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

Interleukin-1 (IL-1) -α and -β are potent regulators of inflammatory responses. The naturally occurring interleukin-1 receptor antagonist (IL-1ra) is effective in vitro and in vivo in modulating biological responses to IL-1. We have previously reported the discovery of IL-1 antagonist peptides from the search of phage display libraries. Further characterization of this group of peptides has led to a 15-mer, AF12198, Ac-FEWTPGWYQJYALPL-NH2 (J represents the unnatural amino acid, 2-azetidine-1-carboxylic acid), with both in vitro and in vivo IL-1 antagonist activity. AF12198 selectively binds the human type I IL-1 receptor but not the human type II receptor or the murine type I receptor. In vitro, AF12198 inhibits IL-1-induced IL-8 production by human dermal fibroblasts with a half-maximal inhibition concentration or IC50 of 25 nM and IL-1-induced intercellular adhesion molecule-1 (ICAM-1) expression by endothelial cells with an IC50 of 9 nM. When given as an intravenous infusion to cynomolgus monkeys, AF12198 blocks ex vivo IL-1 induction of IL-6 and down modulates in vivo induction of IL-6. This is the first small molecule to show IL-1 receptor antagonist activity in vivo. Interleukin-1 (IL-1) -α and -β are potent regulators of inflammatory responses. The naturally occurring interleukin-1 receptor antagonist (IL-1ra) is effective in vitro and in vivo in modulating biological responses to IL-1. We have previously reported the discovery of IL-1 antagonist peptides from the search of phage display libraries. Further characterization of this group of peptides has led to a 15-mer, AF12198, Ac-FEWTPGWYQJYALPL-NH2 (J represents the unnatural amino acid, 2-azetidine-1-carboxylic acid), with both in vitro and in vivo IL-1 antagonist activity. AF12198 selectively binds the human type I IL-1 receptor but not the human type II receptor or the murine type I receptor. In vitro, AF12198 inhibits IL-1-induced IL-8 production by human dermal fibroblasts with a half-maximal inhibition concentration or IC50 of 25 nM and IL-1-induced intercellular adhesion molecule-1 (ICAM-1) expression by endothelial cells with an IC50 of 9 nM. When given as an intravenous infusion to cynomolgus monkeys, AF12198 blocks ex vivo IL-1 induction of IL-6 and down modulates in vivo induction of IL-6. This is the first small molecule to show IL-1 receptor antagonist activity in vivo. INTRODUCTIONA wide range of immune and inflammatory responses are influenced by the cytokine IL-1 1The abbreviations used are: IL-1interleukin-1IL-1rainterleukin-1 receptor antagonistIL-1Rinterleukin-1 receptorJl-azetidine-2-carboxylic acidHPLChigh performance liquid chromatographyHUVEChuman umbilical vein endothelial cellsELISAenzyme-linked immunosorbent assayICAM-1intercellular adhesion molecule-1IL-8interleukin-8IL-6interleukin-6. (1Dinarello C.A. FASEB J... 1994; 8: 1314-1325Google Scholar). The two agonists IL-1α and IL-1β 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 (2Arend W.P. Dayer J.-M. Arthritis & Rheum. 1995; 33: 151-160Crossref Scopus (927) Google Scholar) and inflammatory bowel disease (3Casini-Raggi V. Kam L. Chong Y.J.T. Fiocchi C. Pizarro T.T. Cominelli F. J. Immunol. 1995; 154: 2434-2440PubMed Google Scholar). The naturally occurring antagonist IL-1ra plays an important role in physiological regulation of IL-1-induced activities. The three IL-1 ligands, IL-1α, IL-1β and IL-1ra, bind to the two IL-1 receptors (IL-1R), type I and type II (1Dinarello C.A. FASEB J... 1994; 8: 1314-1325Google Scholar). Only the type I receptor induces signaling upon binding of IL-1α or -β. 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 (4Sims J.E. Gayle M.A. Slack J.L. Alderson M.R. Bird T.A. Giri J.G. Colotta F. Re F. Mantovani A. Shanebeck K. Grabstein K.H. Dower S.K. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6155-6159Crossref PubMed Scopus (544) Google Scholar). The type II receptor released from cells selectively binds IL-1β or -α over IL-1ra, therefore having little effect on the inhibitory action of the antagonist (5Symons J.A. Young P.R. Duff G.W. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1714-1718Crossref PubMed Scopus (179) Google Scholar).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 (6Makarov S.S. Olsen J.C. Johnston W.N. Anderle S.K. Brown R.R. Baldwin Jr., A.S. Haskill J.S. Schwab J.H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 402-406Crossref PubMed Scopus (197) Google Scholar), antigen-induced rheumatoid arthritis in rabbits (7Lewthwaite J. Blake S. Thompson R.C. Hardingham T.E. Henderson B. Ann. Rheum. Dis. 1995; 54: 591-596Crossref PubMed Scopus (20) Google Scholar), and lung fibrosis in mice (8Piquet P.F. Vesin C. Grau G.E. Thompson R.C. Cytokine. 1993; 5: 57-61Crossref PubMed Scopus (190) Google Scholar). 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 (9Yanofsky S.D. Baldwin D.N. Butler J.H. Holden F.R. Jacobs J.W. Balasubramanian P. Chinn J.P. Cwirla S.E. Peters-Bhatt E. Whitehorn E.A. Tate E.H. Akeson A. Bowlin T.L. Dower W.J. Barrett R.W. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7381-7386Crossref PubMed Scopus (105) Google Scholar). AF12198, Ac-FEWTPGWYQJYALPL-NH2, 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.DISCUSSIONIn our search for small IL-1 antagonists we have screened diverse recombinant peptide libraries and found a peptide family that specifically binds with high affinity to the human type I IL-1 receptor. We have characterized in vitro and in vivo activities of one of the most potent of these peptides, AF12198, Ac-FEWTPGWYQJYALPL-NH2. AF12198 inhibits IL-1-induced in vitro events at an effective molar concentration 1000-fold higher than for IL-1ra. Like IL-1ra, AF12198 has no agonist activity.This is the first example of a small, low molecular weight IL-1R antagonist with in vivo antagonist activity. When we followed in vivo activity in cynomolgus monkeys, we found that infusion of the peptide did reduce IL-1 induction of IL-6 in four of six animals. However, AF12198 infusion did not affect the response of two of the test animals, both low responders to IL-1. Several factors seem to be contributing to the marginal in vivo activity of AF12198 in this system. First, rapid metabolism limited the effective plasma concentration of AF12198. Analysis of plasma steady state levels of AF12198 revealed that the peptide was rapidly degraded by endopeptidase activity to two nonactive metabolites, Ac-FEWTPGWYQJY-OH and Ac-FEWTPG-OH. This metabolism was also reflected in the rapid decline in plasma concentrations of AF12198 once infusion was ended. As a consequence of the rapid metabolism, we were able to achieve a plasma concentration of active peptide of only 4 to 5 nmol/ml. When IL-1ra was administered with the same protocol, at a concentration of IL-1ra which completely blocked the response to the IL-1β bolus, steady state plasma concentrations of 35 pmol/ml were attained, and at the end of the infusion IL-1ra levels declined much more slowly than AF12198. If the results from the in vitro cell-based assays are an accurate predictor of in vivo efficacy, we needed to achieve a plasma peptide molar concentration 1000-fold higher than IL-1ra to get comparable activity. Therefore, we would need to achieve a plasma concentration of AF12198 of 35 nmol/ml, 7-fold higher than detected in the in vivo system. Unfortunately, the poor solubility of the peptide limited the dose that could be administered in this model system.We were able to show that AF12198 does bind IL-1R in vivo, by using an ex vivo format, where cynomolgus monkeys could be treated with an infusion of the same dose of AF12198, but administered over a shorter period. With this protocol, a mean AF12198 plasma concentration of 72 nmol/ml was reached. This concentration completely blocked IL-1-induced activity ex vivo. Again, the two metabolites, 11-mer and 6-mer, were detected in plasma samples at the end of the 15-min infusion. Because we were able to deliver a high volume of AF12198 with a short infusion, the plasma concentration of active peptide was sufficient to block the functional effects of IL-1 binding. We are currently testing AF12198 analogs with modifications designed to limit proteolysis and increase solubility.IL-1 has shown no species specificity for receptor binding although some differences in induction of biological activity have been reported (14Dinarello C.A. Blood. 1991; 77: 1627-1652Crossref PubMed Google Scholar). Human IL-1 clearly binds murine type I IL-1R and induces a variety of biological activities. However, analysis of a series of IL-1β muteins has shown different binding affinities and activation characteristics for human and murine receptors (15Grütter M.G. van Oostrum J. Priestle J.P. Edelmann E. Joss U. Feige U. Vosbeck K. Schmitz A. Protein Eng. 1994; 7: 663-671Crossref PubMed Scopus (30) Google Scholar). Surface regions involved in binding appear to be distinct, suggesting that the complementary sites on each of the receptors are also distinct. This may explain the receptor selectivity of AF12198. The site where AF12198 binds to the human type I IL-1R is either not present on the human type II and murine type I receptors or is not essential for binding of ligand. That AF12198 does not bind the human type II receptor is a pharmacological advantage. The soluble type II IL-1R binds pro-IL-1β and IL-1β with high affinity and IL-1α with lower affinity but binds IL-1ra with greatly reduced activity (5Symons J.A. Young P.R. Duff G.W. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1714-1718Crossref PubMed Scopus (179) Google Scholar). This provides a mechanism for blocking activation by limiting circulating levels of IL-1β but not IL-1ra. Since AF12198 does not bind the type II receptor, the peptide does not compromise this regulatory interaction.Recent mutagenesis studies (16Labriola-Tompkins E. Chandran C. Kaffka K.L. Biondi D. Graves B.J. Hatada M. Madison V.S. Karas J. Kilian P.L. Ju G. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 11182-11186Crossref PubMed Scopus (73) Google Scholar, 17Evans R.J. Bray J. Childs J.D. Vigers G.P.A. Brandhuber B.J. Skalicky J.J. Thompson R.C. Eisenberg S.P. J. Biol. Chem. 1995; 270: 11477-11483Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar) and structural information (18Priestle J.P. Schar H.P. Grütter M.G. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 9667-9671Crossref PubMed Scopus (160) Google Scholar, 19Graves B.J. Hatada M.H. Hendrickson W.A. Miller J.K. Madison V.S. Satow Y. Biochemistry. 1990; 29: 2679-2684Crossref PubMed Scopus (130) Google Scholar, 20Schreuder H.A. Rondeau J.M. Tradif C. Soffientini A. Sarubbi E. Akeson A. Bowlin T.L. Yanofsky S. Barrett R.W. Eur. J. Biochem. 1995; 227: 838-847Crossref PubMed Scopus (52) Google Scholar, 21Vigers G.P.A. Caffes P. Evans R.J. Thompson R.C. Eisenberg S.P. Brandhuber B.J. J. Biol. Chem. 1994; 269: 12874-12879Abstract Full Text PDF PubMed Google Scholar) about IL-1 ligands and receptors have shown that ligand residues' key to binding are in discontinuous, nonlinear arrays. The agonists, IL-1β and -α, possess two main surface sites, A and B, which interact with the receptors (17Evans R.J. Bray J. Childs J.D. Vigers G.P.A. Brandhuber B.J. Skalicky J.J. Thompson R.C. Eisenberg S.P. J. Biol. Chem. 1995; 270: 11477-11483Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). It is proposed that site B is essential for agonist activity. IL-1ra has only site A and thus has no agonist activity. Residues within site A identified by Evans et al. (17Evans R.J. Bray J. Childs J.D. Vigers G.P.A. Brandhuber B.J. Skalicky J.J. Thompson R.C. Eisenberg S.P. J. Biol. Chem. 1995; 270: 11477-11483Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar) as critical for IL-1ra binding are Trp-16, Gln-20, Tyr-34, Gln-36, and Tyr-147.We (20Schreuder H.A. Rondeau J.M. Tradif C. Soffientini A. Sarubbi E. Akeson A. Bowlin T.L. Yanofsky S. Barrett R.W. Eur. J. Biochem. 1995; 227: 838-847Crossref PubMed Scopus (52) Google Scholar) and others (21Vigers G.P.A. Caffes P. Evans R.J. Thompson R.C. Eisenberg S.P. Brandhuber B.J. J. Biol. Chem. 1994; 269: 12874-12879Abstract Full Text PDF PubMed Google Scholar) have reported that the crystal structure of IL-1ra like that of IL-1β and IL-1α forms a six-stranded β-barrel which is closed on one side by three β-hairpin loops. In the three-dimensional models of the ligands, the site A residues are clustered at the open end of the β-barrel. We have also been successful in the co-crystallization of IL-1ra and the soluble extracellular domain of the human type I IL-1R (22Schreuder H.A. Tradif C. Soffientini A. Sarubbi E. Akeson A. Bowlin T.L. Yanofsky S. Barrett R.W. FEBS Lett. 1995; 373: 39-40Crossref PubMed Scopus (6) Google Scholar). Analysis of the model of bound IL-1R derived from the co-crystal show that the Trp-16, Gln-20, Tyr-34, Gln-36, and Tyr-147, identified by Evans et al. (17Evans R.J. Bray J. Childs J.D. Vigers G.P.A. Brandhuber B.J. Skalicky J.J. Thompson R.C. Eisenberg S.P. J. Biol. Chem. 1995; 270: 11477-11483Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar), are in direct contact with the receptor.Analyses of peptide mutagenesis libraries designed to determine residues directly involved in AF12198 binding to the type I IL-1R have shown that QPY is essential (9Yanofsky S.D. Baldwin D.N. Butler J.H. Holden F.R. Jacobs J.W. Balasubramanian P. Chinn J.P. Cwirla S.E. Peters-Bhatt E. Whitehorn E.A. Tate E.H. Akeson A. Bowlin T.L. Dower W.J. Barrett R.W. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7381-7386Crossref PubMed Scopus (105) Google Scholar). Furthermore, the immediate amino-terminal residues of active AF12198 analogs are most frequently aromatics. Computer modeling of the WYQJY core of AF12198 indicates that the peptide can adopt a conformation similar to the A site-critical residues of IL-1ra. The identification of AF12198 from screening large libraries, combined with structural information derived from crystallography and NMR of the human type I receptor and molecular modeling, provides powerful tools for structure-based drug design. We are currently designing small peptidomimetic molecules as potential antagonists based on the model of the bound conformation of AF12198.With AF12198 we have shown that a small molecule can block in vivo IL-1-induced activity. However, direct therapeutic use of AF12198 or related analogs will be limited due to rapid metabolism and poor pharmacokinetics. The value of AF12198 is as a tool in understanding pharmacophores important for antagonist activity and as a model for rational structure-based design of low molecular weight peptidomimetic antagonists. INTRODUCTIONA wide range of immune and inflammatory responses are influenced by the cytokine IL-1 1The abbreviations used are: IL-1interleukin-1IL-1rainterleukin-1 receptor antagonistIL-1Rinterleukin-1 receptorJl-azetidine-2-carboxylic acidHPLChigh performance liquid chromatographyHUVEChuman umbilical vein endothelial cellsELISAenzyme-linked immunosorbent assayICAM-1intercellular adhesion molecule-1IL-8interleukin-8IL-6interleukin-6. (1Dinarello C.A. FASEB J... 1994; 8: 1314-1325Google Scholar). The two agonists IL-1α and IL-1β 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 (2Arend W.P. Dayer J.-M. Arthritis & Rheum. 1995; 33: 151-160Crossref Scopus (927) Google Scholar) and inflammatory bowel disease (3Casini-Raggi V. Kam L. Chong Y.J.T. Fiocchi C. Pizarro T.T. Cominelli F. J. Immunol. 1995; 154: 2434-2440PubMed Google Scholar). The naturally occurring antagonist IL-1ra plays an important role in physiological regulation of IL-1-induced activities. The three IL-1 ligands, IL-1α, IL-1β and IL-1ra, bind to the two IL-1 receptors (IL-1R), type I and type II (1Dinarello C.A. FASEB J... 1994; 8: 1314-1325Google Scholar). Only the type I receptor induces signaling upon binding of IL-1α or -β. 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 (4Sims J.E. Gayle M.A. Slack J.L. Alderson M.R. Bird T.A. Giri J.G. Colotta F. Re F. Mantovani A. Shanebeck K. Grabstein K.H. Dower S.K. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6155-6159Crossref PubMed Scopus (544) Google Scholar). The type II receptor released from cells selectively binds IL-1β or -α over IL-1ra, therefore having little effect on the inhibitory action of the antagonist (5Symons J.A. Young P.R. Duff G.W. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1714-1718Crossref PubMed Scopus (179) Google Scholar).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 (6Makarov S.S. Olsen J.C. Johnston W.N. Anderle S.K. Brown R.R. Baldwin Jr., A.S. Haskill J.S. Schwab J.H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 402-406Crossref PubMed Scopus (197) Google Scholar), antigen-induced rheumatoid arthritis in rabbits (7Lewthwaite J. Blake S. Thompson R.C. Hardingham T.E. Henderson B. Ann. Rheum. Dis. 1995; 54: 591-596Crossref PubMed Scopus (20) Google Scholar), and lung fibrosis in mice (8Piquet P.F. Vesin C. Grau G.E. Thompson R.C. Cytokine. 1993; 5: 57-61Crossref PubMed Scopus (190) Google Scholar). 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 (9Yanofsky S.D. Baldwin D.N. Butler J.H. Holden F.R. Jacobs J.W. Balasubramanian P. Chinn J.P. Cwirla S.E. Peters-Bhatt E. Whitehorn E.A. Tate E.H. Akeson A. Bowlin T.L. Dower W.J. Barrett R.W. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7381-7386Crossref PubMed Scopus (105) Google Scholar). AF12198, Ac-FEWTPGWYQJYALPL-NH2, 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.