A novel high-affinity peptide antagonist to the insulin receptor Lauge Schäffer * , Christian L. Brand, Bo F. Hansen, Ulla Ribel, Allan C. Shaw, Rita Slaaby, Jeppe Sturis Diabetes Research Unit, Novo Nordisk A/S, Novo Nordisk Park G8.1.77, DK-2760 Maaloev, Denmark article info Article history: Received 28 August 2008 Available online 7 September 2008 Keywords: Insulin receptor Antagonist Peptide antagonist Hypoglycemia S661 S961 abstract In this publication we describe a peptide insulin receptor antagonist, S661, which is a single chain peptide of 43 amino acids. The affinity of S661 for the insulin receptor is comparable to that of insulin and the selectivity for the insulin receptor versus the IGF-1 receptor is higher than that of insulin itself. S661 is also an antagonist of the insulin receptor of other species such as pig and rat, and it also has considerable affinity for hybrid insulin/IGF-1 receptors. S661 completely inhibits insulin action, both in cellular assays and in vivo in rats. A biosynthetic version called S961 which is identical to S661 except for being a C-ter- minal acid seems to have properties indistinguishable from those of S661. These antagonists provide a useful research tool for unraveling biochemical mechanisms involving the insulin receptor and could form the basis for treatment of hypoglycemic conditions. Ó 2008 Elsevier Inc. All rights reserved. Antagonists exist for most peptide hormone receptors, often truncated or otherwise modified analogs of the peptide hormone itself. One exception has been the insulin receptor to which no such antagonists derived from insulin have ever been identified, despite the fact that hundreds of insulin analogs have been de- scribed. Thus, so far the only available insulin antagonists have been insulin receptor antibodies [1], and due to the dimeric nature of antibodies their antagonistic properties sometimes depend on the context that they are used in. In previous publications, we have described the identification of peptides that bind to two different sites on the insulin receptor and shown that heterodimers of two such peptides can be either ago- nists or antagonists depending on the orientation of the peptides [2,3]. In the present work, we describe the optimization of one such antagonist into an insulin receptor antagonist with high affinity and selectivity for the insulin receptor. Such an insulin receptor antagonist can be used as a biochemical research tool, and could also provide the basis for medical treatment of conditions such as hyperinsulinaemia caused by insulinoma as well as neonatal hyperinsulinaemic hypoglycemia. Materials and methods Peptide synthesis. S661 was synthesized by Fmoc chemistry on a Tentagel S RAM resin with a loading of about 0.25 mmol/g using automated, microwave-assisted synthesis on a Liberty Peptide Synthesizer (CEM Corp., Matthews, NC, USA). The couplings were performed with diisopropylcarbodiimide (DIC) and 1-hydroxy-7- azabenzotriazole (HOAt) in N-methylpyrrolidone (NMP) for 5 min at up to 70 °C. Deprotection was performed with 20% piperidine in NMP for 3 min at up to 75 °C. After completion of synthesis the peptide was cleaved with trifluoroacetic acid/triisopropylsi- lane/water (92.5/5/2.5) for 2 h and precipitated with an excess of diethylether. The peptide was dissolved in 0.1 M Tris (pH 7) at about 5 mg/ml and 1.1 equivalent of [Pt(IV)ethylenedia- mine 2 Cl 2 ]Cl 2 was added to oxidize the two cysteines to form an intramolecular disulfide bond [4]. After at least 1 h the solution was purified by standard RP-HPLC on a C18 column with TFA/ace- tonitrile. The fractions containing S661 were combined and lyophilized. S961 was expressed in Escherichia coli as a fusion protein with an affinity tag. Further details will be published elsewhere (Shaw, unpublished results). Receptor binding assays. Human insulin receptor (A and B iso- form) was partially purified from transfected baby hamster kidney cells by wheat germ agglutinin (WGA) chromatography. Hybrids of insulin receptors and IGF-1 receptors were prepared as described elsewhere. Rat insulin receptor cDNA was obtained from Barry J. Goldstein, Jefferson University, and WGA-purified rat and pig insu- lin receptors were kindly supplied by Asser S. Andersen, Novo Nor- disk. Two competition assays were used, a SPA (scintillation proximity assay) assay in microplates and a classical PEG precipi- tation assay in glass tubes. To analyze binding to the human insulin receptor SPA PVT Anti-mouse beads (Amersham Biosciences, UK) were incubated with IR antibody 83-7 [1] and insulin receptor for 5 h at room temperature. The SPA beads were washed twice with buffer 125 I-insulin (from Novo Nordisk A/S) was added. A two- fold dilution series of human insulin, S661 or S961 was prepared in 0006-291X/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2008.08.151 * Corresponding author. E-mail address: lsc@novonordisk.com (L. Schäffer). Biochemical and Biophysical Research Communications 376 (2008) 380–383 Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc