• asymmetric induction
• biocatalysis
• cancer
• Carbonic anhydrases
• directed evolution
• enantioselectivity
• indole
• isoxazole
• LmrR
• MAO-B
• microtubules
• neurodegenerative diseases
• neuroinflammation
• photobiocatalysis
• photocatalysis
• photochemistry
• stop codon suppression
• tetrahydroquinazolinone
• thioredoxin reductase
• triazine
• TSPO
• tubuline
• tumor

This PhD thesis focuses on the development of small molecules to modulate biological processes involved in neurodegenerative diseases (NDs) and cancer, and enzyme-catalyzed reactions.
The thesis is organized into five research projects: two dedicated to the treatment of NDs and two to cancer therapy, conducted at the Department of Pharmacy of the University of Pisa, and one exploring enzyme engineering for catalytic applications conducted during a research period abroad at the Department of Science and Engineering of the University of Groningen.
NDs are characterized by a progressive neuronal loss, often associated with neuroinflammation and dysregulated neurotransmitter levels. The research activity of this thesis focuses on modulating key proteins associated with neuroinflammation and degenerative processes. Among these, the translocator protein (TSPO), an 18 kDa mitochondrial protein responsive to neuroinflammation, facilitates the synthesis of neurosteroids endowed with anti-inflammatory properties, making it a promising target for the treatment of NDs. Additionally, carbonic anhydrases (CAs), through their enzymatic activity, can enhance synaptic strength, representing another target in the context of neuroprotection. Finally, monoamine oxidase B (MAO-B), an enzyme involved in the oxidative deamination of biogenic amines including neurotransmitters like dopamine, has a key role in neurodegenerative conditions.
On the other hand, cancer is a multifactorial disease characterized by a network of oncogenic mechanisms and resistance to therapies. Despite decades of advancements in treatment and early diagnosis, it remains the second leading cause of death globally, reflecting the need for targeted anticancer therapies. In the context of this thesis, the research activity focuses on the development of anticancer agents targeting two of the numerous cancer targets: spindle formation and thioredoxin reductase (TrxR) system involved in cell growth and proliferation.
The first project aimed to develop a library of 2-phenylindole-based dual TSPO/CA modulators to counter neuroinflammation and promote neuroprotection. Specifically, dual TSPO/CA modulators were synthesized and biologically evaluated, resulting in promising biological activity, particularly with a lead compound showing no cytotoxicity and capable of stimulating steroidogenesis, activating the cerebral hCA VII, and upregulating the gene expression of the brain-derived neurotrophic factor (BDNF).
The second project focused on the synthesis and preliminary biological evaluation of 2-(phenylamino)-7,8-dihydroquinazolinone derivatives as MAO-B inhibitors. These derivatives exhibited high selectivity and potency against MAO-B, with molecular modeling supporting favorable interactions within the active site.
The third project explored the synthesis of 3,4-dihydrobenzoimidazo[1,2-a][1,3,5]triazine (BIT) derivatives as potential anticancer agents. Evaluation on colorectal adenocarcinoma cell lines revealed significant antiproliferative effects, with promising derivatives displaying a similar mechanism of action to that of paclitaxel, identifying them as microtubule stabilizing agents (MSAs).
In the fourth project, auranofin, a gold-based drug with established use in the treatment of rheumatoid arthritis, was modified to develop an anticancer analogue. A novel complex was synthesized by coupling PIGA, a TSPO ligand, with the auranofin-derived cationic fragment. This design enabled the compound to specifically deliver its gold-based pharmacophore to mitochondria, where it can inhibit the thioredoxin reductase system (Trx/TrxR), a key mitochondrial component in cancer cells.
Finally, the fifth project was conducted in the field of biocatalysis, with a distinct focus from pharmacological applications. This study explored enzyme engineering to achieve asymmetric induction in radical reactions which is challenging because of the high reactivity of radical species. Using a thiophenol-based non-canonical amino acid (ncAA) within the Lactococcal multidrug resistance regulator (LmrR), a cyclopropane ring-opening cycloaddition was investigated, leveraging the ability of thiophenol to form thiyl radicals under UV-light irradiation. This project thus introduced a novel method for stereoselective radical reactions, merging radical reactivity with the precision of enzyme catalysis.
Overall, this thesis underscores different applications of small molecule design in NDs, tumor biology, and biocatalysis, demonstrating both therapeutic and catalytic innovation across these areas. By addressing specific molecular targets and reaction challenges, the research projects collectively highlighted the potential of small molecules as versatile modulators in both a disease-related and a chemical biology context. Additionally, stereochemistry plays a critical role in drug discovery, as the biological activity and therapeutic efficacy of small molecules often depend on their specific spatial arrangement. This highlights the importance of stereoselective strategies in improving both drug development and catalytic methods.