Antiparasitic Drug Nitazoxanide Inhibits the Pyruvate Oxidoreductases of Helicobacter pylori , Selected Anaerobic Bacteria and Parasites, and Campylobacter jejuni

Abstract

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 M) 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.Nitazoxanide [2-acetyloxy-N-(5-nitro-2-thiazolyl) benzamide] (NTZ) is a broad-spectrum drug that is efficacious for the treatment of infections caused by amitochondriate luminal parasites and helminths (8,15,21,26) and that shows promise as an alternative therapy for treating infections caused by Clostridium difficile (20). More generally, NTZ appears to be an effective treatment for persistent diarrhea (35) and even infections caused by rotavirus (26). NTZ shares some structural similarities with thiamine pyrophosphate (TPP) (Fig. 1) and exhibits a spectrum of activity similar to those of metronidazole (MTZ) and nitrofuran drugs (22), but several lines of evidence suggest that this drug is mechanistically different from the redox-active prodrugs (8,18,28). For example, NTZ does not increase the mutation frequency or produce DNA damage in Helicobacter pylori (28), and there is no cross-resistance with MTZ, as MTZ-resistant strains of Trichomonas vaginalis and H. pylori remain susceptible to NTZ (1,18,28,34). Unlike MTZ, resistance to NTZ has not been observed clinically or generated by in vitro methods commonly used to isolate MTZ r mutants of H. pylori (18). Despite the ever widening spe...

Thiazolides, a New Class of Antiviral Agents Effective against Rotavirus Infection, Target Viral Morphogenesis, Inhibiting Viroplasm Formation

“…The molecular mechanism at the basis of the antiinfective activity of thiazolides is still not completely understood. In anaerobic organisms, such as Giardia intestinalis or anaerobic bacteria, nitazoxanide acts by inhibiting pyruvate:ferredoxin oxidoreductase (PFOR), an enzyme that is essential for the pathogen energy metabolism but that is not conserved in mammals (32). In the case of M. tuberculosis infection, inhibition of mTORC1 signaling and stimulation of autophagy were observed after thiazolide treatment, and the human quinone oxidoreductase NQO1 was proposed to be a drug target (33).…”

Flavodoxin:Quinone Reductase (FqrB): a Redox Partner of Pyruvate:Ferredoxin Oxidoreductase That Reversibly Couples Pyruvate Oxidation to NADPH Production in Helicobacter pylori and Campylobacter jejuni

“…Both C. jejuni and H. pylori utilize organic acids and amino acids as primary carbon and energy sources and rely on gluconeogenic pathways to provide the necessary intermediates for biosynthesis of cell wall material, vitamins, and nucleic acids (1,11,17,19,27,39). Other general features of these pathogens include (i) a strictly respiratory form of metabolism; (ii) reliance on molecular hydrogen as a key energy source (14,17,18,20,28); (iii) a preference for NADPH, rather than NADH, as the primary electron donor; and (iv) pyruvate oxidation via a reversible pyruvate:ferredoxin oxidoreductase (PFOR) (6,20). While the PFOR of H. pylori is composed of four subunits encoded by the porGDAB operon (20,23,37), the PFOR of C. jejuni is the product of a single gene, similar to the PFORs of anaerobic bacteria and amitochondriate protozoan parasites (21).…”

Calculation of Wiener Indices of Thiazolides: The Potent Inhibitors of Hepatitis B Virus and Hepatitis C Virus Replication

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