On the Utility of Chemical Strategies to Improve Peptide Gut Stability

Inherent susceptibility of peptides to enzymatic degradation in the gastrointestinal tract is a key bottleneck in oral peptide drug development. Here, we present a systematic analysis of (i) the gut stability of disulfide-rich peptide scaffolds, orally administered peptide therapeutics, and well-known neuropeptides and (ii) medicinal chemistry strategies to improve peptide gut stability. Among a broad range of studied peptides, cyclotides were the only scaffold class to resist gastrointestinal degradation, even when grafted with non-native sequences. Backbone cyclization, a frequently applied strategy, failed to improve stability in intestinal fluid, but several site-specific alterations proved efficient. This work furthermore highlights the importance of standardized gut stability test conditions and suggests defined protocols to facilitate cross-study comparison. Together, our results provide a comparative overview and framework for the chemical engineering of gut-stable peptides, which should be valuable for the development of orally administered peptide therapeutics and molecular probes targeting receptors within the gastrointestinal tract.We thank Marina Kujundzic and Johanna Nemec for their help in collecting some of the stability data and peptide synthesis. We are grateful to the laboratory of Prof. David Craik (The University of Queensland) for providing Vc1.1 and cVc1.1. We thank Prof. Christian F.W. Becker (Institute of Biological Chemistry, University of Vienna) for his support of this work. We also thank Dr. Martin Zehl and the Mass Spectrometry Centre at the University of Vienna (a member of Vienna Life-Science Instruments) for assistance with HR-MS analysis. We are grateful to the NIMH PDSP (National Institute of Mental Health’s Psychoactive Drug Screening Program, Contract # HHSN-271-2018-00023-C), which is directed by Bryan L. Roth at the University of North Carolina at Chapel Hill and Project Officer Jamie Driscoll at NIMH, Bethesda MD, USA, for providing agonist functional activity data of OT variants. This research was supported by the European Research Council under the European Union’s Horizon 2020 research and innovation program (grant agreements no. 714366), by the Australian Research Council Discovery Project (DP190101667) and by the Vienna Science and Technology Fund (WWTF) through project LS18-053. T. Kremsmayr was supported by the Austrian Academy of Sciences through a DOC Fellowship (25139). J.B.B.-C. thanks the Spanish Ministry of Science and Innovation (RTI2018-096323-B-100).Peer reviewe