Systematic Design of Trypsin Cleavage Site Mutated Exendin4-Cysteine 1, an Orally Bioavailable Glucagon-Like Peptide-1 Receptor Agonist

Abstract

Abstract:Exendin-4 is a strong therapeutic candidate for the treatment of metabolic syndrome. Related receptor agonist drugs have been on the market since 2005. However, technical limitations and the pain caused by subcutaneous injection have severely limited patient compliance. The goal of the study is to investigate a biologically active exendin-4 analog could be administered orally. Using intraperitoneal glucose tolerance tests, we discovered that exendin4-cysteine administered by oral gavage had a distinct hypoglycemic effect in C57BL/6J mice. Using Rosetta Design and Amber, we designed and screened a series of exendin4-cysteine analogs to identify those that retained biological activity while resisting trypsin digestion. Trypsin Cleavage Site Mutated Exendin4-cysteine 1 (TSME-1), an analog whose bioactivity was similar to exendin-4 and was almost completely resistant to trypsin, was screened out. In addition, TSME-1 significantly normalized the blood glucose levels and the availability of TSME-1 was significantly higher than that of exendin-4 and exendin4-cysteine. Collectively orally administered TSME-1, a trypsin-resistant exendin-4 analog obtained by the system, is a strong candidate for future treatments of type 2 diabetes.

An orally available hypoglycaemic peptide taken up by caveolae transcytosis displays improved hypoglycaemic effects and body weight control in db/db mice

“…Exendin-4 analogues were designed as described in the previous report (Sai et al, 2017). Briefly, the free energy of single amino acid mutation at the enzymic cleavage site of exendin-4 was calculated by Rosetta design online service.…”

“…Encouraged by this unexpected finding, we aimed to further improve the oral bioavailability of exendin-4 by optimizing its enzymic resistance. In an earlier study (Sai et al, 2017), we established a computer-aided method to design GLP-1 receptor agonist peptides and were able to demonstrate that mutation of the trypsin cleavage sites in exendin-4 yielded peptides with greater oral absorption than the parent peptide. Here, we have expanded the use of the computeraided method and obtained a new peptide, OHP2, which was resistant not only to trypsin but also to chymotrypsin and elastase ( Table 1).…”

“…Although absorption enhancers or drug delivery systems can increase the absorption of therapeutic peptides by opening tight junctions or increasing plasma membrane permeability (Steinert et al, 2009), enzymic degradation remains a significant obstacle to oral absorption of peptide due to the large amount of proteases present in the GIT. In the previous study, we have established a computer-aided method to design GLP-1 receptor agonist peptides that would be resistant to trypsin hydrolysis (Sai et al, 2017). We were able to synthesize a peptide almost completely resistant to trypsin, with similar bioactivity to exendin-4.…”

Exendin‐4 from Heloderma suspectum venom: From discovery to its latest application as type II diabetes combatant

“…Although oral administration of exendin‐4 is less likely used as one of the delivery options due to first‐pass effects, oral ingestion is still the most convenient way for drug administration for better patient compliance. Therefore, a new exendin‐4 analogue, Trypsin Cleavage Site Mutated Exendin‐4‐Cysteine 1 (TSME‐1), has been designed with similar biological activity with exendin‐4, but completely resistant to trypsin digestion and having higher bioavailability than that of exendin‐4 . The modifications of existing exendin‐4 suggest the new exendin‐4 analogue as a potential therapeutic candidate for T2DM in the future.…”

Exendin 4-Hapten Conjugate Capable of Binding with Endogenous Antibodies for Peptide Half-life Extension and Exerting Long-Acting Hypoglycemic Activity

“…However, one major challenge of using GLP-1-based peptide biologics as antidiabetes drugs in clinic is their relatively short half-life in vivo due to the inherent characteristics of the peptide, such as susceptibility to enzymatic degradation and rapid renal clearance; thus, they require high dosages and increased administration frequencies to achieve a therapeutic effect in clinic. To address this key issue, several elegant strategies have been successfully developed to increase the circulation time of GLP-1-based peptide drugs, such as peptide sequence optimization, , peptide post-translational modification with small functional molecules (e.g., lipidation , and dicoumarinization), polymer conjugation (e.g., PEGylation and carbohydrate conjugation , ), protein fusion (album or Fc fusion), and sustained delivery systems. Generally, increasing the hydrodynamic radius by reducing renal elimination and steric shielding to reduce metabolic degradation are the two classical mechanisms of peptide half-life extension.…”