Ligand supplementation restores the cancer therapy efficacy of the antirheumatic drug auranofin from serum inactivation

Abstract

Auranofin, an FDA-approved antirheumatic gold drug, has gained ongoing interest in clinical studies for treating advanced or recurrent tumors. However, gold ion's dynamic thiol exchange nature strongly attenuates its bioactivity due to the fast formation of covalent albumin-gold adducts. Here we report that newly-added thiols can modulate the dynamic albumin-gold binding and recover the therapeutic efficacy. Initially, we find that auranofin supplemented with its own thiol ligand, TGTA (1-thio-β-D-glucose tetraacetate), significantly restores the anticancer activities in cells and patient-derived xenograft models. Then, screening a collection of ligand fragments followed by machine learning evaluation unveils diverse synergizing thiols, including pantethine, that effectuate auranofin at a low dosage for rheumatoid arthritis. Interestingly, the thiol exchange inside cells accounts for a cuproptosis-like phenotype that auranofin induces. Together, we believe the ligand-enabled dynamic modulation strategy is of value to researchers and clinicians contemplating metallodrugs and ligand-like molecules in cancer therapy.

Fig. 1. Thiol exchange in auranofin-related clinical trials.

a a timeline summary of auranofin-repurposing clinical trials on cancer therapy in recent years. b drugs used in clinical trial NCT02770378 for glioblastoma. c the chemical structures of the indicated drugs. d cellular gold uptake affected by the co-treatment with thiol-containing drugs in U87 cells. 3 μM of auranofin was used in all groups with or without DSF (570 μM) or Cap (156 μM) for 10 min at 37 °C. Data are shown as mean ± s. d. of three independent experiments. e mass spectrometry identified the formation of a DEDT-gold-phosphine molecule after an in vitro incubation of auranofin (2 μM), captopril (4 μM), and disulfiram (2 μM) for 30 min at 37 °C in methanol. The spectrum was extracted from 463.0000 to 467.0000 m/z [M + H]⁺ values with the predicted m/z labeled in red. The result is representative of three independent experiments. Source data are provided as a Source Data file.

Fig. 2. TGTA restores the anticancer activity of albumin-bound auranofin.

a a scheme for harnessing the dynamic thiol exchange to restore anticancer efficacy from albumin-bound gold. TGTA, thioglucose tetraacetate. Created in BioRender. Xiong, X. (2025) https://BioRender.com/zpgjm50. b HPLC analysis at 214 nm on the thiol exchange reaction in vitro. 1 mM of auranofin was added to 1.0 mL of 4 mM solution of BSA and then reacted at 37 °C for 60 min in a shaker. 5 mM of TGTA (5 equivalents) was used. The peak of auranofin was marked by a dotted red line. c cellular gold uptake of HCT116 cells under indicated conditions of FBS for 1 h. 3 μM of auranofin was used in all groups with or without TGTA (50 μM). The values were normalized by the protein amount extracted. Data are shown as mean ± s. d. of three independent experiments. Significance was calculated by an unpaired, two-tailed t-test. Significance was defined as p < 0.05. d dose-dependent cytotoxicity boosting of auranofin by glucose homologs after 24-hour treatments. GPA, Glucose pentaacetate. e, measuring cellular thioredoxin reductase (TrxR) activity using HCT116 upon indicated treatments. HCT116 cells in RPMI + 30% FBS were incubated with 0.5 μM auranofin and/or 50 μM TGTA for 1 h before cell lysis for TrxR activity detection. For d, e, mean ± s. d., 3 biological replicates. f mouse models to evaluate the in vivo tumor suppression of auranofin. Drugs were injected intraperitoneally 6 times per week, and the initial administration day was marked by black arrows. An HCT116 xenograft and two patient-derived xenografts (non-small cell lung carcinoma and triple-negative breast cancer) were conducted. HCT116 tumors: Vehicle, n = 7 mice; TGTA, n = 6 mice; AF, n = 6 mice; AF + TGTA, n = 9 mice. NSCLC PDX tumors: Vehicle, n = 7 mice; TGTA, n = 6 mice; AF, n = 6 mice; AF + TGTA, n = 7 mice. TNBC PDX tumors: Vehicle, n = 9 mice; TGTA, n = 9 mice; AF, n = 8 mice; AF + TGTA, n = 8 mice. Data were shown as mean ± s. e. m. Source data are provided as a Source Data file.

Fig. 3. Ligand screening in synergy with auranofin.

a PC9 cell viability in response to co-treatments using 5 μM auranofin with 50 μM ligands from a chemical library containing 252 small molecules. Ligands with values under 0.3 (dotted line in pink) were picked for the next experiments. b a counter-screening on the self-toxicity of the picked ligands. Ligands with less than 10% toxicity were picked as the effective synergistic ones. c percentages of sulfur- (S) or phospho- (P) containing molecules in the original and effective ligands were compared. d a workflow evaluating the synergy-associated chemical features using random forest classification based on 354 descriptors calculated by MOE software. The top 10 descriptors ranked by importance were listed with their MeanDecreaseGini values. e plotting the thiol ligands by their values of two descriptors, FASA_P and ASA_H. Effective, ineffective, and untested molecules were colored as indicated. Names of some ligands were shown. Source data are provided as a Source Data file.

Fig. 4. Synergistic effect of pantethine on auranofin.

a a small-scale cell line profiling using indicated agents. The color bar represents the mean value of relative survival (24 h) in three independent experiments. 5 μM auranofin was used. Except for pantethine (200 μM), other thiol ligands were all used at 400 μM. All the cells were cultured in HPLM + 30% FBS. b the dose-dependent synergy of pantethine with auranofin. The viability after a 24-hour treatment was measured. Data are shown as mean ± s. d. of three independent experiments. Significance was calculated by unpaired, two-tailed t-test. c cellular gold uptake of HCT116 cells under 3 μM auranofin with or without 200 μM pantethine for 1 h. The values were normalized by the amount of protein extracted. Data are shown as mean ± s. d. of three independent experiments. Significance was calculated by unpaired, two-tailed t-test. Significance was defined as p < 0.05. d the comparison of the synergistic activities with auranofin endowed by different pantothenate derivatives using HCT116 cells in a 24-hour treatment. Except for pantethine (200 μM), its derivatives were all used at 400 μM. Data are shown as mean ± s. d. of 3 biologically independent experiments. Significance was calculated by unpaired, two-tailed t-test. e mouse models using oral gavage on an HCT116 xenograft. Drugs were administered on the indicated days. f an A549 xenograft model to compare the tumor suppression of auranofin-pantethine combo and cisplatin monotherapy. Drug administration was conducted two times per week with indicated concentrations. For e, f, data were shown as mean ± s. e. m.. HCT116 tumors: Vehicle, n = 8 mice; pantethine, n = 7 mice; AF, n = 6 mice; AF + pantethine, n = 8 mice. A549 tumors: Vehicle, n = 6 mice; cisplatin, n = 6 mice; AF + pantethine, n = 4 mice. Source data are provided as a Source Data file.

Fig. 5. Auranofin induces cuproptosis-like cell death by thiol exchange.

a mass spectrometry identification of the products after thiol exchange during the 0.5-hour incubation of auranofin (100 μM) and lipoic acid (1 mM) in the cell lysate (500 million cells to 400 μL lysates in ddH₂O). Created in BioRender. Xiong, X. (2025) https://BioRender.com/i289oxu. b analysis of the cuproptosis markers (LIAS and FDX1) in HCT116 cells after a 24-hour auranofin treatment in the presence of different concentrations of FBS. The samples derive from the same experiment but different gels for anti-LIAS and anti-FDX1 antibodies were processed in parallel. c qRT-PCR analysis of the mRNA levels of indicated genes after a 12-hour treatment with 4 μM auranofin. Data are shown as mean ± s. d. of four independent experiments. Significance was calculated by unpaired, two-tailed t-test. Significance was defined as p < 0.05. d DLAT aggregation measurement by reduced and non-reduced blots under indicated doses of auranofin in 24-hour treatments. A red arrow marked the band of aggregated DLAT proteins. e measuring the lipoylation of DLAT protein under indicated doses of auranofin in 24-hour treatments. The DLAT protein in the treated cells was enriched by its cognate antibody before the detection of lipoylated DLAT by the anti-lipoylation antibody. For c–e, treatments were performed in RPMI + 10% FBS. f the molecular markers of cuproptosis under auranofin (5 μM) mono or combined treatments (TGTA, 20 μM; pantethine, 100 μM) in RPMI + 30% FBS. The samples derive from the same experiment, but different gels for anti-LIAS and anti-FDX1 antibodies were processed in parallel. For b, d–f, each blot is a representative of three biologically independent experiments. g cell viability assay comparing the boost effect of 2 μM elesclomol (ES) towards copper chloride (2 μM) and auranofin (2 μM) on HCT116 after a 24-hour incubation. Data are shown as mean ± s. d. of six independent experiments. Significance was calculated by unpaired, two-tailed t-test. h Effects of tetrathiomolybdate (50 μM) on cytotoxicity of copper chloride and auranofin treatments on HCT116 after a 24-hour incubation. The fold change of IC₅₀ to that of the control group was shown. For g, h, treatments were performed in RPMI + 10% FBS, and data were presented as mean ± s. d. from 3 biologically independent replicates. Significance was calculated by an unpaired, two-tailed t-test. Significance was defined as p < 0.05. Source data are provided as a Source Data file.