• antivirulence agents
• biofilm
• COVID-19
• Docking
• dual inhibitors
• Electrostatic complementarity (EC)
• ESKAPE bacteria
• Gram-negative bacteria
• Healthcare-associated infections (HCAIs)
• LasB
• Main protein
• Mpro
• Nosocomial Infections (NIs)
• Papain-like protein
• PLpro
• Pseudolysin
• Pseudomonas aeruginosa
• SARS-CoV-2
• Thermolysin
• Virulence factors
• Zinc metalloproteases

The development of novel antibacterial and antiviral agents is necessary to treat two recent medical issues: healthcare-associated infections (HCAIs) and the transmission of COVID-19. HCAIs are mainly caused by Gram-negative and Gram-positive bacteria belonging to the multi-drug resistant bacterial strains known as "ESKAPE" (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter species). In fact, these bacteria are not susceptible to classical antibiotics, and novel drugs with a different mechanism of action are needed. Pseudolysin (PLN) and thermolysin (TLN), metalloproteases involved in bacterial nutrition and sporulation, represent potential targets to block bacterial development and counteract the phenomenon of multidrug resistance (MDR). LM-2, a hydroxamic acid previously developed by our research group, has shown a good inhibitory activity against these two enzymes (Ki-PLN: 4.5 ± 0.1 µM and Ki-TLN: 7.4 ± 0.2 µM) and a series of racemic and optically pure derivatives were developed. The best compounds were selected and their overall anti-proteolytic activity against the PaO1 strain of P. aeruginosa was also evaluated. It is noteworthy that P. aeruginosa secretes four types of proteases: three metalloproteases (PLN, LasA and alkaline protease) and one serine protease (protease IV). Finally, the inhibitory activity specifically directed against PLN has been evaluated on culture filtrates of PaO1 after treatment with the compounds. Hence, further investigations will be conducted to clarify the inhibitory activity of these compounds against LasA, and the alkaline protease and their ability to reduce biofilm formation, leading to a potential new generation of chemotherapeutics that act as adjuvants to classical antibiotics.
The COVID-19 pandemic, which emerged in December 2019, was initially treated with mRNA and adenovirus-based vaccines approved by the FDA, targeting the viral spike protein to prevent host cell entry. In addition, antiviral development has been focused on key SARS-CoV-2 components such as RdRp (RNA-dependent RNA polymerase), Mpro (main protein), and PLpro (papain-like protein). Moreover, recent studies have suggested the involvement of PLpro in the COVID-19 cytokine storm. Herein, my effort has been directed towards the development of dual covalent inhibitors of Mpro and PLpro using olefins conjugated with different sulfone fragments as electrophilic warheads to interact with cysteine residues present in the catalytic sites of both viral proteases. This strategy could counteract SARS-CoV-2 infection by reducing viral replication and inflammation. Therefore, a first series of potential Mpro inhibitors was developed from two already reported COX-2 enzyme inhibitors. The best compound of this series displayed an IC50 against PLpro of 100 µM and a 10% inhibition of Mpro at 100 µM as concentration. The latter was then further modified by computational studies to improve its dual activity leading to a series of [1,8]-naphthyridine derivatives. These derivatives were also synthesized and tested against Mpro by a fluorometric assay. One of the new compounds synthesized showed an inhibition of 20% of Mpro at a concentration of 2 µM, representing a 100-fold improvement in inhibitory activity with respect to the best compound of the first series, thus validating the structure-based optimization approach. The intrinsic fluorescence of the newly developed compounds has hindered the evaluation of inhibitory activity against PLpro, and further enzymatic assays will be developed to fully characterize the most promising derivatives.