Recombinant Expression and Stapling of a Novel Long-Acting GLP-1R Peptide Agonist

Sam Lear,Zaid Amso,Lei Lei,Hyosuk Seo,Huafei Zou,Candy Lee,David Huang,Vân T B Nguyen-Tran,Zhihong Zhou,Weijun Shen

doi:10.3390/molecules25112508

Abstract

Owing to their pleiotropic metabolic benefits, glucagon-like peptide-1 receptor (GLP-1R) agonists have been successfully utilized for treating metabolic diseases, such as type 2 diabetes and obesity. As part of our efforts in developing long-acting peptide therapeutics, we have previously reported a peptide engineering strategy that combines peptide side chain stapling with covalent integration of a serum protein-binding motif in a single step. Herein, we have used this strategy to develop a second generation extendin-4 analog rigidified with a symmetrical staple, which exhibits an excellent in vivo efficacy in an animal model of diabetes and obesity. To simplify the scale-up manufacturing of the lead GLP-1R agonist, a semisynthesis protocol was successfully developed, which involves recombinant expression of the linear peptide followed by attachment of a polyethylene glycol (PEG)-fatty acid staple in a subsequent chemical reaction step.

Highlights

Glucagon-like peptide-1 receptor (GLP-1R) agonists have attained blockbuster status as therapeutics in the area of metabolic diseases
We have demonstrated a robust semisynthetic protocol for the production of a long-acting exenatide analog, Ex-4[M14L, E17C, E24C]-S2, involving recombinant expression of the linear precursor peptide followed by stapling using Cys-reactive chemistry
The methionine residue susceptible to oxidation in the native exenatide sequence was replaced by leucine, and the newly developed symmetric staple S2 was used in order to circumvent regioisomer formation occuring during synthesis of the first-generation long-acting S1-stapled exenatide previously reported

Summary

Introduction

Glucagon-like peptide-1 receptor (GLP-1R) agonists have attained blockbuster status as therapeutics in the area of metabolic diseases. In addition to the above metabolic outcomes, GLP-1R agonists are able to induce sustained weight loss through appetite suppression and delayed gastric emptying, leading to the commercialization of the once daily liraglutide for chronic weight management in patients with obesity [4]. GLP-1R agonists have shown promise in the clinical studies for management of nonalcoholic steatohepatitis, a liver disease with no approved therapies to date [5,6]. As part of our efforts in developing long-acting therapies for chronic diseases, we have previously utilized the in-house developed polyethylene glycol (PEG)-fatty acid stapling technology to produce ”E6”, a potent, long-acting GLP-1R agonist exhibiting excellent efficacy in animal models of diabetes and obesity [7].

Methods

Results

Conclusion