Identification and Biochemical Characterization of Pyrrolidinediones as Novel Inhibitors of the Bacterial Enzyme MurA

Abstract

To develop novel antibiotics, targeting the early steps of cell wall peptidoglycan biosynthesis seems to be a promising strategy that is still underutilized. MurA, the first enzyme in this pathway, is targeted by the clinically used irreversible inhibitor fosfomycin. However, mutations in its binding site can cause bacterial resistance. We herein report a series of novel reversible pyrrolidinedione-based MurA inhibitors that equally inhibit wild type (WT) MurA and the fosfomycin-resistant MurA C115D mutant, showing an additive effect with fosfomycin for the inhibition of WT MurA. For the most potent inhibitor 46 (IC50 = 4.5 μM), the mode of inhibition was analyzed using native mass spectrometry and protein NMR spectroscopy. The compound class was nontoxic against human cells and highly stable in human S9 fraction, human plasma, and bacterial cell lysate. Taken together, this novel compound class might be further developed toward antibiotic drug candidates that inhibit cell wall synthesis.

Cannabidiol Interactions with Outer Membrane Proteins in Salmonella Typhimurium LT2

“…While the formation of UDP-MurNAc-peptide and other intermediates requires the activation/catalysis of enzymes such as MurC, MurD, MurE, MurF, and MurG. Lipid II is an important peptidoglycan precursor, and lipid II synthesis are modulated by enzymes such as Phospho-N-acetylmuramoyl-pentapeptide-transferase (MraY) and UDP-N-acetylglucosamine transferase (MurG) (Fathalla et al, 2022). In the periplasmic space (inner membrane) are where the penicillin-binding proteins (PBPs) are found.…”

Targeting the binding pocket of the fluorophore 8‐anilinonaphthalene‐1‐sulfonic acid in the bacterial enzyme MurA

“…The bacterial transformation protocol started with thawing the competent C41 E. coli BL21 cells from Sigma Aldrich (catalog number CMC0017) on ice, then 50 ng plasmid DNA was added and the cells were incubated on ice for 30 min. The E. coli MurA WT and C115D mutant plasmids were obtained as previously described [ 50 ] and the E. cloacae MurA expression plasmid was generated as described above. The cells were then heat‐shocked for 1 min at 42°C and were again stored on ice for 2 min.…”

“…The bacterial transformation protocol started with thawing the competent C41 E. coli BL21 cells from Sigma Aldrich (catalog number CMC0017) on ice, then 50 ng plasmid DNA was added and the cells were incubated on ice for 30 min. The E. coli MurA WT and C115D mutant plasmids were obtained as previously described [50] and the E.…”

“…E. coli K12 MurA and the C115D mutant enzymes were overexpressed as His-tag fusion proteins in E. coli BL21 cells according to a previously reported procedure. [50] 4. Table S2.…”