DEVELOPMENT OF NOVEL STRATEGIES TO ENHANCE THE AFFINITY OF CYCLIC PEPTIDE LIGANDS FOR INTEGRIN RECEPTORS

The impact of monoclonal antibodies (mAbs) in current pharmaceutical research is due to their unique ability to bind biological targets with very high affinity. On the other hand, there is a considerable interest in the development of small molecule ligands with antibody-like affinities, which may overcome some limitations of mAbs. This PhD work describes the development of general strategies to increase the binding affinity of peptide ligands bearing the Arg-Gly-Asp motif, i.e. the well-known recognition sequence of specific tumor-associated integrin receptors. In our first approach, we designed a bicyclic peptide bearing two RGD motifs that displayed an enhanced inhibition of ECM protein binding to integrin receptors αvβ3 and α5β1 and marked biological effects in U-373 MG glioblastoma cells. Later on, we focused on the 2-hydroxybenzaldehyde tag (2HB), which can engage ϵ‐amino groups of Lys residues in stable imines. After investigating the 2HB installation to different types of reactive handles, we conjugated the 2HB tag to the N-side and on the C-side of a cyclic RGD peptide. The resulting conjugates have been investigated as novel αvβ3 integrin ligands and the nature of the ligand-protein interaction has been investigated performing in silico experiments. For both the 2HB-RGD conjugates, the biological results and the computational outcomes demonstrated to be coherent with each other, proving the feasibility of the reversible covalent engagement of Lys residues with the 2HB tag to enhance the affinity of a well-known small ligand.