization [1, 2], reduce inflammation [3, 4], and as cancer therapeutics [5, 6]. Peptides typically mimic the bioactivity of larger proteins or growth factors, and offer many advantages over traditional protein delivery.…”

Interleukins in Atherosclerosis: Molecular Pathways and Therapeutic Potential

“…Phage display techniques have led to the development of AF12198, a 15-mer peptide with nanomolar affinity for the human type I IL-1 receptor, which does not bind to the human type II receptor (Akeson et al, 1996), and inhibits IL-1-induced ICAM-1 expression by endothelial cells in vitro. Moreover, it downregulates IL-6 induction in cynomolgus monkeys and is thus considered to be the first small molecule to show IL-1 receptor antagonist activity in vivo.…”