SYNTHESIS OF RGD PEPTIDOMIMETIC-DRUG CONJUGATES FOR THE TUMOR-TARGETED DELIVERY OF CYTOTOXIC AGENTS

Integrins are a large family of heterodimeric transmembrane glycoprotein receptors, composed by two non-covalently associated subunits (α and β). Integrins αVβ3 and αVβ5 are overexpressed on blood vessels in human tumors but not on vessels in normal tissues and, for this reason, they have become attractive targets for pharmacological studies in oncology. Thus, in this PhD thesis, the synthesis of new Small Molecule-Drug Conjugates (SMDCs) targeting αvβ3 integrin is described. The structure of such SMDCs consists in the connection of three core components: i) LIGAND - the cyclo[DKP-RGD] peptidomimetic, developed by the Gennari and Piarulli group, has been used as integrin-targeting moiety in all the new constructs; ii) CYTOTOXIC PAYLOAD - three different cytotoxic agents (i.e. paclitaxel, monomethyl auristatin E and monomethyl auristatin F) have been included as anticancer drugs; iii) LINKER - specific functional groups (i.e. peptides) have been used to connect the drug and ligand, aiming at the selective drug release in the intra or extracellular tumor environment. These new SMDCs have been subjected to a panel of biochemical and biological assays, for assessing both their structural features (e.g. stability, kinetics of drug release, etc.) and biological activity (e.g. affinity for the purified integrin αVβ3 receptor, selective cytotoxicity against αvβ3-expressing or non-expressing cells, etc.). As a first project, Chapter II describes the development of a new library of multimeric cyclo[DKP-RGD]-PTX conjugates (monomeric, dimeric, trimeric and tetrameric conjugates) bearing lysosomally-cleavable linkers and their full in vitro biological evaluation. The results achieved with these first compounds prompted the design of next-generation cyclo[DKP-RGD]-PTX conjugates, reported in the following chapters. Chapter III describes the synthesis and in vitro evaluation of cyclo[DKP-RGD]-PTX conjugates bearing extracellularly-cleavable peptide linkers, capable of releasing the payload in the tumor microenvironment, rather than inside cancer cells: this mechanism can be promoted by tumor-associated enzymes present in the tumor stroma (e.g. elastase), which can efficiently cleave the linker and set the drug free to diffuse within the tumor mass. Finally, Chapter IV consists in the development of a small library of conjugates containing the cyclo[DKP-RGD] ligand, a lysosomally-cleavable peptide linker and the highly potent toxins monomethyl auristatin E or F (MMAE and MMAF) as the cytotoxic payloads, which are state-of-the-art tools for targeted anticancer therapy.