Design of Potent and Proteolytically Stable Oxyntomodulin Analogs

Abstract

Incretin-based peptides are effective therapeutics for treating type 2 diabetes mellitus (T2DM). Oxyntomodulin (OXM), a dual agonist of GLP-1R and GCGR, has shown superior weight loss and glucose lowering effects, compared to single GLP-1R agonists. To overcome the short half-life and rapid renal clearance of OXM, which limit its therapeutic potential, both lipid and PEG modified OXM analogs have been reported. However, these approaches often result in reduced potency or PEG-associated toxicity. Herein, we report a new class of cross-linked OXM analogs that show increased plasma stability and higher potency in activating both GLP-1R and GCGR. Moreover, the extended in vivo half-life results in superior antihyperglycemic activity in mice compared to the wild-type OXM.

Figure 1

(a) Sequences of oxyntomodulin, glucagon, GLP-1 and exendin-4. The conserved N-terminal residues crucial for receptor activation are underlined. The sites for cysteine substitution and subsequent cross-linking on oxyntomodulin are colored. (b) Structural model of oxyntomodulin bound to the extracellular domain of GLP-1R (PDB code 3C59), with the cross-linking sites colored. The oxyntomodulin structure was modeled after the crystal structure of glucagon (PDB code: 1GCN) with the octapeptide extension shown as a dotted type II β-helical turn.(22)

Figure 2

Chemical cross-linking extends OXM half-life and efficacy. In vivo stability of the OXM analogs after intravenous (a) and subcutaneous (b) injection of the peptides into mice (n = 3). The peptide concentrations in mouse plasma at the various times were determined using the GLP-1R activation assay. Assay was performed in triplicate. Half-lives of the OXM analogs were calculated by fitting the curve to either two-phase exponential decay (i.v) or one-phase exponential decay (s.c) in Prism 6.0. *No clear distinction was detected between the signal and the background. Cross-linked peptides 9 and 11 show greater activity in the oral glucose tolerance test in mice (n = 4). Mice were injected with the peptides (10 μg/mice) subcutaneously 4 hours prior to the glucose challenge. (c) The glucose concentrations in mouse blood were monitored for up to 150 minutes. (d) Bar graph showing the total amount of glucose in the mice obtained by measuring the area under curve (AUC).