MraY from Pseudomonas aeruginosa is inhibited by uridyl-peptide antibiotics

Introduction
Antibiotic resistance is one of the most serious health threats and the therapeutic options to treat infections caused by multidrug-resistant strains are seriously compromised. Especially for infections caused by Pseudomonas aeruginosa isolates which have emerged resistance towards all carbapenems, aminoglycosides, and fluoroquinolones, novel antibiotics with new targets or unprecedented mechanisms of action are urgently needed. Integral component of the drug development process is the analysis of the mechanism of action of an antibiotic, as well as identification of the molecular target. Without this detailed knowledge, rational drug design is strongly hampered. Uridyl-peptide antibiotics (UPA) are a promising group of nucleoside natural products, which show potent activity against P. aeruginosa. An important molecular target of this compound class is the phospho-N-acetylmuramoyl-pentapeptide-transferase MraY, which catalyses the transfer of phospho-MurNAc pentapeptide to the membrane-standing lipid carrier undecaprenyl phosphate resulting in the formation of Lipid I.

Methods & Results
Using a fluorometric assay with the heterologously expressed MraY from P. aeruginosa, eight UPA's were characterized, and all showed IC₅₀ values in the nanomolar range. Further, a second periplasmatic binding site was identified and confirmed by using site-directed mutagenesis resulting in significant elevation of the IC₅₀ compared to the wildtype MraY, making UPA's the first antibiotics with a dual mode of action against the MraY of P. aeruginosa. To corroborate the data, a bioinformatical approach was used, by calculating the ΔG of the wildtype MraY and the mutants with AutoDock Vina. The theoretical binding showed very similar results to the biological data. Additionally, we could identify LPS synthesis of Pseudomonas as a new target of the UPA's, by establishing an in vitro assay with wbpL of P. aeuruginosa.

Conclusion
We could further characterize UPA's by the identification of a second periplasmatic binding site and a new mode of action by inhibiting LPS biosynthesis, making UPA's a promising new antibotic class.