Cassese, Emilia (2025) Discovery of agents against "superbugs" with new mechanisms of action. [Tesi di dottorato]

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Abstract

Antimicrobial resistance (AMR) is one of the most urgent global health concerns, driven largely by the large bacterial production of β-lactamase enzymes responsible for the hydrolysis and inactivation of the most used β-lactam antibiotics. The β-lactamase are divided into two major classes Serine- and Metallo-β-lactamases (SBL and MBL, respectively), based on their catalytic mechanism. While several inhibitors of SBLs are currently marketed drugs, the treatment of infections caused by bacteria expressing MBLs remains an urgent unmet medical need. MBLs, such as New Delhi Metallo-β-lactamase-1 (NDM-1), catalyse the hydrolysis of almost all β-lactam antibiotics, including carbapenems and except for monobactams, representing a serious threat to human health. Despite extensive research efforts, no MBL inhibitors have yet reached clinical approval; Taniborbactam, active against both SBLs and MBLs, is the most advanced clinical candidate in Phase III for urinary tract infections in combination with Cefepime. In this context, the industrial Ph.D. project in collaboration with Sibylla Biotech introduced three complementary and highly innovative strategies to address antimicrobial resistance: 1) Application of Pharmacological Protein Inactivation by Folding Intermediate Targeting (PPI-FIT) technology to NDM-1 to identify Folding Interfering Small Molecules (FISMs); 2) Rational design and synthesis of broad-spectrum MBL inhibitors through a combination of ligand-based and structure-based strategies; 3) Design of novel antibacterials applying innovative green synthetic methodologies. The first approach was conducted both at Sibylla Biotech and UNINA and focused on the application of Pharmacological Protein Inactivation by Folding Intermediate Targeting (PPI-FIT) technology, a pioneering platform developed by Sibylla Biotech, to NDM-1. PPI-FIT targets protein folding intermediates emerging during the folding process with small molecules designed to stabilize these intermediates and prevent the protein from reaching its native and active state. The misfolded protein is then recognized and degraded by the cellular quality-control machinery. Through this approach, the first Folding Interfering Small Molecules (FISMs) against NDM-1 were identified. The compounds selectively induced NDM-1 degradation without affecting the properly folded enzyme and interfering with the mRNA levels, thereby fulfilling all the criteria defining bona fide FISMs. This discovery represents the first proof-of-concept of a degrader mechanism in prokaryotic cells, paving the way for an entirely new therapeutic paradigm against resistant bacteria. The second part of the project focused on the rational design of broad-spectrum MBL inhibitors through a combination of ligand-based and structure-based strategies. Starting from literature-known zinc-binding motifs and compounds previously developed within our group, new tetrahydro-β-carboline and spiropyrrolidone scaffolds endowed with a thiol group to coordinate Zn(II)-ions of MBL active site were designed and optimized to coordinate the catalytic zinc ions within MBL active sites. Several derivatives displayed potent inhibitory activity against NDM-1, VIM-2, and IMP-1, confirming their potential to inhibit multiple clinically relevant isoforms and restore β-lactam efficacy against multidrug-resistant pathogens. Finally, during the secondment at the Institute of Organic Chemistry – Polish Academy of Sciences, a green synthetic approach was developed for the generation of novel antibacterials through the Passerini multicomponent reaction conducted under micellar conditions. This sustainable methodology enabled the synthesis of peptidomimetic ciprofloxacin derivatives with improved antimicrobial activity and favourable physicochemical properties. All the compounds demonstrated superior potency to ciprofloxacin, particularly against Escherichia coli multidrug-resistant strains and other ESKAPE pathogens, while maintaining low cytotoxicity. Overall, the Ph.D. work introduces three cutting-edge strategies to counteract antimicrobial resistance: (i) the PPI-FIT approach, establishing a new frontier in prokaryotic protein degradation; (ii) the rational design of broad-spectrum MBL inhibitors; and (iii) the development of novel green-synthesized antibiotics via multicomponent reaction. Together, these results contribute to expanding both the conceptual and chemical landscape of antibacterial drug discovery, offering transformative perspectives for the development of next-generation therapeutics against multidrug-resistant bacteria.

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