Phage Selection of Bicyclic Peptide Ligands of the Notch1 Receptor

Abstract

Precise regulation of the Notch signaling pathway is crucial, as increases or deficiencies in signaling are associated with diseases, including a wide range of cancers. Recent studies have demonstrated that monoclonal antibodies that bind and stabilize the structure of the negative regulatory region (NRR) in the extracellular domain of the Notch receptor can inhibit Notch signaling. In this work, we posed the question whether bicyclic peptides, being around 100-fold smaller than antibodies, can also stabilize the NRR and inhibit Notch signaling. Bicyclic peptides that bind the NRR of human Notch1 were isolated from combinatorial libraries by phage display. Affinity maturation yielded ligands with dissociation constant (Kd ) values as low as 150 nM. The bicyclic peptides increased the melting temperature of the NRR by up to 8 °C, thus substantially stabilizing the protein structure, but they did not inhibit Notch signaling in cellular assays. Although Notch signaling could not be inhibited, this work demonstrates that phage-selected bicyclic peptides can stabilize proteins; this capacity of bicyclic peptides may be exploited for modulating the conformation of other disease targets.

Conformational Restriction of Peptides Using Dithiol Bis-Alkylation

“…This library was applied in iterative selections for binding to the plasma protease kallikrein, ultimately producing a low-nanomolar inhibitor which was highly specific for kallikrein over other plasma proteases. Using the same strategy, Heinis and coworkers also discovered a submicromolar bicyclic ligand for the negative regulatory region of the Notch receptor (Urech-Varenne, Radtke, & Heinis, 2015). Thiol bis-alkylation has also been applied to mRNA display libraries (Schlippe, Hartman, Josephson, & Szostak, 2012).…”

Bicyclic Peptides as Next‐Generation Therapeutics

“…Heinis and colleagues screened phage displayed bicyclic peptide libraries against the soluble extracellular domains (ECDs) of the epidermal growth factor receptor Her2 and the Notch receptor. [64,65] Although relatively potent and selective ligands were identified for both ECDs ( K D of 304 and 150 nM, respectively), neither ligand showed agonist or antagonist activity against the respective receptor signaling. Presumably, the bicyclic peptides bound to the ECDs at functionally unimportant sites.…”

Neue Modalitäten für schwierige Zielstrukturen in der Wirkstoffentwicklung

“…B. mithilfe von Split-Mix-Bibliotheken bicyclischer Peptide identifiziert. B. S.-Aureus-Sortase A, [70] Her2, [71] b-Catenin, [72] Urokinase, [73] Kallikrein im Plasma [67a, 74] und Notch1, [75] zu finden. [66] Randomisierte Peptidbibliotheken, die drei konservierte Cysteinreste enthalten, kçnnen auch auf filamentçsen Phagen präsentiert und einer Cyclisierung mittels thiolreaktiver organischer Gerüststrukturen unterzogen werden (Abbildung 5a).…”