Antiparasitic drug nitazoxanide inhibits the pyruvate oxidoreductases of Helicobacter pylori, selected anaerobic bacteria and parasites, and Campylobacter jejuni.
Nitazoxanide (NTZ) exhibits broad-spectrum activity against anaerobic bacteria and parasites and the ulcer-causing pathogen Helicobacter pylori. Here we show that NTZ is a noncompetitive inhibitor (K(i), 2 to 10 microM) of the pyruvate:ferredoxin/flavodoxin oxidoreductases (PFORs) of Trichomonas vaginalis, Entamoeba histolytica, Giardia intestinalis, Clostridium difficile, Clostridium perfringens, H. pylori, and Campylobacter jejuni and is weakly active against the pyruvate dehydrogenase of Escherichia coli. To further mechanistic studies, the PFOR operon of H. pylori was cloned and overexpressed in E. coli, and the multisubunit complex was purified by ion-exchange chromatography. Pyruvate-dependent PFOR activity with NTZ, as measured by a decrease in absorbance at 418 nm (spectral shift from 418 to 351 nm), unlike the reduction of viologen dyes, did not result in the accumulation of products (acetyl coenzyme A and CO(2)) and pyruvate was not consumed in the reaction. NTZ did not displace the thiamine pyrophosphate (TPP) cofactor of PFOR, and the 351-nm absorbing form of NTZ was inactive. Optical scans and (1)H nuclear magnetic resonance analyses determined that the spectral shift (A(418) to A(351)) of NTZ was due to protonation of the anion (NTZ(-)) of the 2-amino group of the thiazole ring which could be generated with the pure compound under acidic solutions (pK(a) = 6.18). We propose that NTZ(-) intercepts PFOR at an early step in the formation of the lactyl-TPP transition intermediate, resulting in the reversal of pyruvate binding prior to decarboxylation and in coordination with proton transfer to NTZ. Thus, NTZ might be the first example of an antimicrobial that targets the "activated cofactor" of an enzymatic reaction rather than its substrate or catalytic sites, a novel mechanism that may escape mutation-based drug resistance.
Hoffman
Paul S
PS
Division of Infectious Diseases and International Health, 409 Lane Road, University of Virginia Health Systems, Charlottesville, VA 22908-1340, USA. psh2n@virginia.edu
Sisson
Gary
G
Croxen
Matthew A
MA
Welch
Kevin
K
Harman
W Dean
WD
Cremades
Nunilo
N
Morash
Michael G
MG
D000105:Acetyl Coenzyme A / Q000037:antagonists & inhibitors / Q000096:biosynthesis; D000818:Animals; D000981:Antiprotozoal Agents / Q000494:pharmacology*; D001421:Bacteria, Anaerobic / Q000187:drug effects / Q000201:enzymology*; D016123:Campylobacter jejuni / Q000187:drug effects / Q000201:enzymology*; D002245:Carbon Dioxide / Q000378:metabolism; D003001:Cloning, Molecular; D003013:Clostridium / Q000187:drug effects / Q000201:enzymology; D003470:Culture Media; D004791:Enzyme Inhibitors; D016480:Helicobacter pylori / Q000187:drug effects / Q000201:enzymology*; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D009574:Nitro Compounds; D010271:Parasites / Q000187:drug effects / Q000201:enzymology*; D011768:Pyruvate Dehydrogenase Complex / Q000037:antagonists & inhibitors*; D019289:Pyruvic Acid / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013835:Thiamine Pyrophosphate / Q000378:metabolism; D013844:Thiazoles / Q000494:pharmacology*
D000981:Antiprotozoal Agents; D003470:Culture Media; D004791:Enzyme Inhibitors; D009574:Nitro Compounds; D011768:Pyruvate Dehydrogenase Complex; D013844:Thiazoles; D002245:Carbon Dioxide; D000105:Acetyl Coenzyme A; D019289:Pyruvic Acid; D007770:L-Lactate Dehydrogenase; D013835:Thiamine Pyrophosphate; C041747:nitazoxanide