Investigations on a potential antimicrobial adjuvant as inhibitor of bacterial non-essential pathway in human and veterinary bacteria

Abstract

The threat of antimicrobial resistance (AMR) is critically worldwide widespread and, in recent years, the search for novel therapeutical strategies to counteract this phenomenon both in human and veterinary medicine is of great interest. Antibacterial adjuvants represent a valid alternative to the use of conventional single antimicrobial molecules, since their activity in combination allows to significantly reduce the therapeutical dosage of the latter, in reason to the synergistic effect of their combination. With this aim, in this PhD thesis was reported the antimicrobial activity of an inhibitor of bacterial non-essential pathway – that is O-acetylserine sulfhydrilase (OASS) inhibitor, UPAR415 – as colistin adjuvant towards six bacterial reference strains of veterinary and human interest, three Gram-negative (Escherichia coli; Salmonella Typhimurium and Klebsiella pneumoniae) and three Gram-positive (Staphylococcus aureus; Methicillin resistant Staphylococcus aureus and Staphylococcus pseudintermedius). UPAR415 represents the most potent OASS inhibitor so far, due to its nanomolar ranged dissociation constant (Kd) and, in addition to the already demonstrated inhibition of the enzyme, it showed a promising activity in combination with colistin, synergistic against almost all Gram-negatives and additive against Gram-positives. Furthermore, association activity was confirmed in a rich medium (Müeller Hinton broth), where cysteine (the biosynthetic product of reductive sulfate assimilation pathway using inorganic sulfur in procaryotes) is abundant. The intracellular target engagement of UPAR415 was confirmed using a S. Typhimurium strain inactivated for OASS-A, supporting the proposed mechanism of action. Moreover, to assess the absence of cell toxicity, UPAR415 and its association with colistin were tested on bovine kidney cells (MDBK) and sheep defibrinated blood, ensuring a good biocompatibility and a very low hemolytic effect. At last, the 3D structure of UPAR415 in complex with OASS was determined, providing the first structural insight about the interaction between the enzyme and this class of competitive inhibitors. The obtained results showed in this PhD thesis could represent a good starting point for the development of antibiotic adjuvants operating on a bacterial non-essential target, such as cysteine biosynthetic pathway, and could pave the way for further research on adjuvant compounds able to overcome the current pressing issue of AMR, both in veterinary and human medicine.