Half-life extension of anti-obesity peptides

Research output: Book/ReportPh.D. thesisResearch

Esben Matzen Bech

As the prevalence of obesity continuous to grow, the development of new, efficacious anti-obesity pharmaceuticals becomes increasingly important. In this context, endogenous peptides with anorexigenic effects are highly interesting. However, peptides have short in vivo half-lives and, thus, half-life extension is essential for the development of anti-obesity peptide pharmaceuticals. Typically, half-life extension is ensured by attachment of peptides to macromolecules (e.g. PEG) or through lipidation, which promotes non-covalent interactions with albumin. However, alternative half-life extension strategies that can add novel properties to peptides or further increase peptide half-lives continues to be attractive. Moreover, half-life extension can affect peptide properties beyond pharmacokinetics, for example distribution, internalization, and pharmacodynamics. Such ‘secondary’ properties of half-life extenders remain relatively understudied despite their importance for peptide pharmaceuticals. This thesis aimed to expand the toolbox of half-life extenders applicable for anti-obesity peptides. For this, two avenues of research were followed. One aiming at the development of a novel half-life extending strategy, and a second aiming to expand the understanding of ‘secondary’ properties conferred to peptides by commonly applied half-life extenders. In project 1, we investigated the effect of mono- or divalent small-molecule albumin binders for half-life extension of peptides. For proof-of-principle, the clinically relevant peptide glucagon-like peptide 1 (GLP-1) was functionalized with diflunisal, indomethacin, or both. In vitro- and biophysical characterization revealed how albumin affinity was significantly increased for divalent analogues, as compared to the monovalent analogues. In lean mice, this translated to a superior biological efficacy and a promising gain in circulatory half-life and absorption time for the divalent GLP-1 analogues. Importantly, the in vivo effects of the divalent GLP-1 analogue were comparable to that of liraglutide, a marketed once-daily GLP-1 analogue. In project 2, we investigated how three commonly applied peptide half-life extenders affected peptide YY3-36 (PYY3-36) in terms of receptor (Y2R) signalling, endocytosis, and membrane affinity. Specifically, we investigated the effects of palmitoylation (C16), lipidation with octadecanedioic acid (C18acid), and PEGylation (PEG20). We report how large PEG-chains inhibited peptide-stimulated endocytosis by promotion of a receptor signalling bias. For the first time, we provide a biophysical explanation for PEG’s promotion of such bias by linking arrestin recruitment to Y2R residence time. In contrast to PEGylation, palmitoylation of PYY3-36 had a negligible impact on Y2R signalling, binding, and endocytosis, while increasing membrane affinity. Meanwhile, C18acid-lipidation minimized internalization, but with negligible effects on Y2R signalling, binding or membrane affinity. Additionally, the site of lipidation could bias peptide activity. Thus, small changes in the lipidation strategy could greatly influence the properties of PYY3-36. This underlines how half-life extending techniques can be applied to tune peptide properties beyond pharmacokinetics.
Original languageEnglish
PublisherDepartment of Chemistry, Faculty of Science, University of Copenhagen
Publication statusPublished - 2019

ID: 219532029