Auranofin

The strategy for pharmacological inhibition of redox regulation systems is as follows (Fig. S1): Piperlongumine (Sigma‐Aldrich, St. Louis, MO) is a plant alkaloid 17 and selectively kills cancer cells over normal cells 12, 18. One of its molecular targets is GSTπ (encoded by the GSTP1 gene) 12, one of the five up‐regulated isoforms in CRC CTOSs (Fig. 1). Auranofin (Sigma‐Aldrich) is a clinically approved drug for rheumatoid arthritis and considered to inhibit TXNRD 9, 11, 19. CB83 (ChemBridge, San Diego, CA) is a newly identified glucose‐6‐phosphate dehydrogenase (G6PD) inhibitor and inhibits PPP activity (G6PD is a critical enzyme for PPP) 10, 20.

The ‘Pathogen Box’ compounds were kindly provided by MMV in 96-well plates containing 10 mM stock solutions dissolved in dimethyl sulfoxide (DMSO). Additional amounts of several compounds were purchased from Sigma Aldrich and dissolved in DMSO (stock concentration given in brackets), including azoxystrobin (31697-100MG; 50 mM), trifloxystrobin (46447-100MG; 50 mM), auranofin (A6733-10MG; 50 mM), buparvaquone (SML1662-25MG; 3 mM), and atovaquone (A7986-10MG; 10 mM). 3-MB-PP1 was purchased from Cayman Chemical (17860; 10 mM). Additional MMV688853 (BKI-1517; 10 mM) was a kind gift from Wes Van Voorhis (University of Washington). Additional MMV024397 was also provided by MMV. The DMSO concentration introduced when using these compounds in assays was < 0.2% (v/v), except MMV688853 when used at the higher concentrations (up to 50 μM) in the Plasmodium assays (up to 0.5% (v/v) DMSO).

Mtb was grown to mid-log phase and diluted to an OD of 0.03 in 7H9 medium. Bacteria were then exposed to 1.5-fold serial dilution of antimicrobial compounds. Optical density was recorded after 14 days and normalized to the corresponding strains without drug treatment. Minimum inhibitory concentration is defined as the lowest concentration of a drug at which bacterial growth was inhibited at least 90%, as compared to the control containing no antimicrobial compounds. Ampicillin, auranofin, D-cycloserine, ebselen, ethambutol, faropenem, hydroxyurea, isoniazid, kanamycin, levofloxacin, meropenem, mitomycin C, moxifloxacin, piperacillin, rifampicin, streptomycin and vancomycin were purchased from Sigma Aldrich, St. Louis, MO. Moenomycin was from Santa Cruz Biotechnology. Bedaquilline was received as a gift from C. Barry.

Auranofin, gambogic acid (GA), disulfiram, menadione, oltipraz, parthenolide, plumbagin from Plumbago indica, PZQ, thonzonium bromide, sanguinarine chloride hydrate, dimethyl sulphoxide (DMSO), percoll and fetal bovine serum (FBS) were from Sigma-Aldrich. The Ro 15–5458 compound was a kind gift from Dr H. Stohler (Hoffman-La Roche, Basel, Switzerland) and oxamniquine was provided by Pfizer, London. Drugs were dissolved in DMSO to obtain stock solutions at 10 mM and were then diluted into culture medium. CellTiter-Glo (CTG) reagent, used in the schistosomula viability luminescence-based assay, and CellTox green dye, used in the schistosomula staining, were from Promega. BioWhittaker Dulbecco-Modified Eagle’s Medium (DMEM) lacking phenol red and containing 4500 mg/l glucose, 1 mM Hepes pH 6.98–7.30, 2 mM L-glutamine, 1x antibiotic-antimycotic reagent (Life Technologies) and 10% heat inactivated FBS, was used as tissue culture medium for schistosomula. Adult worms were cultured in BioWhittaker DMEM containing 4500 mg/l glucose, 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, 0.5 μg/ml amphothericin B and 10% heat inactivated FBS.

Rat cortical neurons were incubated in equilibrated neurobasal media containing (10 or 40 nM) single-chain βlac-TT or variants for 30 min at 37°C. Neurons were washed with Hanks’ balanced salt solution lacking Ca²⁺ and Mg²⁺ (HBSS^−/−) (Life Technologies) and cooled to room temperature (RT) for 10 min followed by loading of media with 2 μM CCF2-AM (Life Technologies) and 1 mM probenecid (Life Technologies), an anion transport inhibitor, in HBSS^−/− for 30 min. Neurons were washed and processed for immunofluorescence. The inhibitor-treated translocation assay was performed as described above, with the following preincubation modifications. Bafilomycin A1 (400 nM), a vesicular ATPase inhibitor (Sigma-Aldrich), was preincubated with neurons in neurobasal media for 30 min at 37°C before aspiration and application of 40 nM single-chain βlac-TT in neurobasal media containing inhibitor at the indicated concentration. Then, auranofin (500 nM), a thioredoxin reductase inhibitor (Sigma), was preincubated with neurons for 30 min at 37°C before its removal and addition of 40 nM single-chain βlac-TT in neurobasal media containing inhibitor at the indicated concentration. After being maintained 30 min with βlac-TT at 37°C, cells were washed and loaded with CCF2 as described above followed by fixation.

Auranofin and tunicamycin were purchased from Sigma-Aldrich (St Louis, MO, United States). TUDCA was purchased from TCI America (Portland, OR, United States). Irradiation was performed at room temperature using a ¹³⁷Cs gamma-ray source, Gammacell 3000, manufactured by Nordion (Ottawa, ON, Canada).