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Ferroptotic cancer therapy has been extensively investigated since the genesis of the ferroptosis concept. However, the therapeutic efficacy of ferroptosis induction in heterogeneous and plastic melanoma has been compromised, because the melanocytic and transitory cell subpopulation is resistant to iron-dependent oxidative stress. Here, we report a phenotype-altering liposomal nanomedicine to enable the ferroptosis-resistant subtypes of melanoma cells vulnerable to lipid peroxidation via senescence induction. The strategy involves the ratiometric coencapsulation of a cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitor (palbociclib) and a ferroptosis inducer (auranofin) within cRGD peptide-modified targeted liposomes. The two drugs showed a synergistic anticancer effect in the model B16F10 melanoma cells, as evidenced by the combination index analysis (<1). The liposomes could efficiently deliver both drugs into B16F10 cells in a targeted manner. Afterward, the liposomes potently induced the intracellular redox imbalance and lipid peroxidation. Palbociclib significantly provoked cell cycle arrest at the G 0 /G 1 phase, which sensitized auranofin-caused ferroptosis through senescence induction. Meanwhile, palbociclib depleted intracellular glutathione (GSH) and reduced nicotinamide adenine dinucleotide phosphate (NADPH), further boosting ferroptosis. The proof-of-concept was also demonstrated in the B16F10 tumor-bearing mice model. The current work offers a promising ferroptosis-targeting strategy for effectively treating heterogeneous melanoma by manipulating the cellular plasticity.

Auranofin, a gold(I)-based complex, is under clinical trials for application as an anticancer agent for the treatment of nonsmall-cell lung cancer and ovarian cancer. In the past years, different derivatives have been developed, modifying gold linear ligands in the search for new gold complexes endowed with a better pharmacological profile. Recently, a panel of four gold(I) complexes, inspired by the clinically established compound auranofin, was reported by our research group. As described, all compounds possess an [Au{P(OMe) 3 }] + cationic moiety, in which the triethylphosphine of the parent compound auranofin was replaced with an oxygen-rich trimethylphosphite ligand. The gold(I) linear coordination geometry was complemented by Cl - , Br - , I - , and the auranofin-like thioglucose tetraacetate ligand. As previously reported, despite their close similarity to auranofin, the panel compounds exhibited some peculiar and distinctive features, such as lower log P values which can induce relevant differences in the overall pharmacokinetic profiles. To get better insight into the P-Au strength and stability, an extensive study was carried out for relevant biological models, including three different vasopressin peptide analogues and cysteine, using 31 P NMR and LC-ESI-MS. A DFT computational study was also carried out for a better understanding of the theoretical fundamentals of the disclosed differences with regard to triethylphosphine parent compounds.

The search for highly selective sensitizers with a novel mechanism for tumor targeting therapy is of considerable interest. In this work, we have developed a series of new biotin-targeted Au(I) complexes. Through systematic biological evaluation and comparison, biotinylated Au(I) complex 3a containing a triphenylphosphine ligand was screened, as it realized both prominent efficient inhibition and selective cytotoxicity to cancer cells, and the effect was better than that of popularly used auranofin. Meanwhile, complex 3a , as a potent radiosensitizer, enhances anticancer effects in vitro and in vivo and has sensitization selectivity. From the action mechanism study, we provide evidence that complex 3a could intervene in redox homeostasis through targeted binding and strong suppression of thioredoxin reductase (TrxR) and induce the ferroptosis death process, enabling it to sensitize tumor cells to radiotherapy. Thus, complex 3a has enormous potential as an efficient and specific radiosensitizing agent in cancer therapy.

Primary amebic meningoencephalitis, caused by brain infection with a free-living ameba, Naegleria fowleri , leads to extensive inflammation of the brain and death within 3-7 days after symptoms begin. Treatment of primary amebic meningoencephalitis relies on amphotericin B in combination with other drugs, but use of amphotericin B is associated with severe adverse effects. Despite a fatality rate of over 97%, economic incentive to invest in development of antiamebic drugs by the pharmaceutical industry is lacking. Development of safe and rapidly acting drugs remains a critical unmet need to avert future deaths. Since FDA-approved anti-inflammatory and antiarthritic drug auranofin is a known inhibitor of selenoprotein synthesis and thioredoxin reductase and the genome of N. fowleri encodes genes for both selenocysteine biosynthesis and thioredoxin reductases, we tested the effect of auranofin against N. fowleri strains of different genotypes from the USA, Europe, and Australia. Auranofin was equipotent against all tested strains with an EC 50 of 1-2 μM. Our growth inhibition study at different time points demonstrated that auranofin is fast-acting, and ∼90% growth inhibition was achieved within 16 h of drug exposure. A short exposure of N. fowleri to auranofin led to the accumulation of intracellular reactive oxygen species. This is consistent with auranofin's role in inhibiting antioxidant pathways. Further, combination of auranofin and amphotericin B led to 95% of growth inhibition with 2-9-fold dose reduction for amphotericin B and 3-20-fold dose reduction for auranofin. Auranofin has the potential to be repurposed for the treatment of primary amebic meningoencephalitis.

Selenoenzymes, containing a selenocysteine (Sec) residue, fulfill important roles in biology. The mammalian thioredoxin reductase selenoenzymes are key regulators of antioxidant defense and redox signaling and are inhibited by methylmercury species and by the gold-containing drug auranofin. It has been proposed that such inhibition is mediated by metal binding to Sec in the enzyme. However, direct structural observations of these classes of inhibitors binding to selenoenzymes have been few to date. Here we therefore have used extended X-ray absorption fine structure as a direct structural probe to investigate binding to the selenium site in recombinant rat thioredoxin reductase 1 (TrxR1). The results demonstrate for the first time the direct and complete binding of the metal atom of the inhibitors to the selenium atom in TrxR1 for both methylmercury and auranofin, indicating that TrxR1 inhibition indeed can be attributed to such direct metal-selenium binding.

The quantification of cellular deoxyribonucleoside triphosphate (dNTP) levels is important for studying pathologies, genome integrity, DNA repair, and the efficacy of pharmacological drug treatments. Current standard methods, such as enzymatic assays or high-performance liquid chromatography, are complicated, costly, and labor-intensive, and alternative techniques that simplify dNTP quantification would present very useful complementary approaches. Here, we present a dNTP assay based on isothermal rolling circle amplification (RCA) and rapid time-gated Förster resonance energy transfer (TG-FRET), which used a commercial clinical plate reader system. Despite the relatively simple assay format, limits of detection down to a few picomoles of and excellent specificity for each dNTP against the other dNTPs, rNTPs, and dUTP evidenced the strong performance of the assay. Direct applicability of RCA-FRET to applied nucleic acid research was demonstrated by quantifying all dNTPs in CEM-SS leukemia cells with and without hydroxyurea or auranofin treatment. Both pharmacological agents could reduce the dNTP production in a time- and dose-dependent manner. RCA-FRET provides simple, rapid, sensitive, and specific quantification of intracellular dNTPs and has the potential to become an advanced tool for both fundamental and applied dNTP research.

Cell-targeting peptides (CTPs) are increasingly used in the field of cancer research due to their high affinity and specificity to cell or tissue targets. In the search for novel metal-based drug candidates, our research group is particularly focused on bioconjugates by utilizing peptides to increase the selectivity of cytotoxic organometallic compounds. Motivated by the relatively high cytotoxic activity of gold complexes, such as Auranofin (approved to treat rheumatoid arthritis), for the treatment of various diseases, we anticipated that gold peptide bioconjugates would present interesting candidates for novel breast cancer therapies. For this, we investigate the use of the natural compound lipoic acid (Lpa) as a bioconjugation handle to link Au complexes in the oxidation state +III to peptides using the dithiol moiety. Using this strategy, we have synthesized Au(III) complex bioconjugates linked to the linear LTVSPWY peptide and two cyclic DfKRG and KTTHWGFTLG tumor-targeting peptides. Solid-phase peptide synthesis (SPPS) was used to prepare the peptides, with lipoic acid introduced N-terminally as a conjugation handle. After peptide cleavage, the metal complex was introduced in solution by first reducing the internal disulfide bond, followed by reaction with Au(ppy)Cl 2 ( 1 , ppy: 2-phenyl-pyridine), to yield the Au(III)-Lpa-peptide bioconjugates. The new bioconjugates were successfully synthesized, purified by semi-preparative HPLC, and characterized by ESI-MS. Au(III)-peptide bioconjugates were tested as cytotoxic agents against two different human breast cancer cell lines (MCF-7 and MDA-MB-231) and normal human fibroblasts cells (GM5657T) and compared to cisplatin, the parent Au(III) dichloride complex, and metal-free peptides. These in vitro data show that the Au(III)-peptide bioconjugate 5 , possessing the cyclic integrin-targeting RGD-derived peptide sequence in the structure, exhibits improved activity compared to the parent gold(III) compound Au(ppy)Cl 2 ( 1 ) as well as to cisplatin or the metal-free peptide. Moreover, the excellent targeting properties of 5 are supported by the fact that a Au(III)-peptide conjugate with the exact same peptide sequence, but a linear rather than the cyclic form of 5 exhibits 10 times lower cytotoxic activity.

Auranofin, an FDA-approved arthritis drug, has recently been repurposed as a potential antimicrobial agent; it performed well against many Gram-positive bacteria, including multidrug resistant strains. It is, however, inactive toward Gram-negative bacteria, for which we are in dire need of new therapies. In this work, 40 auranofin analogues were synthesized by varying the structures of the thiol and phosphine ligands, and their activities were tested against ESKAPE pathogens. The study identified compounds that exhibited bacterial inhibition (MIC) and killing (MBC) activities up to 65 folds higher than that of auranofin, making them effective against Gram-negative pathogens. Both thiol and the phosphine structures influence the activities of the analogues. The trimethylphosphine and triethylphosphine ligands gave the highest activities against Gram-negative and Gram-positive bacteria, respectively. Our SAR study revealed that the thiol ligand is also very important, the structure of which can modulate the activities of the Au I complexes for both Gram-negative and Gram-positive bacteria. Moreover, these analogues had mammalian cell toxicities either similar to or lower than that of auranofin.

Dinuclear metal complexes have emerged as a promising class of anticancer compounds with the ability to cross-link biomolecular targets. Here, we describe two novel series of phosphine-linked dinuclear ruthenium(II) p-cymene and gold(I) complexes, in which the length of the connecting poly(ethylene glycol) chain has been systematically modified. The impact of the multinuclearity, lipophilicity, and linker length on the antiproliferative activity of the compounds on tumorigenic (A2780 and A2780cisR) and nontumorigenic (HEK-293) cell lines was assessed. The dinuclear ruthenium(II) complexes were considerably more cytotoxic than their mononuclear counterparts, and a correlation between the lipophilicity of the linker and the cytotoxicity was observed, whereas the cytotoxicity of the gold(I) series is independent of these factors.

New gold(I) thiolate complexes have been synthesized and characterized, and their physicochemical properties and anticancer activity have been tested. The coordination of PTA derivatives provides optimal hydrophilicity/lipophilicity properties to the complexes, which present high solution stability. Moreover, the complexes show a high anticancer activity against Caco-2 cells, comparable to that of auranofin, and a very low cytotoxic activity against enterocyte-like differentiated cells. Their activity has been shown to produce cell death by apoptosis and arrest of the cell cycle because of interaction with the reductase enzymes and consequent reactive oxygen species production. Some of these new complexes are also able to decrease the necessary dose of 5-fluorouracil, a drug used for the treatment of colon cancer, by a synergistic mechanism.

Gold phosphine complexes, such as auranofin, have been recognized for decades as antirheumatic agents. Clinical trials are now underway to validate their use in anticancer or anti-HIV treatments. However, their mechanisms of action remain unclear. A challenging question is whether the gold phosphine complex is a prodrug that is administered in an inactive precursor form or rather that the gold atom remains attached to the phosphine ligand during treatment. In this study, we present two novel gold complexes, which we compared to auranofin and to their phosphonium analogue. The chosen ligand is a phosphine-based smart probe, whose strong fluorescence depends on the presence of the gold atom. The in vitro biological action of the gold complexes and the phosphonium derivative were investigated, and a preliminary in vivo study in healthy zebrafish larvae allowed us to evaluate gold complex biodistribution and toxicity. The different analyses carried out showed that these gold complexes were stable and behaved differently from phosphonium and auranofin, both in vitro and in vivo. Two-photon microscopy experiments demonstrated that the cellular targets of these gold complexes are not the same as those of the phosphonium analogue. Moreover, despite similar IC50 values in some cancer cell lines, gold complexes displayed a low toxicity in vivo, in contrast to the phosphonium salt. They are therefore suitable for future in vivo investigations.

Cutaneous leishmaniasis remains ignored in therapeutic drug discovery programs worldwide. This is mainly because cutaneous leishmaniasis is frequently a disease of impoverished populations in countries where funds are limited for research and patient care. However, the health burden of individuals in endemic areas mandates readily available, effective, and safe treatments. Of the existing cutaneous leishmaniasis therapeutics, many are growth inhibitory to Leishmania parasites, potentially creating dormant parasite reservoirs that can be activated when host immunity is compromised, enabling the reemergence of cutaneous leishmaniasis lesions or worse spread of Leishmania parasites to other body sites. To accelerate the identification and development of novel cutaneous leishmaniasis therapeutics, we designed an integrated in vitro and in vivo screening platform that incorporated multiple Leishmania life cycles and species and probed a focused library of pharmaceutically active compounds. The objective of this phenotypic drug discovery platform was the identification and prioritization of bona fide cytotoxic chemotypes toward Leishmania parasites. We identified the Food and Drug Administration-approved drug auranofin, a known inhibitor of Leishmania promastigote growth, as a potent cytotoxic anti-leishmanial agent and inducer of apoptotic-like death in promastigotes. Significantly, the anti-leishmanial activity of auranofin transferred to cell-based amastigote assays as well as in vivo murine models. With appropriate future investigation, these data may provide the foundation for potential exploitation of gold(I)-based complexes as chemical tools or the basis of therapeutics for leishmaniasis. Thus, auranofin may represent a prototype drug that can be used to identify signaling pathways within the parasite and host cell critical for parasite growth and survival.

Gold(I) complexes with 1,3-substituted imidazole-2-ylidene and benzimidazole-2-ylidene ligands of the type NHC-Au-L (NHC = N-heterocyclic carbene L = Cl or 2-mercapto-pyrimidine) have been synthesized and structurally characterized. The compounds were evaluated for their antiproliferative properties in human ovarian cancer cells sensitive and resistant to cisplatin (A2780S/R), as well in the nontumorigenic human embryonic kidney cell line (HEK-293T), showing in some cases important cytotoxic effects. Some of the complexes were comparatively tested as thioredoxin reductase (TrxR) and glutathione reductase (GR) inhibitors, directly against the purified proteins or in cell extracts. The compounds showed potent and selective TrxR inhibition properties in particular in cancer cell lines. Remarkably, the most effective TrxR inhibitors induced extensive oxidation of thioredoxins (Trxs), which was more relevant in the cancerous cells than in HEK-293T cells. Additional biochemical assays on glutathione systems and reactive oxygen species formation evidenced important differences with respect to the classical cytotoxic Au(I)-phosphine compound auranofin.

Seleno-auranofin (SeAF), an analogue of auranofin (AF), the orally active antiarthritic gold drug in clinical use, was synthesized and has been characterized by an array of physical techniques and biological assays. The Mössbauer and extended X-ray absorption fine structure (EXAFS) parameters of the solid compound demonstrate a linear P-Au-Se coordination environment at a gold(I) center, analogous to the structure of auranofin. The (31)P, (13)C, and (1)H NMR spectra of SeAF in chloroform solution closely resemble those of auranofin. The (77)Se spectrum consists of a singlet at 481 ppm, consistent with a metal-bound selenolate ligand. The absence of (2)J(PSe) coupling in the (31)P and (77)Se spectra may arise from dynamic processes occurring in solution or because the (2)J(PSe) coupling constants are smaller than the observed bandwidths. Electrospray ionization mass spectrometry (ESI-MS) spectra of SeAF in 50:50 methanol-water exhibited strong signals for [(Et(3)P)(2)Au](+), [(Et(3)PAu)(2)-mu-Se-tagl](+), and [Au(Se-tagl)(2)](-), which arise from ligand scrambling reactions. Three assays of the anti-inflammatory activity of SeAF allowed comparison to AF. SeAF exhibited comparable activity in the topically administered murine arachadonic acid-induced and phorbol ester-induced anti-inflammatory assays but was inactive in the orally administered carrageenan-induced assay in rats. However, in vivo serum gold levels were comparable in the rat, suggesting that differences between the in vivo metabolism of the two compounds, leading to differences in transport to the inflamed site, may account for the differential activity in the carrageenan-induced assay. Reactions of serum albumin, the principal transport protein of gold in the serum, demonstrated formation of AlbSAuPEt(3) at cysteine 34 and provided evidence for facile reduction of disulfide bonds at cysteine 34 and very rapid formation of Et(3)P=O, a known metabolite of auranofin.

The mechanism of action of auranofin, an antiarthritic gold(I) drug, is unknown, but several studies suggest that oxidation may be important for its biochemical effect. Bulk electrolysis studies on auranofin [(Et(3)P)Au(TATG); TATG = 2,3,4,6-tetraacetyl-1-thio-d-glucopyranosato] at +1.2 and +1.6 V versus Ag/AgCl in 0.1 M Bu(4)NBF(4)/CH(2)Cl(2) results in n values of 0.5 and >2 electrons, respectively. Oxidation of auranofin with the mild oxidant, Cp(2)Fe(+), results in formation of disulfide and a digold(I) cation with a bridging thiolate ligand, [(Et(3)PAu)(2)(mu-TATG)](+) (1). The X-ray structure of the PMe(3) analogue, [(Me(3)PAu)(2)(mu-TATG)](NO(3)) (2), is reported. Compound 2 forms a tetranuclear cluster containing an almost perfect square of four gold atoms with Au.Au distances averaging 3.14 A. The complex crystallizes in the tetragonal space group P4(2)2(1)2 with cell constants a = 26.1758(6) A, b = 26.1758(6) A, c = 9.7781(3) A, alpha = beta = gamma = 90 degrees, V = 6699.7(3) A(3), Z = 4, R1 = 0.0644, and wR2 = 0.1152. A mechanism for oxidation of auranofin and possible biological implications are discussed.