Peptide therapeutics hold significant therapeutic promise, potentially offering patients treatments with high potency, selectivity and reduced off-target toxicity. However, the development of peptide therapeutics is hindered by low oral bioavailability. Orally active peptides are required to be membrane permeable, metabolically stable and active towards therapeutical targets1. Of these, membrane permeation remains the limiting factor. The discovery of the orally bioavailable Cyclosporin A (CsA) led to the chameleonic hypothesis, suggesting solvent-dependent backbone conformation allows passive permeation2. However, this hypothesis fails to guide the design of permeable cyclic peptides targeting GPCRs, which often require polar/charged residues.
The somatostatin receptor (SSTR) family are well-studied GPCRs that are modulated by small cyclic peptides3. There are 5 subtypes in this family, SSTR1-5, which are responsible for a diverse range of physiological functions including hormone regulations. Current therapeutics targeting SSTRs are administered via injections4. Patient compliance and comfort can be greatly improved by oral treatments.
In this work, we aim to improve the passive permeability of an active SSTR2 ligand, MK678, to elucidate the structural properties of permeable cyclic peptides targeting GPCRs. To address this, we synthesised combinatorial libraries of MK678 analogues retaining the active pharmacophore (D-Trp-Lys) to SSTR2. These libraries were then screened for passive permeability in Parallel Artificial Membrane Permeability Assay (PAMPA), followed by deconvoluting the sequence by re-synthesising permeable hits as pure peptides. The SSTR2 activity of pure permeable hits was evaluated using Bioluminescence Resonance Energy Transfer assay (BRET) by measuring the dissociation of Gαi/o and Gβ/γ.
References
(1) Räder, A. F. B.; Weinmüller, M.; Reichart, F.; Schumacher-Klinger, A.; Merzbach, S.; Gilon, C.; Hoffman, A.; Kessler, H. Orally Active Peptides: Is There a Magic Bullet? Angewandte Chemie International Edition 2018, 57 (44), 14414-14438. DOI: https://doi.org/10.1002/anie.201807298.
(2) Corbett, K. M.; Ford, L.; Warren, D. B.; Pouton, C. W.; Chalmers, D. K. Cyclosporin Structure and Permeability: From A to Z and Beyond. Journal of Medicinal Chemistry 2021, 64 (18), 13131-13151. DOI: 10.1021/acs.jmedchem.1c00580.
(3) Günther, T.; Tulipano, G.; Dournaud, P.; Bousquet, C.; Csaba, Z.; Kreienkamp, H. J.; Lupp, A.; Korbonits, M.; Castaño, J. P.; Wester, H. J.; et al. International Union of Basic and Clinical Pharmacology. CV. Somatostatin Receptors: Structure, Function, Ligands, and New Nomenclature. Pharmacol Rev 2018, 70 (4), 763-835. DOI: 10.1124/pr.117.015388 From NLM.
(4) de Herder, W. W. Somatostatin Analogues in Pharmacotherapy. In Somatostatin Analogues, 2015; pp 166-168.