Auranofin exerts broad-spectrum bactericidal activities by targeting thiol-redox homeostasis

Abstract

Infections caused by antibiotic-resistant bacteria are a rising public health threat and make the identification of new antibiotics a priority. From a cell-based screen for bactericidal compounds against Mycobacterium tuberculosis under nutrient-deprivation conditions we identified auranofin, an orally bioavailable FDA-approved antirheumatic drug, as having potent bactericidal activities against both replicating and nonreplicating M. tuberculosis. We also found that auranofin is active against other Gram-positive bacteria, including Bacillus subtilis and Enterococcus faecalis, and drug-sensitive and drug-resistant strains of Enterococcus faecium and Staphylococcus aureus. Our biochemical studies showed that auranofin inhibits the bacterial thioredoxin reductase, a protein essential in many Gram-positive bacteria for maintaining the thiol-redox balance and protecting against reactive oxidative species. Auranofin decreases the reducing capacity of target bacteria, thereby sensitizing them to oxidative stress. Finally, auranofin was efficacious in a murine model of methicillin-resistant S. aureus infection. These results suggest that the thioredoxin-mediated redox cascade of Gram-positive pathogens is a valid target for the development of antibacterial drugs, and that the existing clinical agent auranofin may be repurposed to aid in the treatment of several important antibiotic-resistant pathogens.

Keywords: Gram-positive; MRSA; auranofin; tuberculosis.

Fig. 1.

Auranofin displays potent activity against replicating and nonreplicating M. tuberculosis. (A) Structure of auranofin. (B) Auranofin activity against replicating and nonreplicating M. tuberculosis H37Ra constitutively expressing bacterial luciferase. Nonreplicating bacteria were starved in PBS for 24 h before treatment. Replicating cultures were grown in 7H9 medium. Cultures were treated for 24 h with auranofin and then assayed for luminescence. (C) Auranofin (AF) shows potent bactericidal activity against nonreplicating M. tuberculosis H37Ra relative to INH and RMP. Starvation-induced nonreplicating M. tuberculosis was treated with auranofin or the front-line anti-TB drugs INH and RMP for 5 d, followed by enumeration of viable colonies. (D) Combination treatment of M. tuberculosis H37Ra in growth assays in 7H9 medium with auranofin in combination with either INH or RMP. One compound was diluted along the ordinate of a 96-well plate and the other along the abscissa, resulting in a checkerboard of the two compounds.

Fig. 2.

Auranofin inhibits bacterial TrxR. (A) Purified recombinant M. tuberculosis TrxB2 was preincubated with NADPH and DTNB with or without auranofin for 15 min. Reactions were initiated by the addition of TrxC. Shown is a dose–response plot for the initial rate of TrxB2 activity in the presence or absence of auranofin. (Inset) Representative progress plot of TrxB2 activity in the presence or absence of auranofin used for generating the dose–response plot. The colors of the lines on the progress plot correspond to the same color points on the dose–response plot. The purple line on the progress plot corresponds to the untreated control, which is not graphed on the dose-response plot. Auranofin concentrations tested were 800, 400, 200, 100, 50, and 25 nM. (B) Dose–response plots for S. aureus TrxB activity with and without a 15-min preincubation with auranofin with corresponding progress plots showing that auranofin inhibits TrxB activity. Assays were carried out as with TrxB2. Dose–response plots represent at least three determinations ± SE.

Fig. 3.

Auranofin depletes intracellular thiols and sensitizes S. aureus to oxidizing agents and oxidative stress. (A) S. aureus cultures treated with indicated concentrations of auranofin for 15 min show a decrease in free thiol concentration relative to untreated control. (B) M. tuberculosis mc²6230 cultures treated with auranofin in minimal medium for 3 h at the indicated concentrations also show a similar dose-dependent depletion of thiols. (C) Combination treatment of S. aureus with auranofin and diamide has synergistic antimicrobial activities. S. aureus cultures were treated for 3 h with the indicated concentrations of diamide and 700 nM auranofin, alone or in combination. (D) Combined treatment of S. aureus with auranofin and paraquat affords synergistic antimicrobial activity. S. aureus cultures were treated for 3 h with the indicated concentrations of paraquat and 700 nM auranofin, alone or in combination. (E) Loss of GSH synthesis sensitizes E. coli to auranofin. WT or E. coli lacking GSH (gshA) or TrxR (trxB) were treated with the indicated concentrations of auranofin for 6 h, and growth was assayed by absorbance.

Fig. 4.

Auranofin shows efficacy in a murine peritonitis MRSA infection model. Survival of mice with an acute i.p. infection induced with an initial inoculum of 2 × 10⁹ CFU/mouse MRSA Sanger 252 (n = 8 per group). Mice were treated with one dose at 1 h post infection, followed by once-daily treatment with i.p. auranofin. P < 0.01, Mantel–Cox test.