Notch signaling is a highly conserved developmental pathway that plays important roles in the regulation of cellular processes including cell fate decisions and stem cell maintenance. A tight regulation of the Notch cascade ensures proper tissue homeostasis in adult organs, in which the outcome is context-dependent in terms of tissue and cells. Aberrant activation of Notch signaling is implicated in many cancers, including T-cell acute lymphoblastic leukemia (T-ALL). The majority of T-ALL patients carry mutations in the negative regulatory region (NRR) or in the PEST sequence of NOTCH1. Notch NRR plays a crucial role in maintaining the signaling shut down by preventing catalytic activation of the receptor, and mutations in this domain lead to the constitutive activation of the cascade and the subsequent development of T-ALL. The extensive implication of Notch signaling in diseases highlights the need for the development of new therapeutics targeting this pathway. One aim of my thesis was to develop Notch inhibitors in the format of bicyclic peptides, which have raised more and more interest as therapeutics. Indeed, bicyclic peptides combine advantages from large proteins, namely high affinity and specificity, and small molecules, namely accessible chemical synthesis and good tissue penetration. Use of phage display technology allows rapid isolation of binding molecules from large combinatorial libraries containing billion of phage peptides. In Christian Heinisâ laboratory, bicyclic peptides have mainly been developed for protease inhibition as they easily fit into catalytic pockets. In my research, I developed bicyclic peptides that bound the Notch1 NRR. Following affinity maturation, a peptide binding the protein with a Kd of 150 nM was isolated. This peptide homed the ability to stabilize the target protein against catalytic activation and thermal denaturation. However, it was not potent enough to prevent activation of the pathway in cell-based assays. Even though the peptidic ligands developed here did not lead to the expected outcome, we were able, for the first time, to demonstrate their ability to stabilize a complex domain of a receptor. A second objective of my thesis was to perform high-throughput screening (HTS) of chemical compound libraries in order to identify novel modulators of Notch. HTS is widely used in biology and chemistry for drug discovery and allows the use of functional assays to screen libraries of million of molecules. Three libraries were screened in the frame of this project, using a luciferase-reporter gene assay known as the co-culture assay, leading to the identification of thousands of potential modulators. From the 1953 potential inhibitors isolated during ï¿Œthe primary HTS, âŒ200 molecules were left following a secondary screen; they now need to be further characterized in vitro and in vivo for specific Notch inhibition, mechanism of action and pharmaco-kinetic properties.