Giorgio Milli

FPC5 – Giorgio Milli

Department of Drug Sciences, University of Pavia, Viale Taramelli 12, Pavia 27100, Italy

giorgio.milli01@universitadipavia.it

Expanding the Chemical Space for LsrK Kinase Modulators: Hit Identification and Biological Validation for AI-2 Quorum-Sensing Quenching and Biofilm Formation Inhibition
Milli Giorgio¹, Ambrosio Francesca Alessandra², Bellan Menegussi Elisa³, Pagano Katiuscia⁴, Ragona Laura⁴, Costa Giosuè², Alcaro Stefano²,Pietrocola Giampiero³, Linciano Pasquale¹, Collina Simona¹. ¹ Department of Drug Sciences, University of Pavia, Viale Taramelli 12, Pavia 27100, Italy ² Department of Health Sciences, University “Magna Græcia” of Catanzaro, Viale Europa, 88100 Catanzaro, Italy ³ Department of Molecular Medicine, Biochemistry Unit, University of Pavia, Viale Taramelli 3/b, Pavia 27100, Italy ⁴ NMR Laboratory, Istituto di Scienze e Tecnologie Chimiche “Giulio Natta”, CNR, via Alfonso Corti, 12, Milano 20133, Italy
Abstract Antimicrobial resistance (AMR) poses a critical global health crisis by undermining existing treatments and threatening public health. Consequently, identifying novel targets and antimicrobials with innovative mechanisms of action has become essential to combat AMR [1]. In this context, disrupting quorum sensing (QS) and biofilm formation emerges as a promising strategy. LsrK, a kinase that initiates QS and promotes biofilm development through phosphorylation of 4,5-dihydroxy-2,3-pentanedione (DPD) – a ubiquitous signaling molecule in both Gram-positive and Gram-negative bacteria – represents a critical component of QS pathways and its inhibition could be a winning strategy for fighting AMR [2]. To this end, structure-based virtual screening of commercial libraries identified a panel of hit molecules, which were evaluated in biological, following an experimental pathway previously optimized [3], to confirm their ability to inhibit biofilm formation in three bacterial models: Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli. We developed a dedicated synthesis strategy to obtain sufficient quantities of the most promising compounds for comprehensive biological and microbiological evaluations. Their ability to interfere with quorum sensing was assessed in Vibrio harveyi reporter assays, while interaction with LsrK was confirmed by biophysical studies, including Saturation Transfer Difference (STD) NMR. These efforts identified two lead compounds, CzdDPD 8 and CzDPD 10, with a Minimum Biofilm Inhibitory Concentration 50% (MBIC₅₀) values of 121 and 183 μg mL⁻¹ against E. coli, and 55 and 166 μg mL⁻¹ against P. aeruginosa, respectively. Mechanistic studies validated QS disruption and LsrK binding, marking a significant advance against AMR.
References [1] World Health Organization. Global Antibiotic Resistance Surveillance Report 2025: WHO Global Antimicrobial Resistance and Use Surveillance System (GLASS) – Summary; World Health Organization, 2025. https://doi.org/10.2471/B09585. [2] Linciano, P.; Cavalloro, V.; Martino, E.; Kirchmair, J.; Listro, R.; Rossi, D.; Collina, S. Tackling Antimicrobial Resistance with Small Molecules Targeting LsrK: Challenges and Opportunities. J. Med. Chem. 2020, 63 (24), 15243–15257. https://doi.org/10.1021/acs.jmedchem.0c01282. [3] Milli, G.; Pellegrini, A.; Listro, R.; Fasolini, M.; Pagano, K.; Ragona, L.; Pietrocola, G.; Linciano, P.; Collina, S. New LsrK Ligands as AI-2 Quorum Sensing Interfering Compounds against Biofilm Formation. J. Med. Chem. 2024, 67 (20), 18139–18156. https://doi.org/10.1021/acs.jmedchem.4c01266.