Benzothioxalone derivatives as novel inhibitors of UDP-N-acetylglucosamine enolpyruvyl transferases (MurA and MurZ).
OBJECTIVE
We sought to identify and characterize new inhibitors of MurA and MurZ, which are enzymes involved in the early stages of bacterial peptidoglycan synthesis.


METHODS
A library of ∼650 000 compounds was screened for inhibitors of Escherichia coli MurA in an endpoint assay measuring release of inorganic phosphate from phosphoenolpyruvate. Hits were validated by determining the concentrations required for 50% inhibition (IC(50)) of MurA from E. coli and MurA/MurZ from Staphylococcus aureus. The mode of action of selected inhibitors was explored by examining the reversibility of MurA inhibition, the binding of a radiolabelled inhibitor to MurA proteins and through docking studies. Inhibitors were further characterized by determining their antibacterial activity against E. coli and S. aureus.


RESULTS
Benzothioxalone derivatives were identified that inhibited MurA from E. coli and MurA/MurZ from S. aureus with IC(50) values between 0.25 and 51 µM. Several inhibitors also exhibited activity against S. aureus with MICs in the range 4-128 mg/L. Inhibition of MurA was irreversible and a radiolabelled inhibitor from this compound class displayed stoichiometric binding to the enzyme, which was displaced by dithiothreitol. Binding was undetectable with a C115D mutant MurA protein.


CONCLUSIONS
The results suggest a mode of action for the benzothioxalones that involves the formation of a disulfide bond with MurA/MurZ, via attack from an active site cysteine on the thioxalone ring carbonyl group, followed by ring opening to yield an S-acylated protein. The proposed covalent mode of action may prove useful in the design of new antibacterial agents.
Miller
Keith
K
Antimicrobial Research Centre, University of Leeds, Leeds, UK.
Dunsmore
Colin J
CJ
Leeds
Jennifer A
JA
Patching
Simon G
SG
Sachdeva
Meena
M
Blake
Katy L
KL
Stubbings
William J
WJ
Simmons
Katie J
KJ
Henderson
Peter J F
PJ
De Los Angeles
Joe
J
Fishwick
Colin W G
CW
Chopra
Ian
I
D019883:Alkyl and Aryl Transferases / Q000037:antagonists & inhibitors*; D000900:Anti-Bacterial Agents / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors; D004791:Enzyme Inhibitors / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology; D004926:Escherichia coli / Q000187:drug effects* / Q000201:enzymology; D057166:High-Throughput Screening Assays; D006801:Humans; D007783:Lactones / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology; D008826:Microbial Sensitivity Tests; D010457:Peptidoglycan / Q000096:biosynthesis; D013211:Staphylococcus aureus / Q000187:drug effects* / Q000201:enzymology; D013329:Structure-Activity Relationship
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D007783:Lactones; D010457:Peptidoglycan; C009474:thioxolone; D019883:Alkyl and Aryl Transferases; C022566:UDP-N-acetylglucosamine 1-carboxyvinyltransferase