Nicola Pontillo

This thesis explores strategies to revive aminoglycoside (AG) antibiotics in the face of growing antimicrobial resistance (AMR). AGs are potent bactericidal drugs, but their clinical use is compromised by toxicity and resistance mechanisms such as enzymatic modification and ribosomal methylation. The work is organized into six chapters:

Chapter 1 – Background and Rationale Provides an overview of AG antibiotics, their mechanism of action, and the major resistance pathways, including aminoglycoside-modifying enzymes (AMEs) and 16S rRNA methyltransferases. It establishes the urgent need for innovative approaches to restore AG efficacy.

Chapter 2 – Overcoming AAC(3)-Mediated Resistance Focuses on N-acetyltransferase AAC(3)-IIIa, a key AME. Through structural and kinetic studies, the chapter demonstrates that minimal chemical modifications such as 3-N-alkylation of neomycin can prevent acetylation while preserving ribosomal binding, validated by cryo-EM analysis.

Chapter 3 – Photopharmacology and Dynamic Control Introduces photo-switchable aminoglycoside dimers incorporating azobenzene linkers. These compounds enable reversible modulation of antibiotic activity using light, offering spatiotemporal control and reducing selective pressure for resistance.

Chapter 4 – Site-Selective Modifications Against APH Enzymes Investigates structural modifications at the 3’-OH position of kanamycin and paromomycin to evade phosphorylation by APH(3’)-Ia. Several derivatives retained antibacterial activity and acted as competitive inhibitors. X-ray crystallography revealed steric hindrance at the ATP binding site, explaining resistance evasion.

Chapter 5 – In-Silico Design of ArmA Inhibitors Explores fragment-based and AI-assisted computational approaches to identify inhibitors of ArmA methyltransferase, which confers pan-aminoglycoside resistance. Two lead scaffolds were designed for synthetic accessibility, and preliminary synthesis confirmed feasibility despite solubility challenges.

Chapter 6 – General Discussion and Conclusions Summarizes the findings, discusses their scientific and technical implications, and places the research in a broader societal and translational context. It addresses limitations, future directions, and the cultural aspects of antibiotic innovation.

Overall thi thesis demonstrates a multidisciplinary approach, combining synthetic chemistry, structural biology, and computational design, to address AMR. While experimental validation and optimization remain ongoing, these findings provide a foundation for next-generation aminoglycosides and resistance inhibitors.