Assembly of high-affinity insulin receptor agonists and antagonists from peptide building blocks

Abstract

Insulin is thought to elicit its effects by crosslinking the two extracellular ␣-subunits of its receptor, thereby inducing a conformational change in the receptor, which activates the intracellular tyrosine kinase signaling cascade. Previously we identified a series of peptides binding to two discrete hotspots on the insulin receptor. Here we show that covalent linkage of such peptides into homodimers or heterodimers results in insulin agonists or antagonists, depending on how the peptides are linked. An optimized agonist has been shown, both in vitro and in vivo, to have a potency close to that of insulin itself. The ability to construct such peptide derivatives may offer a path for developing agonists or antagonists for treatment of a wide variety of diseases.I nsulin is one of the most studied peptide hormones because of its importance in maintaining glucose homeostasis. This 51-aa hormone is very well characterized with regard to its structure, both in crystal form and in solution. The insulin receptor (IR) is a transmembrane ␣ 2 ␤ 2 glycoprotein whose intracellular tyrosine kinase domain is activated by binding of insulin, leading to a cascade of intracellular signaling events. The kinase domain of the IR (1) and an extracellular fragment of the related receptor for insulin-like growth factor I (IGF-IR; ref. 2) have been crystallized, but the structure of the insulin binding domain of the IR is not known, and the mechanism for the transmission of a signal through its transmembrane domain is not well understood. A model for the binding and activation has been proposed in which insulin uses two different sites on its surface to crosslink the two ␣-subunits of the IR, thus inducing a conformational change that activates the receptor (refs. 3 and 4; Fig. 1).In a previous report (5), we panned random, highly diverse peptide display libraries against the IR. By using this approach, we identified a large number of peptides binding to the IR and competing for insulin binding with micromolar or submicromolar affinity, although these peptides had no sequence homology with insulin. These peptides bound to two discrete hotspots on the receptor (designated site 1 and site 2), and these hotspots appeared to correspond to the two contact sites involved in insulin binding predicted by the crosslinking model (ref. 3 and J.B., unpublished results). At least two different sequence motifs were found for site 1 peptides, and some of these were full agonists but of low affinity. Other site 1 peptides were antagonists, whereas site 2 peptides were either antagonists or inactive. The mechanism behind the agonism of the site 1 peptides is not known, but it has been speculated that site 1 binding may be important for receptor activation, whereas the role of the site 2 interaction may be more related to affinity and selectivity. In addition to these two families of peptides, a third group was identified, but no further work has been done on this group. In the present work, we have used site 1 and site 2 peptides as building blocks ...

Mass Spectrometry Characterization of a Novel Insulin Mimetic Peptide s597

“…Therefore the difference in mitogenitic response between s597 and insulin results from different signaling more than from cross-talking with IGF-1R. This is supported by the finding that s597 had no effect on phosphorylation of the IGF-1R, ERK1/2 or PKB in untransfected L6 cells [6] and by studies of the gene expression profile in IRtransfected L6 myoblasts after IR activation by insulin and s597, using microarray technology [7]. It was shown that almost half of the genes differentially regulated by s597 and insulin were related with cell proliferation and growth.…”

Activation of the Insulin Receptor by Insulin and a Synthetic Peptide Leads to Divergent Metabolic and Mitogenic Signaling and Responses

“…3,4 Furthermore we have earlier published on other peptides that bind to the insulin receptor with regard to affinity for the receptor and ability to stimulate lipogenesis in mouse adipocytes (8,9), but intracellular signaling was not investigated.…”

“…Recently homodimers and heterodimers of the site 1 and 2 peptides have been generated. Many of these can activate the insulin receptor with high potency and specificity, possibly through a mechanism similar to the binding of insulin (9). One of these peptides was furthermore found to lower the blood glucose level in rats with equipotency to insulin.…”

How to make a functional β-cell

“…These studies prompted the idea that blocking the insulin receptor with a small molecule or peptide antagonist could recapitulate these effects, including the induction of β-cell replication. In fact, treatment with one such insulin receptor antagonist, a novel peptide named S961, results in hyperinsulinemia in rats (Schäffer et al, 2003;Schäffer et al, 2008;Vikram and Jena, 2010).…”

“…These studies prompted the idea that blocking the insulin receptor with a small molecule or peptide antagonist could recapitulate these effects, including the induction of β-cell replication. In fact, treatment with one such insulin receptor antagonist, a novel peptide named S961, results in hyperinsulinemia in rats (Schäffer et al, 2003;Schäffer et al, 2008;Vikram and Jena, 2010).Furthermore, an additional study of the LIRKO model revealed that β-cells were specifically induced to replicate in this context with no effect on α-cells or other non-pancreatic tissues (El DEVELOPMENT 2480 Ouaamari et al, 2013). This study also demonstrated that the induced replication could occur in a normal parabiotic partner (with normal blood glucose and insulin levels) if that animal was joined with a LIRKO mouse partner, suggesting both that a systemic factor was at work and that it could work independently of blood glucose levels.…”