ETD

• bioorthogonal chemistry
• click chemistry
• drug delivery
• Listeria Monocytogenes
• melanoma

In recent years, the use of bacteria as cancer vaccine has been very successful. Several studies have explored the potential of bacteria, particularly attenuated Listeria Monocytogenes (Lmat), as intelligent drug delivery systems. Lmat has been widely used for its ability to infect even the most hypoxic regions, spread within and stimulate the immune system to fight cancer cells, promoting tumour regression. Thanks to its intrinsic tumour-targeting properties, Lmat can deliver therapeutic payloads selectively to the Tumor Microenvironment (TME), combining its immunostimulatory effects with the chemotherapeutic action of the cargo.
Bio-orthogonal chemistry is a widely used approach in cell biology and microbiology that enables chemical reactions to occur in living cells and bacteria without interfering with natural biological processes or damaging cellular components.
Cutaneous melanoma is a skin cancer which originates from the uncontrolled proliferation of melanocytes and often spreads to distant sites. DNA mutations, induced for example by UV rays, play a key role in the development of this tumor; the most commons occur in the BRAF proto-oncogene which regulates the Mitogen Activated Protein Kinase (MAPK) pathway, thereby controlling cell growth and proliferation.
The therapies currently used to treat cancer patients include targeted therapy and immunotherapy. The first one employs drugs that specifically block the molecular alterations driving tumour growth. In melanoma, targeted therapy focuses on MAPK signalling pathway, particularly on key proteins such as BRAF and MEK. Examples of selective inhibitors are Vemurafenib and Dabrafenib as BRAF inhibitors (BRAFi), and Cobimetinib, Trametinib or Mirdametinib, still in phase 2 clinical trial, as MEK inhibitors (MEKi). However, chronic exposure to these agents leads to the emergence of resistance mechanisms that interfere with their efficacy, including increased pigmentation. Therefore, the co-administration of BRAFi or MEKi with pigmentation inhibitors (PIGMi) could represent a valid strategy to counteract the development of resistance. The aim of this thesis project is to conjugate Mirdametinib and Phenylthiourea (PTU), i.e. a MEKi and a PIGMi, on the modified peptidoglycan of Lmat, via bio-orthogonal chemistry. This approach exploits the natural tropism of Lmat for the TME, allowing it to function as a selective drug delivery system. In this way, the conjugated drugs can be delivered directly within the melanoma microenvironment, reducing their secondary effects on healthy tissues.
The workflow of the project involved first the synthesis of two molecules, PTU as pigmentation inhibitor, and Mirdametinib as MEK inhibitor.
Then, both of them were conjugated to: (i) an azido moiety by means of a short polyethylene glycol (PEG)-based linker, to allow the click reaction on the bacteria surface and (ii) to a dye molecule, represented by nitrobenzoxadiazole (NBD), as detection system to easily follow the outcome of the reaction.
Following the achievement of these derivatives, two click chemistry reaction protocols were developed in collaboration with Dr. Laura Poliseno’s group at the Clinical Physiology Institute, National Research Council (IFC-CNR) of Pisa, with the purpose of obtaining a high yield of conjugated Lmat while, above all, maintaining its fundamental vital functions and fitness. Bacteria cell wall was modified employing two different strategies. In the first approach, the peptidoglycan of Lmat is metabolically labelled through the incorporation of an alkyne-bearing D-alanine dipeptide; in the second, the peptidoglycan is chemically modified via covalent attachment of a molecule bearing a strained alkyne (i.e. endo-BCN-PEG4-NHS ester). These groups are then reacted with the azido compounds through two different click chemistry reactions: Copper-Catalysed Azido-Alkyne Cycloaddition (CuAAC) or Strained-Promoted Azido-Alkyne Cycloaddition (SPAAC). These reactions not only allow substantial yields to be achieved in few hours and with a simple setup, but they can also be carried out in a biocompatible environment and under physiological conditions, such as pH, temperature and non-toxic solvent.
Finally, PTU- and Mirdametinib-loaded bacteria were tested to evaluate their viability and their proliferative capacity compared to the unmodified Lmat. This biological characterization is still in its early stages, and further testing in in vitro and in vivo melanoma cell models will be required to determine whether the reactions assure the balance between Lmat fitness and loading efficiency, while enhancing the cytotoxic potential of drug-loaded listeria. Additional protocol optimisation may ultimately be necessary.