A reprofiled drug, auranofin, is effective against metronidazole-resistant Giardia lamblia

Abstract

Giardiasis is one of the most common causes of diarrheal disease worldwide. Treatment is primarily with 5-nitro antimicrobials, particularly metronidazole. Resistance to metronidazole has been described, and treatment failures can occur in up to 20% of cases, making development of alternative antigiardials an important goal. To this end, we have screened a chemical library of 746 approved human drugs and 164 additional bioactive compounds for activity against Giardia lamblia. We identified 56 compounds that caused significant inhibition of G. lamblia growth and attachment. Of these, 15 were previously reported to have antigiardial activity, 20 were bioactive but not approved for human use, and 21 were drugs approved for human use for other indications. One notable compound of the last group was the antirheumatic drug auranofin. Further testing revealed that auranofin was active in the low (4 to 6)-micromolar range against a range of divergent G. lamblia isolates representing both human-pathogenic assemblages A and B. Most importantly, auranofin was active against multiple metronidazole-resistant strains. Mechanistically, auranofin blocked the activity of giardial thioredoxin oxidoreductase, a critical enzyme involved in maintaining normal protein function and combating oxidative damage, suggesting that this inhibition contributes to the antigiardial activity. Furthermore, auranofin was efficacious in vivo, as it eradicated infection with different G. lamblia isolates in different rodent models. These results indicate that the approved human drug auranofin could be developed as a novel agent in the armamentarium of antigiardial drugs, particularly against metronidazole-resistant strains.

Fig 1

Retesting of selected antigiardial compounds from a chemical library. The indicated compounds were tested quantitatively for activity (pEC₅₀) against G. lamblia WB in a 48-h growth assay using ATP measurements as a readout. Data are shown as mean + SD (n = 3). The dashed horizontal line represents the highest tested drug concentration.

Fig 2

Activity of auranofin against divergent metronidazole-sensitive and -resistant G. lamblia isolates. Metronidazole (MZ) and auranofin (AF) were tested for activity (pEC₅₀) against the depicted G. lamblia lines. WB-M1 and WB-M2 are metronidazole-resistant derivatives of the parental WB line, 713-M3 is a metronidazole-resistant derivative of the parental 713 line, 2ID10 and 106-C17 are metronidazole-resistant and C17-resistant (C17 is a 5-nitroimidazole related to metronidazole) lines, respectively, of the parental 106 isolate, and 1279-MR is a metronidazole-resistant derivative of the parental 1279 isolate. WB, 713, and 106 belong to assemblage A, while 1279 belongs to assemblage B. (A and B) Representative examples of the concentration-response curves for the effects of AF (A) and MZ (B) on Giardia survival and growth, relative to maximal growth (as determined by total ATP levels) in solvent-treated control cultures. (C and D) Summary of data from several independent experiments for AF (C) and MZ (D). All data are shown as mean ± standard error of the mean (SEM) for panels A and B (one representative experiment) and mean + SD (n = 3 experiments) for panels C and D, *, P < 0.05 versus the respective parental isolates.

Fig 3

Inhibition of G. lamblia TrxR by auranofin. (A) In vitro activity of purified, recombinant TrxR from G. lamblia was determined with the substrate DTNB in the presence of increasing concentrations of auranofin (AF) or DPI. Data are expressed relative to the control activity without drugs and are shown as mean ± SD (n = 3). (B) The antigiardial activities of DPI, AF, and metronidazole (MZ) were determined by 48-h growth assay and are shown as pEC₅₀ (mean + SD). (C) The effects of the indicated drugs on the morphology of G. lamblia WB were determined with the aid of a spinning-disk confocal microscope. MZ-treated cells exhibited marked slowing of flagellar beating, since flagella could be easily captured by the camera (black arrowhead), whereas the flagella in untreated cells could barely be detected under these conditions due to their rapid movement. By comparison, AF and DPI treatment caused only modest flagellar slowing but instead led to marked cellular blebbing (white arrowheads).

Fig 4

In vivo efficacy of auranofin in rodent models of giardiasis. Suckling mice (5 to 7 days old), adult mice, or adult gerbils were infected with the indicated G. lamblia isolates. After 2 days (mice) or 7 days (gerbils), animals were given one daily dose of auranofin (AF) for 5 days or were given solvent alone (2.5 to 25% ethanol in PBS) as a control. Afterwards, live trophozoites in the small intestine were enumerated. Data are mean + SD for 3 to 6 mice or 4 gerbils per group; P < 0.05 by rank sum test versus PBS-treated controls. The dashed horizontal line represents the detection limit of the assay.