TrxR Inhibition Research Articles

Discovery of novel organoarsenicals as robust thioredoxin reductase inhibitors for oxidative stress mediated cancer therapy

Targeting overexpressed thioredoxin reductase (TrxR) in cancer cells to induce oxidative stress has been proved to be an effective strategy for cancer therapy. However, the treatment was hindered by the low efficiency and frequent administration of TrxR inhibitors, and hence more potent TrxR inhibitors were urgently needed. Herein, we designed and synthesized a series of TrxR inhibitors based on arsenicals. Among these, compound 1d inhibited the proliferation of a variety of cancer cells at low micromolar concentrations and exhibited low toxicity to normal cells. Importantly, compound 1d induced the accumulation of reactive oxygen species (ROS) by inhibiting the TrxR activity, further causing the collapse of the redox system, mitochondrial dysfunction, endoplasmic reticulum (ER) stress, and DNA damage, followed by oxidative stress-induced cell apoptosis. In vivo data showed that, compared with the clinical TrxR inhibitor auranofin (AUR), compound 1d could more effectively eliminate tumors by 90 % at a dose of 1.5 mg/kg without any obvious side effects. These results indicated that compound 1d was a potent TrxR inhibitor against cancer.

Activation and Denitrosylation of Procaspase-3 in KA-induced Excitotoxicity.

It has been reported that activation of glutamate kainate receptor subunit 2 (GluK2) subunit-containing glutamate receptors and the following Fas ligand(FasL) up-regulation, caspase-3 activation, subsequently results in delayed apoptosis-like neuronal death in hippocampus CA1 subfield after cerebral ischemia. Nitric oxide-mediated S-nitrosylation might inhibit the procaspase activation, whereas denitrosylation might contribute to cleavage and activation of procaspases. The study aimed to elucidate the molecular mechanisms underlying procaspase-3 denitrosylation and activation following kainic acid (KA)-induced excitotoxicity in rat hippocampus. S-nitrosylation of procaspase-3 was detected by biotin-switch method. Activation of procaspase-3 was shown as cleavage of procaspase-3 detected by immunoblotting. FasL expression was detected by immunoblotting. Cresyl violets and TdT-mediated dUTP Nick-End Labeling (TUNEL) staining were used to detect apoptosis-like neuronal death in rat hippocampal CA1 and CA3 subfields. KA led to the activation of procaspase-3 in a dose- and time-dependent manner, and the activation was inhibited by KA receptor antagonist NS102. Procaspase-3 was denitrosylated at 3 h after kainic acid administration, and the denitrosylation was reversed by SNP and GSNO. FasL ASODNs inhibited the procaspase-3 denitrosylation and activation. Moreover, thioredoxin reductase (TrxR) inhibitor auranofin prevented the denitrosylation and activation of procaspase-3 in rat hippocampal CA1 and CA3 subfields. NS102, FasL AS-ODNs, and auranofin reversed the KAinduced apoptosis and cell death in hippocampal CA1 and CA3 subfields. KA led to denitrosylation and activation of procaspase-3 via FasL and TrxR. Inhibition of procaspase-3 denitrosylation by auranofin, SNP, and GSNO played protective effects against KA-induced apoptosis-like neuronal death in rat hippocampal CA1 and CA3 subfields. These investigations revealed that the procaspase-3 undergoes an initial denitrosylation process before becoming activated, providing valuable insights into the underlying mechanisms and possible treatment of excitotoxicity.

May 1,2-Dithiolane-4-carboxylic Acid and Its Derivatives Serve as a Specific Thioredoxin Reductase 1 Inhibitor?

Thioredoxin reductase is an essential enzyme that plays a crucial role in maintaining cellular redox homeostasis by catalyzing the reduction of thioredoxin, which is involved in several vital cellular processes. The overexpression of TrxR is often associated with cancer development. A series of 1,2-dithiolane-4-carboxylic acid analogs were obtained to verify the selectivity of 1,2-dithiolane moiety toward TrxR. Asparagusic acid analogs and their bioisoters remain inactive toward TrxR, which proves the inability of the 1,2-dithiolane moiety to serve as a pharmacophore during the interaction with TrxR. It was found that the Michael acceptor functionality-containing analogs exhibit higher inhibitory effects against TrxR compared to other compounds of the series. The most potent representatives exhibited micromolar TrxR1 inhibition activity (IC50 varied from 5.3 to 186.0 μM) and were further examined with in vitro cell-based assays to assess the cytotoxic effects on various cancer cell lines and cell death mechanisms.

Auranofin Modulates Thioredoxin Reductase/Nrf2 Signaling in Peripheral Immune Cells and the CNS in a Mouse Model of Relapsing-Remitting EAE.

Multiple sclerosis (MS) is one of the most prevalent chronic inflammatory autoimmune diseases. It causes the demyelination of neurons and the subsequent degeneration of the central nervous system (CNS). The infiltration of leukocytes of both myeloid and lymphoid origins from the systemic circulation into the CNS triggers autoimmune reactions through the release of multiple mediators. These mediators include oxidants, pro-inflammatory cytokines, and chemokines which ultimately cause the characteristic plaques observed in MS. Thioredoxin reductase (TrxR) and nuclear factor erythroid 2-related factor 2 (Nrf2) signaling plays a crucial role in the regulation of inflammation by modulating the transcription of antioxidants and the suppression of inflammatory cytokines. The gold compound auranofin (AFN) is known to activate Nrf2 through the inhibition of TrxR; however, the effects of this compound have not been explored in a mouse model of relapsing-remitting MS (RRMS). Therefore, this study explored the influence of AFN on clinical features, TrxR/Nrf2 signaling [heme oxygenase 1 (HO-1), superoxide dismutase 1 (SOD-1)] and oxidative/inflammatory mediators [IL-6, IL-17A, inducible nitric oxide synthase (iNOS), myeloperoxidase (MPO), nitrotyrosine] in peripheral immune cells and the CNS of mice with the RR type of EAE. Our results showed an increase in TrxR activity and a decrease in Nrf2 signaling in SJL/J mice with RR-EAE. The treatment with AFN caused the amelioration of the clinical features of RR-EAE through the elevation of Nrf2 signaling and the subsequent upregulation of the levels of antioxidants as well as the downregulation of oxidative/pro-inflammatory mediators in peripheral immune cells and the CNS. These data suggest that AFN may be beneficial in the treatment of RRMS.

Thioredoxin Reductase and Organometallic Complexes: A Pivotal System to Tackle Multidrug Resistant Tumors?

Cancers classified as multidrug-resistant (MDR) are a family of diseases with poor prognosis despite access to increasingly sophisticated treatments. Several mechanisms explain these resistances involving both tumor cells and their microenvironment. It is now recognized that a multi-targeting approach offers a promising strategy to treat these MDR tumors. Inhibition of thioredoxin reductase (TrxR), a key enzyme in maintaining redox balance in cells, is a well-identified target for this approach. Auranofin was the first inorganic gold complex to be described as a powerful inhibitor of TrxR. In this review, we will first recall the main results obtained with this metallodrug. Then, we will focus on organometallic complexes reported as TrxR inhibitors. These include gold(I), gold(III) complexes and metallocifens, i.e., organometallic complexes of Fe and Os derived from tamoxifen. In these families of complexes, similarities and differences in the molecular mechanisms of TrxR inhibition will be highlighted. Finally, the possible relationship between TrxR inhibition and cytotoxicity will be discussed and put into perspective with their mode of action.

Thioredoxin 1 and Thioredoxin Reductase 1 Redox System Is Dysregulated in Neutrophils of Subjects with Autism: In Vitro Effects of Environmental Toxicant, Methylmercury.

Autism spectrum disorder (ASD) is a complex developmental disorder in children that results in abnormal communicative and verbal behaviors. Exposure to heavy metals plays a significant role in the pathogenesis or progression of ASD. Mercury compounds pose significant risk for the development of ASD as children are more exposed to environmental toxicants. Increased concentration of mercury compounds has been detected in different body fluids/tissues in ASD children, which suggests an association between mercury exposure and ASD. Thioredoxin1 (Trx1) and thioredoxin reductase1 (TrxR1) redox system plays a crucial role in detoxification of oxidants generated in different immune cells. However, the effect of methylmercury and the Nrf2 activator sulforaphane on the Trx1/TrxR1 antioxidant system in neutrophils of ASD subjects has not been studied previously. Therefore, this study examined the effect of methylmercury on Trx1/TrxR1 expression, TrxR activity, nitrotyrosine, and ROS in neutrophils of ASD and TDC subjects. Our study shows that Trx1/TrxR1 protein expression is dysregulated in ASD subjects as compared to the TDC group. Further, methylmercury treatment significantly inhibits the activity of TrxR in both ASD and TDC groups. Inhibition of TrxR by mercury is associated with upregulation of the Trx1 protein in TDC neutrophils but not in ASD neutrophils. Furthermore, ASD neutrophils have exaggerated ROS production after exposure to methylmercury, which is much greater in magnitude than TDC neutrophils. Sulforaphane reversed methylmercury-induced effects on neutrophils through Nrf2-mediated induction of the Trx1/TrxR1 system. These observations suggest that exposure to the environmental toxicant methylmercury may elevate systemic oxidative inflammation due to a dysregulated Trx1/TrxR1 redox system in the neutrophils of ASD subjects, which may play a role in the progression of ASD.

Gold complex compounds that inhibit drug-resistant Staphylococcus aureus by targeting thioredoxin reductase

IntroductionThere is a significant need for new antimicrobial compounds that are effective against drug-resistant microbes. Thioredoxin reductase (TrxR) is critical in redox homeostasis and was identified as a potential drug target and confirmed through inhibition by compounds auranofin and Bay11-7085.MethodsAdditional TrxR inhibitors were designed and found to exhibit antimicrobial activity against Gram-positive (Enterococcus faecium and Staphylococcus aureus) and glutathione-deficient bacteria (Helicobacter pylori). Investigational compounds were tested for antimicrobial activity, anti-biofilm efficacy, target impact, and cytotoxicity.ResultsThe first-generation molecules AU1 and AU5 inhibited TrxR activity and inhibited methicillin-resistant S. aureus strain MW2 with minimal inhibitory concentrations (MIC) of 0.125 and 0.5 μg/mL, respectively. In an S. aureus enzymatic assay, AU1 inhibited TrxR enzymatic activity in a dose-dependent manner causing a decrease in intracellular free thiols. In addition, biofilm studies demonstrated that AU1 and AU5 reduced biofilm formation at 1X MIC and disrupted mature biofilms at 4X MIC. Cytotoxicity profiles were created using human cell lines and primary cells with LD50 exceeding MICs by at least 12X.DiscussionThus, AU1 and AU5 were TrxR inhibitors that yielded low-concentration antimicrobial activity impacting S. aureus in planktonic and biofilm forms with limited toxic liability.

Novel starting points for fragment-based drug design against mycobacterial thioredoxin reductase identified using crystallographic fragment screening.

The increasing number of people dying from tuberculosis and the existence of extensively drug-resistant strains has led to an urgent need for new antituberculotic drugs with alternative modes of action. As part of the thioredoxin system, thioredoxin reductase (TrxR) is essential for the survival of Mycobacterium tuberculosis (Mtb) and shows substantial differences from human TrxR, making it a promising and most likely selective target. As a model organism for Mtb, crystals of Mycobacterium smegmatis TrxR that diffracted to high resolution were used in crystallographic fragment screening to discover binding fragments and new binding sites. The application of the 96 structurally diverse fragments from the F2X-Entry Screen revealed 56 new starting points for fragment-based drug design of new TrxR inhibitors. Over 200 crystal structures were analyzed using FragMAXapp, which includes processing and refinement by largely automated software pipelines and hit identification via the multi-data-set analysis approach PanDDA. The fragments are bound to 11 binding sites, of which four are positioned at binding pockets or important interaction sites and therefore show high potential for possible inhibition of TrxR.

Enzymatic Redox Properties and Cytotoxicity of Irreversible Nitroaromatic Thioredoxin Reductase Inhibitors in Mammalian Cells.

NADPH:thioredoxin reductase (TrxR) is considered a potential target for anticancer agents. Several nitroheterocyclic sulfones, such as Stattic and Tri-1, irreversibly inhibit TrxR, which presumably accounts for their antitumor activity. However, it is necessary to distinguish the roles of enzymatic redox cycling, an inherent property of nitroaromatics (ArNO2), and the inhibition of TrxR in their cytotoxicity. In this study, we calculated the previously unavailable values of single-electron reduction potentials of known inhibitors of TrxR (Stattic, Tri-1, and 1-chloro-2,4-dinitrobenzene (CDNB)) and inhibitors identified (nitrofuran NSC697923 and nitrobenzene BTB06584). These calculations were according to the rates of their enzymatic single-electron reduction (PMID: 34098820). This enabled us to compare their cytotoxicity with that of model redox cycling ArNO2. In MH22a and HCT-116 cells, Tri-1, Stattic, CDNB, and NSC697023 possessed at least 10-fold greater cytotoxicity than can be expected from their redox cycling activity. This may be related to TrxR inhibition. The absence of enhanced cytotoxicity in BTB06548 may be attributed to its instability. Another known inhibitor of TrxR, tetryl, also did not possess enhanced cytotoxicity, probably because of its detoxification by DT-diaphorase (NQO1). Apart from the reactions with NQO1, the additional mechanisms influencing the cytotoxicity of the examined inhibitors of TrxR are their reactions with cytochromes P-450. Furthermore, some inhibitors, such as Stattic and NSC697923, may also inhibit glutathione reductase. We suggest that these data may be instrumental in the search for TrxR inhibitors with enhanced cytotoxic/anticancer activity.

Synthesis, cytotoxicity, antioxidant activity and molecular modeling of new NSAIDs-EBS derivatives

Two series of NSAIDs-EBS derivatives (5a-j and 9a-i) based on the hybridization of nonsteroidal anti-inflammatory drugs (NSAIDs) skeleton and Ebselen moiety were synthesized. Their cytotoxicity was evaluated against five types of human cancer cell lines, BGC-823 (human gastric cancer cell line), SW480 (human colon adenocarcinoma cells), MCF-7 (human breast adenocarcinoma cells), HeLa (human cervical cancer cells), A549 (human lung carcinoma cells). Moreover, the most active compound 5j showed IC50 values below 3 μM in all cancer cell lines and with remarkable anticancer activity against MCF-7 (1.5 μM) and HeLa (1.7 μM). The redox properties of the NSAIDs-EBS derivatives prepared herein were conducted by 2, 2-didiphenyl-1-picrylhydrazyl (DPPH), bleomycin dependent DNA damage and glutathione peroxidase (GPx)-like assays. Finally, TrxR1 inhibition activity assay and molecular docking study revealed NSAIDs-EBS derivatives could serve as potential TrxR1 inhibitor.

Heterocyclic Iminoquinones and Quinones from the National Cancer Institute (NCI, USA) COMPARE Analysis.

This review uses the National Cancer Institute (NCI) COMPARE program to establish an extensive list of heterocyclic iminoquinones and quinones with similarities in differential growth inhibition patterns across the 60-cell line panel of the NCI Developmental Therapeutics Program (DTP). Many natural products and synthetic analogues are revealed as potential NAD(P)H:quinone oxidoreductase 1 (NQO1) substrates, through correlations to dipyridoimidazo[5,4-f]benzimidazoleiminoquinone (DPIQ), and as potential thioredoxin reductase (TrxR) inhibitors, through correlations to benzo[1,2,4]triazin-7-ones and pleurotin. The strong correlation to NQO1 infers the enzyme has a major influence on the amount of the active compound with benzo[e]perimidines, phenoxazinones, benz[f]pyrido[1,2-a]indole-6,11-quinones, seriniquinones, kalasinamide, indolequinones, and furano[2,3-b]naphthoquinones, hypothesised as prodrugs. Compounds with very strong correlations to known TrxR inhibitors had inverse correlations to the expression of both reductase enzymes, NQO1 and TrxR, including naphtho[2,3-b][1,4]oxazepane-6,11-diones, benzo[a]carbazole-1,4-diones, pyranonaphthoquinones (including kalafungin, nanaomycin A, and analogues of griseusin A), and discorhabdin C. Quinoline-5,8-dione scaffolds based on streptonigrin and lavendamycin can correlate to either reductase. Inhibitors of TrxR are not necessarily (imino)quinones, e.g., parthenolides, while oxidising moieties are essential for correlations to NQO1, as with the mitosenes. Herein, an overview of synthetic methods and biological activity of each family of heterocyclic imino(quinone) is provided.

Prdx5 in the Regulation of Tuberous Sclerosis Complex Mutation-Induced Signaling Mechanisms.

(1) Background: Tuberous sclerosis complex (TSC) mutations directly affect mTORC activity and, as a result, protein synthesis. In several cancer types, TSC mutation is part of the driver mutation panel. TSC mutations have been associated with mitochondrial dysfunction, tolerance to reactive oxygen species due to increased thioredoxin reductase (TrxR) enzyme activity, tolerance to endoplasmic reticulum (ER) stress, and apoptosis. The FDA-approved drug rapamycin is frequently used in clinical applications to inhibit protein synthesis in cancers. Recently, TrxR inhibitor auranofin has also been involved in clinical trials to investigate the anticancer efficacy of the combination treatment with rapamycin. We aimed to investigate the molecular background of the efficacy of such drug combinations in treating neoplasia modulated by TSC mutations. (2) Methods: TSC2 mutant and TSC2 wild-type (WT) cell lines were exposed to rapamycin and auranofin in either mono- or combination treatment. Mitochondrial membrane potential, TrxR enzyme activity, stress protein array, mRNA and protein levels were investigated via cell proliferation assay, electron microscopy, etc. (3) Results: Auranofin and rapamycin normalized mitochondrial membrane potential and reduced proliferation capacity of TSC2 mutant cells. Database analysis identified peroxiredoxin 5 (Prdx5) as the joint target of auranofin and rapamycin. The auranofin and the combination of the two drugs reduced Prdx5 levels. The combination treatment increased the expression of heat shock protein 70, a cellular ER stress marker. (4) Conclusions: After extensive analyses, Prdx5 was identified as a shared target of the two drugs. The decreased Prdx5 protein level and the inhibition of both TrxR and mTOR by rapamycin and auranofin in the combination treatment made ER stress-induced cell death possible in TSC2 mutant cells.

Synthesis and evaluation of Piperine analogs as thioredoxin reductase inhibitors to cause oxidative stress-induced cancer cell apoptosis

Inhibiting thioredoxin reductase (TrxR) to disrupt the redox equilibrium and induce tumor cell apoptosis is a significant tumor therapeutic strategy. Piperine, a natural product from black pepper, has been demonstrated to suppress tumor cell proliferation by enhancing reactive oxygen species (ROS), subsequently leading to cell death. However, the development of Piperine as an active molecule is hampered by its weak cytotoxicity. To develop a compound with higher activity, we synthesized 22 Piperine analogs and evaluated their pharmacological properties. Ultimately, B5 was screened by the results of cytotoxicity and inhibition of TrxR activity. In contrast to Piperine, B5 had significant cytotoxicity with a 4-fold increase. The structure–activity relationship demonstrated that the introduction of an electron-withdrawing group into the benzene ring adjacent to the amino group, particularly in the meta-position, was positive and that shortening the olefin double bond had no appreciable impact on cytotoxicity. Further investigating the physiological activity of B5 in HeLa cells, we found that B5 selectively inhibits the activity of TrxR by binding to Sec residues on TrxR. B5 then induces cellular oxidative stress and finally leads to apoptosis. As a result, the study of B5 paved the way for further investigation into the modification and function of Piperine analogs as TrxR inhibitors.

Diselenide Covalent Allosteric Inhibitors of Glutaminase with Strong In Vivo Anticancer Activity.

Allosteric glutaminase inhibitors demonstrate inhibition of glutamine-dependent cancer cells with low general drug toxicity, but have issues with efficacy in vivo. Here, we designed a series of diselenide compounds with 6 atoms in the middle, aiming to target the allosteric site of kidney type glutaminase (KGA) with a covalent linkage to strengthen the interaction. Proteomic analysis demonstrated that the diselenide compounds cross-linked with the Lys320 residue at the KGA allosteric site; this was confirmed by the KGA K320A mutant which showed essentially no binding to the diselenide. Further, structure-activity relationship (SAR) analysis demonstrated that growth inhibition correlated well with KGA inhibition and was enhanced by thioredoxin reductase (TrxR) inhibition. Interestingly, diselenide compounds showed no inhibition of glutamate dehydrogenase (GDH), indicating some enzyme selectivity. Importantly, the designed novel diselenides are glutaminase allosteric inhibitors that showed in vivo efficacy and survival in the xenograft animal model.

Anti-Tumoural [NHC(thiolato)] Gold(I) Complexes Derived from HIF-1α Inhibitor AC1-004 Target the Mitochondrial Redox System and Show Antiangiogenic Effects in vivo.

AC1-004 is a potent inhibitor of the hypoxia-inducible factor alpha (HIF-1α) pathway, essential for tumour growth, angiogenesis and metastasis. We modelled a series of gold(I) complexes on AC1-004, retaining its 5-carboalkoxybenzimidazole as an NHC ligand while replacing its 2-aryloxymethyl residue with modified thiolato gold(I) fragments. The intention was to augment a potential HIF-1α inhibition by conducive effects typical of NHC gold complexes, such as an inhibition of tumoural thioredoxin reductase (TrxR), an increase in reactive oxygen species (ROS), and cytotoxic and antiangiogenic effects. We report on the synthesis and biological effects of twelve such N,N'-dialkylbenzimidazol-2-ylidene gold(I) complexes, obtained in average yields of 65 % for the thiophenolato and 45 % for the novel 4-(adamant-2-yl)benzenethiol complexes. The structure of one complex was validated via single-crystal X-ray diffraction. Structure-activity relationships (SAR) were derived by variation of the N-substituents (Me, Et, iPr, pentyl, Bn) and the thiolato ligand. Their cytotoxicity against various human cancer cell lines of different entities reached IC50 values in the single-digit micromolar range. The complexes were also assayed for the induction of tumour cell apoptosis (activation of caspase-3/7), TrxR inhibition and antiangiogenic effects in zebrafish. Cyclopropene-bearing congeners were employed in click reactions to examine the subcellular accumulation of the complexes.

High-throughput fluorescent screening of thioredoxin reductase inhibitors to inhibit Mycobacterium tuberculosis

Tuberculosis (TB) is a chronic infectious disease, which is caused by the pathogen Mycobacterium tuberculosis (Mtb) and reemerged as a global health risk with a significant proportion of multi-drug resistant and extensively drug resistant TB cases. It is very urgent to find some novel high-confidence drug targets in Mtb for discovering the effective anti-TB agents. Thioredoxin reductase (TrxR) has been identified to be a highly viable target for anti-TB drugs for its important role in protecting the pathogen from thiol-specific oxidizing stress, regulating intracellular dithiol/disulfide homeostasis and DNA replication and repair. In the present work, a near-infrared (NIR) fluorescent probe DDAT was developed for the detection of TrxR activity and used to high-throughput screen the TrxR inhibitors from natural products. Two screened TrxR inhibitors from Sappan Lignum and microbial metabolites that were further used to inhibit Mycobacterium tuberculosis. All the results indicate that DDAT is a practical fluorescent molecular tool for the discovery of potential anti-TB drugs.

Modulating the Chemical Reactivity of Gold Complexes in Living Systems: From Concept to Biomedical Applications.

ConspectusOver the past few decades, research on the chemistry of gold has progressed rapidly, encompassing topics like catalysis, supramolecular chemistry, molecular recognition, etc. These chemical properties are of great value in developing therapeutics or orthogonal catalysts in biology. However, the presence of concentrated nucleophiles and reductants, particularly thiol-containing serum albumin in blood and glutathione (GSH) inside cells that can strongly bind and quench the active gold species, makes it difficult to translate the chemistry of gold from test tubes into living systems. In this regard, modulating the chemical reactivity of gold complexes to conquer nonspecific interactions with thiols and meanwhile to controllably activate their reactivity in a spatiotemporal manner is of pivotal importance to develop gold complexes for biomedical applications. In this account, we aim to highlight the concept of developing stimuli-activatable gold complexes with masked chemical properties, the bioactivity of which can be spatiotemporally activated at the target site by leveraging approaches from classic structure design to recently emerged photo- and bioorthogonal-activation.A straightforward approach to tuning the reactivity of gold complexes is based on structure modification. This is achieved by introducing strong carbon donor ligands, such as N-heterocyclic carbene, alkynyl, and diphosphine, to improve the stability of gold(I) complexes against off-target thiols. Likewise, GSH-responsive gold(III) prodrug and supramolecular Au(I)-Au(I) interaction have been harnessed to keep a reasonable stability against serum albumin and confer tumor-targeted cytotoxicity by inhibiting thiol- and selenol-containing thioredoxin reductase (TrxR) for potent cancer treatment in vivo. To achieve better spatiotemporal controllability, photoactivatable prodrugs are developed. These complexes are equipped with cyclometalated pincer-type ligands and carbanion or hydride as ancillary ligands, rendering high thiol-stability in the dark, but upon photoirradiation, the complexes can undergo unprecedented photoinduced ligand substitution, β-hydride elimination, and/or reduction to release active gold species for TrxR inhibition at the diseased tissue. To further improve the therapeutic activity, an oxygen-dependent conditional photoreactivity of gold(III) complexes by evolving from photodynamic into photoactivated chemotherapy has been achieved, resulting in highly potent antitumor efficacy in tumor-bearing mice. Of equal importance is harnessing the bioorthogonal activation approach by chemical inducers, as exemplified by a palladium-triggered transmetalation reaction to selectively activate the chemical reactivities of gold including its TrxR inhibition and catalytic activity in living cells and zebrafish. Collectively, strategies to modulate gold chemistry in vitro and in vivo are emerging, and it is hoped that this Account will spur the creation of better approaches to advance gold complexes closer to clinical application.

Abstract 4905: Anticancer activity and paraptosis induction of novel bipyridine-silver(I) compounds against resistant colorectal and ovarian cancer cell lines

Abstract Background: Platinum-based anticancer drugs (e.g. Carboplatin and Oxaliplatin) are frequently applied in chemotherapy regimens against several types of cancer including ovarian carcinoma (OC) and colorectal carcinoma (CRC). However, intrinsic or acquired tumor resistance severely limit their clinical application. Here, we investigated the anticancer activity of several novel silver(I) 2,2’-bipyridine derivatives, containing either triphenylphosphane (PPh3) or 1,2-bis(diphenylphosphino)ethane (dppe) ligands and tested their potential to overcome platinum resistance. Methods: Cytotoxicity and cross resistance profiles of the newly synthesized compounds were tested against two OC models (SKOV-3 and MES-OV), a CRC model (HCT-116) and their corresponding sublines resistant to carboplatin or oxaliplatin, as well as non-malignant fibroblasts (F331) via an MTT-based cell viability assay after 72 h continuous drug exposure. Moreover, the intracellular levels of silver were measured via ICP-MS. In addition, thioredoxin reductase (TrxR) inhibition properties, impact on cell cycle distribution and apoptosis induction potential were investigated via multiple molecular biological methods. Morphological changes induced by the silver compounds were characterized via spinning disk confocal microscopy. Results: All tested compounds displayed pronounced anticancer activity and cancer cell selectivity. Moreover, cell intrinsic resistance to carboplatin or oxaliplatin was not impacting on the cytotoxic activity of the compounds. Noteworthy, MES-OV and HCT-116 cells showed exceptional sensitivity to several silver(I) 2,2’-bipyridine derivatives with a 1,2-bis(diphenylphosphino)ethane (dppe) (AP-compounds). This sensitivity was not based on enhanced drug uptake, pronounced TrxR inhibition nor apoptosis induction or cell cycle arrest. In contrast, hypersensitive cells displayed pronounced endoplasmic reticulum (ER)-derived vesicles together with multiple others hallmarks of paraptotic cell death. Conclusion: Our findings did not only demonstrate that the new silver compounds were unaffected by cancer cell intrinsic resistance mechanisms to platinum-based chemotherapy, but also indicated a high vulnerability of tumor (sub)types towards these bipyridine silver complexes. This hypersensitivity is based on the induction of paraptosis, a novel form of programmed cell death. To summarize, these silver compounds represent a new class of promising anticancer drugs, which demand further preclinical development. Citation Format: Alessia Stefanelli, Ricardo G. Teixeira, Adhan Pilon, Rebecca Warmers, Ingo Ott, Christian R. Kowol, Anamaria Brozovic, Ana Isabel Tomaz, Andreia Valente, Petra Heffeter. Anticancer activity and paraptosis induction of novel bipyridine-silver(I) compounds against resistant colorectal and ovarian cancer cell lines. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4905.

SERD-NHC-Au(I) complexes for dual targeting ER and TrxR to induce ICD in breast cancer

The development of selective estrogen receptor degraders (SERDs) has brought new ideas for the clinical treatment of ER-positive advanced breast cancer. The successful application of combinational therapy inspired the exploration of other targets to prevent breast cancer progression. Thioredoxin reductase (TrxR) is an important enzyme that can regulate redox balance in cells and it was considered as a potential target for anticancer treatment. In this study, we firstly combine a clinical SERD candidate——G1T48 (NCT03455270), with a TrxR inhibitor——N-heterocyclic carbene gold(I) [NHC-Au(I)] to form dual targeting complexes that can regulate both signaling pathways. The most efficacious complex 23 exhibited significant antiproliferative profile through degrading ER and inhibiting TrxR activity. Interestingly, it can induce immunogenic cell death (ICD) caused by ROS. This is the first evidence to elucidate the role of ER/TrxR-ROS-ICD axis in ER positive breast cancer and this research may inspire new drug development with novel mechanisms. The in vivo xenograft study demonstrated that complex 23 had excellent antiproliferative activity toward MCF-7 cells in mice model.

Auranofin Synergizes with the PARP Inhibitor Olaparib to Induce ROS-Mediated Cell Death in Mutant p53 Cancers

Auranofin (AF) is a potent, off-patent thioredoxin reductase (TrxR) inhibitor that efficiently targets cancer via reactive oxygen species (ROS)- and DNA damage-mediated cell death. The goal of this study is to enhance the efficacy of AF as a cancer treatment by combining it with the poly(ADP-ribose) polymerase-1 (PARP) inhibitor olaparib (referred to as 'aurola'). Firstly, we investigated whether mutant p53 can sensitize non-small cell lung cancer (NSCLC) and pancreatic ductal adenocarcinoma (PDAC) cancer cells to AF and olaparib treatment in p53 knock-in and knock-out models with varying p53 protein expression levels. Secondly, we determined the therapeutic range for synergistic cytotoxicity between AF and olaparib and elucidated the underlying molecular cell death mechanisms. Lastly, we evaluated the effectiveness of the combination strategy in a murine 344SQ 3D spheroid and syngeneic in vivo lung cancer model. We demonstrated that high concentrations of AF and olaparib synergistically induced cytotoxicity in NSCLC and PDAC cell lines with low levels of mutant p53 protein that were initially more resistant to AF. The aurola combination also led to the highest accumulation of ROS, which resulted in ROS-dependent cytotoxicity of mutant p53 NSCLC cells through distinct types of cell death, including caspase-3/7-dependent apoptosis, inhibited by Z-VAD-FMK, and lipid peroxidation-dependent ferroptosis, inhibited by ferrostatin-1 and alpha-tocopherol. High concentrations of both compounds were also needed to obtain a synergistic cytotoxic effect in 3D spheroids of the murine lung adenocarcinoma cell line 344SQ, which was interestingly absent in 2D. This cell line was used in a syngeneic mouse model in which the oral administration of aurola significantly delayed the growth of mutant p53 344SQ tumors in 129S2/SvPasCrl mice, while either agent alone had no effect. In addition, RNA sequencing results revealed that AF- and aurola-treated 344SQ tumors were negatively enriched for immune-related gene sets, which is in accordance with AF's anti-inflammatory function as an anti-rheumatic drug. Only 344SQ tumors treated with aurola showed the downregulation of genes related to the cell cycle, potentially explaining the growth inhibitory effect of aurola since no apoptosis-related gene sets were enriched. Overall, this novel combination strategy of oxidative stress induction (AF) with PARP inhibition (olaparib) could be a promising treatment for mutant p53 cancers, although high concentrations of both compounds need to be reached to obtain a substantial cytotoxic effect.