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Following the discovery of nitric oxide (NO) and carbon monoxide (CO), hydrogen sulfide (H

A diselenide/disulfide unit was introduced into camptothecin (CPT), and two selenoprodrugs (e.g., CPT-Se3 and CPT-Se4) were identified to show improved potency in killing cancer cells and inhibiting tumor growth in vivo. Interestingly, the intrinsic fluorescence of CPT was severely quenched by the diselenide bond. Both the selenoprodrugs were activated by glutathione with a nearly complete recovery of CPT's fluorescence. The activation of prodrugs was accompanied by the production of selenol intermediates, which catalyzed the constant conversion of glutathione and oxygen to oxidized glutathione and superoxides. The diselenide unit is widely employed in constructing thiol-responsive materials. However, the selenol intermediates were largely ignored in the activation process prior to this study. Our work verified that integration of the diselenide unit may further enhance the parent drug's efficacy. Also, the discovery of the fluorescence quenching property of the diselenide/disulfide bond further shed light on constructing novel theranostic agents.

Metabolic reprogramming is critical for tumorigenesis. Pyruvate kinase M2 (PKM2) is overexpressed in lung carcinoma cells and plays a critical role in the Warburg effect, making the enzyme a research hotspot for anticancer drug development. Cynaropicrin (CYN), a natural sesquiterpene lactone compound from artichoke, has received increasing consideration due to its consumable esteem and pharmacological properties. Our data reveal that CYN not only inhibited the purified PKM2 activity but also decreased the cellular PKM2 expression in A549 cells. The inhibition of PKM2 leads to the upregulation of p53 and the downregulation of the DNA repair enzyme poly (ADP-ribose) polymerase (PARP), and subsequently causes the cell cycle arrest. Additionally, CYN inhibits the interaction of PKM2 and Nrf2, resulting in the impairment of cellular antioxidant capacity, induction of oxidative stress, and mitochondrial damages. Overexpression of PKM2 attenuates the CYN-induced DNA damage, mitochondrial fission, and cell viability. Thus, targeting PKM2 provides an original mechanism for understanding the pharmacological impact of CYN and assists in the further development of CYN as an anticancer agent.

Significance: Thioredoxin (Trx) and thioredoxin reductase are two core members of the Trx system. The system bridges the gap between the universal reducing equivalent NADPH and various biological molecules and plays an essential role in maintaining cellular redox homeostasis and regulating multiple cellular redox signaling pathways. Recent Advance: In recent years, the Trx system has been well documented as an important regulator of many diseases, especially tumorigenesis. Thus, the development of potential therapeutic molecules targeting the system is of great significance for disease treatment. Critical Issues: We herein first discuss the physiological functions of the Trx system and the role that the Trx system plays in various diseases. Then, we focus on the introduction of natural small molecules with potential therapeutic applications, especially the anticancer activity, and review their mechanisms of pharmacological actions via interfering with the Trx system. Finally, we further discuss several natural molecules that harbor therapeutic potential and have entered different clinical trials. Future Directions: Further studies on the functions of the Trx system in multiple diseases will not only improve our understanding of the pathogenesis of many human disorders but also help develop novel therapeutic strategies against these diseases. Antioxid. Redox Signal. 34, 1083-1107.

Desmoid fibromatosis (DF) is a locally aggressive connective-tissue tumor arising in deep soft tissues. Although multiple therapeutic modalities have been demonstrated effective for DF, there is no standard systemic treatment for progressive and recurrent DF. As a part of systemic treatment, tyrosine kinase inhibitors have shown promising activity against DF with tolerable toxicity profiles. Thus, the aim of this study was to investigate the efficacy and safety of apatinib, a novel multi-target angiogenesis inhibitor, in patients with DF. We retrospectively analyzed the medical records of patients with advanced extremity DF regularly treated with apatinib between October 2017 and January 2020 in our center. Apatinib was initially administered with a dose of 250 mg daily and the dose was adjusted according to the toxicity. Tumor response was assessed by the Response Evaluation Criteria in Solid Tumors 1.1 criteria. The primary endpoint was progression-free survival (PFS); objective response rates and drug-related adverse events were also evaluated. A total of 22 (6 male, 16 female) patients with advanced extremity DF were included. The mean medication time was 17 months. None of the patients reached a complete response, but ten (45.5%) patients achieved partial response, and 11 patients (50%) achieved stable disease. One (4.5%) patient developed progressive disease, and the 1-year PFS rate was 95.2%. The disease control rate was 95.4% (21/22) and the objective response rate was 45.5% (10/22). Meanwhile, 18 (81.8%) patients with a tumor shrinkage were accompanied by a decreased signal intensity of lesions in T2-weighted magnetic resonance imaging. The most frequent adverse events included hand-foot syndrome (n = 7, 31.8%), fatigue (n = 6, 27.2%), local pain (n = 4, 18.1%), diarrhea (n = 4, 18.1%). Apatinib is an effective and well-tolerated option for patients with advanced extremity DF. Indeed, further prospective, randomized studies with larger cases are required to fully explore the clinical utility of apatinib in DF.

Thioredoxin reductase (TrxR) is a pivotal regulator of redox homeostasis, while dysregulation of redox homeostasis is a hallmark for cancer cells. Thus, there is considerable potential to inhibit the aberrantly upregulated TrxR in cancer cells to discover selective cancer therapeutic agents. Nevertheless, the structural types of TrxR inhibitors presented currently are still relatively limited. We herein report that PACMA 31, previously reported to inhibit protein disulfide isomerase (PDI), is a potent TrxR inhibitor. PACMA 31 possesses a pharmacophore scaffold that is structurally different from the announced TrxR inhibitors and exhibits effective cytotoxicity against cervical cancer cells. Our results reveal that PACMA 31 selectively inhibits TrxR over the related glutathione reductase (GR) and in the presence of reduced glutathione (GSH). Further studies with mutant enzyme and molecular docking suggest that the propynamide fragment of PACMA 31 interacts covalently with the selenocysteine residue of TrxR. Moreover, PACMA 31 effectively and selectively curbs TrxR activity in cells and further stimulates the production of reactive oxygen species (ROS) at low micromolar concentrations, which in turn triggers the accumulation of oxidized thioredoxin (Trx) and GSSG in cells. Follow-up studies demonstrate that PACMA 31 targets TrxR in cells to induce oxidative stress-mediated cancer cell apoptosis. Our results provide a new structural type of TrxR inhibitor that may serve as a useful probe for investigating the biology of TrxR-implicated pathways, and uncover a new target of PACMA 31 that contributes to it becoming a candidate for cancer treatment.

Inhibition of thioredoxin reductase (TrxR) is a crucial strategy for the discovery of antineoplastic drugs and radiosensitizers. As an anticancer candidate derived from Michelia, micheliolide (MCL) is converted readily from parthenolide (PTL), and has better stability and solubility than PTL. However, the anticancer mechanism of MCL has not been fully dissected. We present here for the first time that MCL-targeted inhibition of TrxR not only promotes oxidative stress-mediated HeLa cell apoptosis but also sensitizes ionizing radiation (IR) treatment. Further mechanistic studies demonstrate that MCL covalently binds to Sec at position 498 of TrxR to restrain the biological function of TrxR. It exhibits the inhibition of TrxR activity, enhancement of oxidized Trx, and sensitization of IR in the cellular environment, accompanied by the accumulation of reactive oxygen species (ROS) and the collapse of the intracellular redox balance. In addition, HeLa-shTrxR1 cells with knockdown of TrxR were more sensitive than the HeLa-shNT cells to either MCL-treated or IR-induced cytotoxicity, ROS, and apoptosis, suggesting that inhibition of TrxR by MCL is likely responsible for increased cytotoxicity and enhanced radiation response. These findings further establish the mechanistic understanding and preclinical data to support the further investigation of MCL's potential as a prospective radiosensitizer and cancer chemotherapeutic agent.

Elevated intracellular reactive oxygen species (ROS) and antioxidant defense systems have been recognized as one of the hallmarks of cancer cells. Compared with normal cells, cancer cells exhibit increased ROS to maintain their malignant phenotypes and are more dependent on the "redox adaptation" mechanism. Thus, there are two apparently contradictory but virtually complementary therapeutic strategies for the regulation of ROS to prevent or treat cancer. The first strategy, that is, chemoprevention, is to prevent or reduce intracellular ROS either by suppressing ROS production pathways or by employing antioxidants to enhance ROS clearance, which protects normal cells from malignant transformation and inhibits the early stage of tumorigenesis. The second strategy is the ROS-mediated anticancer therapy, which stimulates intracellular ROS to a toxicity threshold to activate ROS-induced cell death pathways. Therefore, targeting the regulation of intracellular ROS-related pathways by small-molecule candidates is considered to be a promising treatment for tumors. We herein first briefly introduce the source and regulation of ROS, and then focus on small molecules that regulate ROS-related pathways and show efficacy in cancer therapy from the perspective of pharmacophores. Finally, we discuss several challenges in developing cancer therapeutic agents based on ROS regulation and propose the direction of future development.

A smart fluorescent probe DPAS-Cys has been rationally designed based on a typical AIEgen DPAS and an acrylate moiety. The probe DPAS-Cys not only can be used for the detection of cysteine (Cys) selectively with large Stokes shift (200 nm) and relatively low detection limit (2.4 μM), but also shows lipid droplets (LDs) targeting property. The response mechanism for Cys was carefully verified. Importantly, due to the aggregation-induced emission characteristic, the introduction of considerable percentage of traditional organic solvent is avoidable, which makes it suitable for bioimaging in physiological systems. In addition, the confocal fluorescence imaging demonstrates that DPAS-Cys is able to detect Cys in LDs of different cell lines with universality. Our study opens a new avenue to understand the importance of LDs in biosystem, for which the gap between the essential biothiol Cys and the energy storage organelle LDs was bridged for the first time.

Upregulation of the selenoprotein thioredoxin reductase (TrxR) is of pathological significance in maintaining tumor phenotypes. Thus, TrxR inhibitors are promising cancer therapeutic agents. We prepared different amino-substituted phenylarsine oxides and evaluated their cytotoxicity and inhibition of TrxR. Compared with our reported

The ubiquitous thioredoxin reductase (TrxR, encoded by TXNRD gene) enzymes are essential to maintain the cellular redox homeostasis. The most outstanding function of TrxR is to keep its native subs...

Clonorosins A (

Nonsmall cell lung cancer (NSCLC) has been a fatal and refractory disease worldwide. Novel therapeutic developments based on fundamental investigations of anticancer mechanisms underlie substantial foundations to win the fight against cancer diseases. In this study, we isolated a natural product fusaricide (FCD) from an endophytic fungus of Lycium barbarum, identified as Epicoccum sp. For the first time, we discovered that FCD potently inhibited proliferation in a variety of human NSCLC cell lines, with relatively less toxicity to normal cells. Our study exhibited that FCD induced apoptosis, caused DNA damage and cell cycle arrest in G0/G1 phase, and activated caspase-3 as well as other apoptosis-related factors in human NSCLC NCI-H460 cells. FCD was proven to be an iron chelator that actively decreased levels of cellular labile iron pool in NCI-H460 cells in our study. FeCl3 supplement reversed FCD-induced apoptosis. The upregulation of transferrin receptor 1 (TfR1) and downregulation of ferritin heavy chain (FTH) expression were observed after FCD treatment. In summary, our study highlighted the potential anticancer effects of FCD against human NSCLCs and demonstrated that the FCD-mediated apoptosis depended on binding to intracellular iron.

Six new polyketides, alternaritins A–D [(±)-1–4] and isoxanalteric acid I (8), and 25 known Alternaria toxins were isolated from the culture of an endophytic fungi Alternaria sp. MG1. 3 is a rare fungal metabolite. 6 is a new natural product, and 5, 7, and 9 are known previously but their absolute configurations have not been determined. Three enantiomers [(±)-1, (±)-7, and (±)-15] were separated via chiral HPLC resolution. The structures of those polyketides (1–9) were elucidated by spectrometric analysis using MS and NMR. The absolute configurations were established using X-ray diffraction analysis and statistical comparative analysis of the experimental ECD and OR data, in conjunction with quantum mechanical calculations. All of the compounds were evaluated for their bioactivities. Known compound 27 exerted the most potent cytotoxic activities against HT-1080 and NCI-H1299 cell lines. The new compounds, 2 and 3, showed moderate inhibition on COX-2, while a pair of isomers, 8 and 9, exhibited medium activity on COX-2 and uropathogenic Escherichia coli.

Finding specific small molecule probes of a biological target is extremely desired but remains a big challenge. We reported herein a highly selective fluorescent probe derivatized from the nile blue fluorophore, NBL-SS, for thioredoxin (Trx), a ubiquitous redox-regulating protein essentially involved in cell growth, differentiation, and death. NBL-SS displayed multiple favorable properties, such as red emission, fast response, and high fluorescence signal, which enabled the probe to readily image Trx functions in live cells and in vivo. The fluorophore-dependent selectivity indicates that manipulation of weak interactions between probes and their target biomacromolecules could further improve the probes' specificity. In addition, our discovery, i.e., the preference reduction of simple disulfide bonds by Trx over glutathione, also advances the development of disulfide cleavage-based probes, prodrugs, and theranostic agents.

Small molecule probes are indispensable tools to explore diverse cellular events. However, finding a specific probe of a target remains a high challenge. Here we report the discovery of Fast-TRFS, a specific and superfast fluorogenic probe of mammalian thioredoxin reductase, a ubiquitous enzyme involved in regulation of diverse cellular redox signaling pathways. By systematically examining the processes of fluorophore release and reduction of cyclic disulfides/diselenides by the enzyme, structural factors that determine the response rate and specificity of the probe are disclosed. Mechanistic studies reveal that the fluorescence signal is switched on by a simple reduction of the disulfide bond within the probe, which is in stark contrast to the sensing mechanism of published probes. The favorable properties of Fast-TRFS enable development of a high-throughput screening assay to discover inhibitors of thioredoxin reductase by using crude tissue extracts as a source of the enzyme., Thioredoxin reductase (TrxR) plays a crucial part in regulating cellular redox homeostasis. Here, the authors developed a fluorescent probe composed of a five-membered disulphide, a coumarin fluorophore and a urea linker that detects TrxR activity with fast response and high selectivity.

The selenoprotein thioredoxin reductase (TXNRD) is a promising therapeutic target for cancer. To discover novel TXNRD inhibitors, a library of α, β-unsaturated carbonyl compounds were applied in structure-based virtual screening for the selection of hit compounds. Fifteen top-ranked compounds were further validated experimentally, exhibiting potent inhibition of TXNRD and remarkable cytotoxicity to cancer cells. The further binding mode analysis indicated that multiple noncovalent interactions between the inhibitors and the active pocket of TXNRD facilitated the formation of covalent bonds between the Sec498 on TXNRD and the α, β-unsaturated carbonyl groups on inhibitors. Results from both simulations and experiments demonstrated that Sec498 is the prime interaction site for the inhibition of TXNRD. Taking compound 7 as an example, the inhibition of TXNRD by compounds promoted oxidative stress-mediated apoptosis of cancer cells. Given these findings, novel TXNRD inhibitors may be discovered and introduced to the growing fields of small molecule drugs against TXNRD.

Ibrutinib (IBT), the first-in-class inhibitor of Bruton's tyrosine kinase (BTK), has demonstrated clinical activity against various B-cell malignancies. Aside from its therapeutic mechanism through BTK inhibition, IBT has other target sites reported for cancer therapy, leading us to investigate whether IBT has unreported targets. Our study revealed that IBT can inhibit SMMC-7721 cells through irreversible inhibition of mammalian thioredoxin reductase enzymes. Further study demonstrated that IBT can cause cellular reactive oxygen species elevation and induce cancer cell apoptosis. The discovery of a new target of IBT sheds light on better understanding its anticancer mechanisms and provides a theoretical foundation for its further use in clinical therapy.

Isolation and identification of natural products from plants is an essential approach for discovering drug candidates. Herein we report the characterization of three sesquiterpene lactones from a new genus Shangwua, e.g. onopordopicrin (ONP), C2, and C3, and evaluation of their pharmacological functions in interfering cellular redox signaling. Compared to C2 and C3, ONP shows the most potency in killing cancer cells. Further experiments demonstrate that ONP robustly inhibits thioredoxin reductase (TrxR), which leads to perturbation of cellular redox homeostasis with the favor of oxidative stress. Knockdown of the TrxR sensitizes cells to the ONP treatment while overexpression of the enzyme reduces the potency of ONP, underpinning the correlation of TrxR inhibition to the cytotoxicity of ONP. The discovery of ONP expands the library of the natural TrxR inhibitors, and the disclosure of the action mechanism of ONP provides a foundation for the further development of ONP as an anticancer agent., Graphical Abstract

A phytochemical study led to the isolation of 25 diterpenoid alkaloids from Aconitum sinomontanum, of which six were described for the first time. Among them compounds 1–3 are anhydrolycoctonine derivatives, rare rearranged aconitine-type C19-diterpenoid alkaloids. To our best knowledge, less than ten of this type of alkaloids were isolated just from the genus Aconitum. The structures of these unreported compounds were elucidated by extensive analysis of NMR spectroscopic data and X-ray diffraction. The biological activities of compounds 1–3, 5–9, and 12–25 were evaluated. Among the tested compounds, compounds 2 and 17 showed potent inhibitory effect on the capsaicin (selective TRPV1 agonist) mediated activation of transient receptor potential vanilloid 1 (TRPV1) channels expressed in HEK-293 cells with inhibition rate of 31.78% and 30.94% at the concentration of 10 μM. Compounds 1–3, 5–9, 13, and 18–25 exhibited weak cytotoxic activity against human tumor cell lines NCI–H226 and MDA-MB-231 with inhibition rate over 10% at the concentration of 10 μM. Compound 16 showed most inhibitory effect on the expression of Nrf2 (NF–E2-related factor-2)-regulated gene with inhibition rate of 25% at the concentration of 20 μM.

Reactive oxygen species (ROS) and reactive sulfur species (RSS) participate in many physiological activities and help maintaining the redox homeostasis in biological system. The complicated intrinsic connection between specific ROS/RSS needs to be further explored. Herein, a novel fluorescent probe (MB-NAP-N

Thioredoxin reductase (TrxR) enzymes are critical in regulating redox homeostasis in cells. We report the first two-photon fluorescent probe of mammalian TrxR (TP-TRFS). TP-TRFS retains high specificity in recognizing TrxR. More importantly, the two-photon absorbing character of TP-TRFS enables it to be used in vivo. With the aid of TP-TRFS, a remarkable decline of the TrxR function was observed in the brain of a mouse model of stroke for the first time, providing a mechanistic link of TrxR dysfunction with stroke.

Altered redox homeostasis as a hallmark of cancer cells is exploited by cancer cells for growth and survival. The thioredoxin (Trx), an important regulator in maintaining the intracellular redox homeostasis, is cumulatively recognized as a promising target for the development of anticancer drugs. Herein, we synthesized 72 disulfides and evaluated theirinhibition for Trx and antitumor activity. First, we established an efficient and fast method to screen Trx inhibitors by using the probe NBL-SS that was developed by our group to detect Trx function in living cells. After an initial screening of the Trx inhibitory activity of these compounds, 8 compounds showed significant inhibition activity against Trx. We then evaluated the cytotoxicity of these 8 disulfides, compounds 68 and 69 displayed high cytotoxicity to HeLa cells, but less sensitive to normal cell lines. Next, we performed kinetic studies of both two disulfides, 68 had faster inhibition of Trx than 69. Further studies revealed that 68 led to the accumulation of reactive oxygen species and eventually induced apoptosis of Hela cells via inhibiting Trx. The establishment of a method for screening Trx inhibitors and the discovery of 68 with remarkable Trx inhibition provide support for the development of anticancer candidates with Trx inhibition.

Protein sulfhydryl groups play a vital role in maintaining cellular redox homeostasis and protein functions and have attracted increasing interests for the selective detection of protein thiols over low-molecular-weight thiols (LMWTs). Herein, we reported a red-emitting and environment-sensitive probe (FM-red) for detecting and labeling protein thiols. The probe contains a maleimide unit as a thiol receptor and an environment-sensitive fluorophore as a sensor. The emission signal of the probe was exclusively switched on by binding to protein sulfhydryl groups through the twisted intramolecular charge transfer mechanism, while negligible fluorescence was observed when FM-red reacted with LMWTs. Various experiments verified that FM-red possessed fast responsivity (∼10 min) and high selectivity to sense protein thiols over LMWTs with a red emission (∼655 nm). These favorable properties enable the probe to image protein sulfhydryl groups in live cells and in vivo. In addition, as FM-red has a relatively high molecular weight (MW 688), it is able to separate the labeled proteins from the unlabeled ones after FM-red derivatization via routine protein electrophoresis, which may be applied to determine the redox states of thioredoxin, a small redox protein ubiquitous in all cells. With the aid of the probe, we demonstrated a significant decrease in the protein thiols and the accumulation of oxidized thioredoxin in a cellular model of Parkinson's disease.

Vicinal dithiol-containing proteins (VDPs) play an important role in maintaining the structures and functions of proteins mainly through the conversion between dithiols and disulfide bonds. The content of VDPs also reflects the redox status of an organism. To specifically and expediently detect VDPs, we developed a turn-on monoarsenical fluorescent probe (NEP) based on the intramolecular charge transfer mechanism. Naphthalimide was chosen as a fluorophore and linked with the receptor moiety (cyclic dithiarsolane) via carbamate segment. In the presence of VDPs, NEP displays a strong green fluorescence signal produced by the cyclic dithiarsolane cleavage and subsequent intramolecular cyclization to liberate the fluorophore. Furthermore, NEP exhibits high selectivity toward VDPs over other protein thiols and low molecular weight thiols. The favorable properties of NEP enable it readily to detect VDPs in live cells and in vivo. In addition, a remarkable decrease of VDPs in parkinsonism was disclosed for the first time, highlighting that regulating VDPs level has a therapeutic potential for parkinsonism.

The first β-allyl carbamate fluorescent probe, AC-green, was reported for specifically imaging vicinal dithiol proteins (VDPs) in living systems. The probe displays low toxicity and features high sensitivity and fast response towards VDPs. A drastic loss of VDPs in a Parkinson's model was disclosed by AC-green, linking the fluctuation of VDP levels to Parkinson's disease for the first time. The structural novelty and favorable properties of AC-green will advance the development of novel VDP probes.

Objectives Herpes simplex virus 1 (HSV-1) is one of the most prevalent viruses in humans worldwide. Owing to limited therapeutic options mainly with acyclovir (ACV) and analogues and the emergence of ACV-resistant strains, new drugs with different modes of action and low toxicity are required. The aim of this study was to determine the anti-HSV-1 effect and mechanism of action of the flavonoid compound dihydromyricetin (DHM) from Ampelopsis grossedentata. Methods The HSV-1 inhibitory effect of DHM was evaluated by measuring plaque formation and generation of progeny virus as well as expression of HSV-1-related genes in Vero cells. The molecular mechanism of the antiviral activity of DHM against HSV-1 was explored by real-time quantitative PCR and ELISA. Results DHM presented a significant inhibitory effect on HSV-1 plaque formation and generation of progeny virus, with an EC50 (50% effective concentration) of 12.56 μM in Vero cells. Furthermore, expression of HSV-1 immediate-early genes (ICP4 and ICP22), early genes (ICP8 and UL42) and late genes (gB, VP1/2) was decreased by DHM at concentrations of 16 μM and 32 μM. DHM specifically suppressed mRNA levels of Toll-like receptor 9 (TLR9), leading to inhibition of the inflammatory transcriptional factor NFκB and a decrease in TNFα. Conclusion These findings indicate that the effective inhibitory activity of DHM was achieved by suppressing TNFα production in a TLR9-dependent manner. Although further studies are needed to better characterise the activity of DHM in vivo, the results suggest this extract as a promising new anti-HSV-1 agent.

The selenoprotein thioredoxin reductase (TrxR) enzymes are the only identified proteins that maintain thioredoxin (Trx) proteins in a reduced state under physiological conditions, and consequently play a pivotal role in the regulation of various cellular redox signaling pathways involved in cell differentiation, growth, and death. The elevated expression of TrxR enzymes has been observed in different types of cancer cells, and this overexpression is of pathological significance in maintaining tumor phenotypes, such as uncontrolled proliferation and resistance to apoptosis. Herein, we discuss recent advances in the study of TrxR, including classic assays of TrxR, the emerging chemical tools of TrxR, and small molecules that target TrxR as potential anticancer agents.

Xanthatin (XT), a naturally occurring sesquiterpene lactone presented in cocklebur ( Xanthium strumarium L.), is under development as a potential anticancer agent. Despite the promising anticancer effect of XT, the molecular mechanism underlying its cellular action has not been well elucidated. The mammalian thioredoxin reductase (TrxR) enzymes, the essential seleno-flavoproteins containing a penultimate selenocysteine (Sec) residue at the C-terminus, represent a promising target for cancer chemotherapeutic agents. In this study, XT inhibits both the purified TrxR and the enzyme in cells. The possible binding mode of XT with the TrxR protein is predicted by the covalent docking method. Mechanism studies reveal that XT targets the Sec residue of TrxR and inhibits the enzyme activity irreversibly. Simultaneously, the inhibition of TrxR by XT promotes the oxidative stress-mediated apoptosis of HeLa cells. Importantly, the knockdown of the enzyme sensitizes the cells to XT treatment. Targeting TrxR thus discloses a novel molecular mechanism in accounting for the cellular action of XT and provides insights into the development of XT as an anticancer agent.

Elevated reactive oxygen species and antioxidant defense systems have been recognized as one of the hallmarks of cancer cells. As a major regulator of the cellular redox homeostasis, the selenoprotein thioredoxin reductase (TrxR) is increasingly considered as a promising target for anticancer drug development. The current approach to inhibit TrxR predominantly relies on the modification of the selenocysteine residue in the C-terminal active site of the enzyme, in which it is hard to avoid the off-target effects. By conjugating the anticancer drug gemcitabine with a 1,2-dithiolane scaffold, an unprecedented prodrug strategy is disclosed that achieves a specific release of gemcitabine by TrxR in cells. As overexpression of TrxR is frequently found in different types of tumors, the TrxR-dependent prodrugs are promising for further development as cancer chemotherapeutic agents.

Plumbagin (PLB), a natural naphthoquinone from the traditional folk medicines Plumbago zeylanica, Dionaea muscipula, or Nepenthes gracilis, has been documented possessing a wide variety of pharmacological activities. Although PLB demonstrates anticancer activity in multiple types of malignant cells, the cellular targets of PLB have not been well defined and remained only partially understood. We reported here that PLB selectively inhibits TrxR and elicits reactive oxygen species in human promyelocytic leukemia HL-60 cells, which leads to elevation of GSSG/GSH ratio and decrease of cellular thiol pool. As a consequence, PLB disturbs the cellular redox homeostasis, induces oxidative stress-mediated apoptosis and eventually selectively kills HL-60 cells. Inhibition of TrxR by PLB thus discloses an unprecedented mechanism underlying the anticancer efficacy of PLB, and sheds light in considering the usage of PLB as a promising cancer therapeutic agent.

The gastrointestinal (GI) stability of three flavonoids, dihydromyricetin (DMY), myricetin (MYR), and myricitrin (MYT), was examined in simulated physiological fluids. Several factors that may influence the degradation rate of theses flavonoids were evaluated, including pH and the presence of pepsin and pancreatin enzymes. We found that GI stability followed the order of MYT DMY MYR. These flavonoids were stable in simulated gastric fluids and buffer solutions (pH 1.2), but encountered a pseudo-first-order kinetic degradation in simulated intestinal fluids and buffer solutions (pH 6.8). We conclude that it is the pH, rather than the presence of pepsin or pancreatin, which most strongly influences the stability of these three flavonoids. Further study of the stability of the compounds using a pH range (1.0-8.0) indicated potential instability in the duodenum, small intestine, and colon. Therefore, we conclude that the low bioavailability of these flavonoids may be due to their poor stability in the GI tract.

A dual mode fluorescent probe, which is based on an integration of fluorescein and coumarin fluorophores, was developed for the discrimination of Cys from Hcy and GSH. This probe (2) shows the advantage of quick reaction (5 min) with Cys, resulting in a strong fluorescence turn-on response when excited at 450 nm. Notably, it also demonstrates the ratiometric fluorescence property while excited by a shorter wavelength (332 nm). All of results suggest probe 2 has a high selectivity toward Cys even in the presence of other amino acids, cations and anions. The detection limit of Cys was calculated as 0.084 μM, which was much lower than the intracellular concentration. 1H NMR, MS and DFT calculation were used to reveal the detection mechanism further. Finally, this low cytotoxic probe was successfully applied in bioimaging within HepG2 cells.

Relay recognition of copper(II) ions and biothiols via a fluorescence “on–off–on” cascade was designed and realized as a new sequential combination of cations and small molecules. Probe 1 bearing a fluorescein skeleton was thus synthesized, which performed well in 100% HEPES buffer (pH = 7.0) solution, as a highly sensitive, selective fluorescence sensor for Cu2+. The limit of detection (LOD, 0.017 ppm) was obtained, and this value is much lower than 1.3 ppm, allowed by US EPA. The 1:1 complex generated from fast sensing of Cu2+ when excited at 491 nm, showed good relay recognition for biothiols (i.e., Cys, Hcy and GSH with low detection limits of 0.12 μM, 0.036 μM and 0.024 μM, respectively) via remarkable fluorescence enhancement. The origin of this relay process was disclosed through ESI-MS and corresponding density functional theory (DFT) computations. Notably, probe 1 can be utilized for the construction of a molecular logic gate with the IMPLICATION function by using the above fluorescence changes. Moreover, this relay recognition was also applied to HepG2 cell imaging successfully.

Cancer is considered as one of the highly mortal diseases globally. This is largely due to the lack of efficacious medicines for tumors, and thus development of potent anticancer agents is urgently needed. The thioredoxin (Trx) system is crucial to the survival ability of cells and its expression is up-regulated in many human tumors. Recently, increasing evidence has been established that mammalian thioredoxin reductase (TrxR), a selenocysteine-containing protein and the core component of the thioredoxin system, is a promising therapeutic target. The sesquiterpene lactone compound cynaropicrin (CYN), a major component of Cynara scolymus L., has shown multiple pharmacological functions, especially the anticancer effect, in many experimental models. Most of these functions are concomitant with the production of reactive oxygen species (ROS). Nevertheless, the target of this promising natural anticancer product in redox control has rarely been explored. In this study, we showed that CYN induces apoptosis of Hela cells. Mechanistic studies demonstrated that CYN impinges on the thioredoxin system via inhibition of TrxR, which leads to Trx oxidation and ROS accumulation in HeLa cells. Particularly, the cytotoxicity of CYN is enhanced through the genetic knockdown of TrxR, supporting the pharmacological effect of CYN is relevant to its inhibition of TrxR. Together, our studies reveal an unprecedented mechanism accounting for the anticancer effect of CYN and identify a promising therapeutic agent worthy of further development for cancer therapy.

Alzheimer's disease (AD), an age-dependent neurodegenerative disorder, is the main cause of dementia. Common hallmarks of AD include the amyloid β-peptide (Aβ) aggregation, high levels of hyperphosphorylated tau protein (p-tau) and failure in redox homeostasis. To date, all proposed drugs affecting Aβ and/or p-tau have been failed in clinical trials. A decline in the expression of the transcription factor Nrf2 (nuclear factor-erythroid 2-p45 derived factor 2) and its driven genes (NQO1, HO-1, and GCLC), and alteration of the Nrf2-related pathways have been observed in AD brains. Nrf2 plays a critical role in maintaining cellular redox homeostasis and regulating inflammation response. Nrf2 activation also provides cytoprotection against increasing pathologies including neurodegenerative diseases. These lines of evidence imply that Nrf2 activation may be a novel AD treatment option. Interestingly, recent studies have also demonstrated that Nrf2 interferes with several key pathogenic processes in AD including Aβ and p-tau pathways. The current review aims to provide insights into the role of Nrf2 in AD. Also, we discuss the progress and challenges regarding the Nrf2 activators for AD treatment.

Stenosis of the critical blood vessels, which occurs in a variety of cardiovascular and cerebrovascular diseases, is one of leading causes of death in the world. Vascular stenosis will significantly alter the hemodynamic features in the vessel. Hemodynamic shear stress, one of the most important physical parameters of blood flow, will be dramatically elevated at the stenotic site. When platelets flow through the constricted site, they will sense these abnormally high shear stresses, and then respond by activating, sticking to the vascular wall, and aggregating at these sites. The shear-dependent platelet activation inspired a novel targeting platform-shear stress activated drug targeting delivery. The shear-activated drug delivery systems preferentially release their content under elevated shear stress, providing a novel approach to cure various diseases, in particular, cardiovascular diseases. In this review, we, on one hand, introduced the features of hemodynamic shear stress under both physiological and pathological conditions. On the other hand, we summarized the carriers displaying sensitivity to shear stress, such as liposomes, aggregations, gels, emulsions, in addition to the factors affecting the mechanical properties of them. Lastly, the clinical applications and prospects of this novel drug targeting strategy were discussed. It is hoped that, with a better understanding of shear stress-sensitive carriers and their targeted principle, a novel targeted drug delivery strategy will be one day applied in the clinics of the future.

The first red emission off-on probe, TRFS-red, for thioredoxin reductase was reported. Compared to the previous green emission probe TRFS-green, TRFS-red maintains a high selectivity to the redox enzyme yet with improved response rate and sensitivity.

Mammalian thioredoxin reductase (TrxR) enzymes are homodimeric flavin proteins sharing a unique yet essential selenocysteine residue at their C-terminus. TrxRs, together with their endogenous substrate thioredoxins, play a crucial role in regulating diverse cellular redox events. A wealth of evidence from both clinic observations and bench studies supports that overactivation/dysfunction of TrxRs has a close link to the onset and development of various diseases, such as cancer and neurodegeneration. Thus, an increasing interest has been attracted to find small molecule modulators of TrxRs during the past years. Herein, we briefly discussed the relevance of targeting TrxRs inhibition for cancer treatment, and presented the small molecule inhibitors of mammalian TrxRs published in the nonpatent literatures from 2011 to 2016. The mechanisms of inhibition by different classes of molecules were summarized, and some inhibitors with promising anticancer activity were further discussed. We expect this work would be a comprehensive reference in the medicinal chemistry, and have a broad audience across multiple disciplines.

Several novel series of sesquiterpene mustards (SMs) bearing nitrogen mustard and glutathione (GSH)-reactive α-methylene-γ-butyrolactone groups were successfully prepared for the first time and showed excellent antiproliferative activities in vitro. Among them, compounds 2e and 2g displayed the highest antiproliferative properties with IC50 values ranging from 2.5 to 8.7 μM. The selectivity of these two compounds was evaluated by SRB method against human cancer and normal hepatic cells (HepG2 and L02). The induction of apoptosis and effects on the cell cycle distribution with compounds 2e and 2g were investigated by Hoechst 33,258 staining and flow cytometry, which exhibited that they could induce selective cell apoptosis and cell cycle arrest in HepG2 and L02 cells. In addition, further investigation showed that compounds 2e and 2g could obviously inhibit the proliferation of HepG2 cells by inducing significant DNA cross-linking and depleting GSH in cell media. The good cytotoxicity and selectivity of compounds 2e and 2g pointed them as promising leads for anticancer drug design.

The selenoprotein thioredoxin reductases (TrxRs) are attractive targets for anticancer drugs development. Xanthohumol (Xn), a naturally occurring polyphenol chalcone from hops, has received increasing attention because of its multiple pharmacological activities. We synthesized Xn and its 43 analogues and discovered that compound 13n displayed the highest cytotoxicity toward HeLa cells (IC50 = 1.4 μM). Structure-activity relationship study indicates that the prenyl group is not necessary for cytotoxicity, and introducing electron-withdrawing group, especially on the meta-position, is favored. In addition, methylation of the phenoxyl groups generally improves the potency. Mechanistic study revealed that 13n selectively inhibits TrxR and induces reactive oxygen species and apoptosis in HeLa cells. Cells overexpressing TrxR are resistant to 13n insult, while knockdown of TrxR sensitizes cells to 13n treatment, highlighting the physiological significance of targeting TrxR by 13n. The clarification of the structural determinants for the potency would guide the design of novel potent molecules for future development.

Selenium (Se) is an essential micronutrient element, and the biological significance of Se is predominantly dependent on its incorporation as selenocysteine (Sec), the genetically encoded 21st amino acid in protein synthesis, into the active site of selenoproteins, which have broad functions, ranging from redox regulation and anti-inflammation to the production of active thyroid hormones. Compared to its counterpart Cys, there are only limited probes for selective recognition of Sec, and such selectivity is strictly restricted at low pH conditions. We reported herein the design, synthesis, and biological evaluations of a series of potential Sec probes based on the mechanism of nucleophilic aromatic substitution. After the initial screening, the structural determinants for selective recognition of Sec were recapitulated. The follow-up studies identified that probe 19 (Sel-green) responds to Sec and other selenols with more than 100-fold increase of emission in neutral aqueous solution (pH 7.4), while there is no significant interference from the biological thiols, amines, or alcohols. Sel-green was successfully applied to quantify the Sec content in the selenoenzyme thioredoxin reductase and image endogenous Sec in live HepG2 cells. With the aid of Sel-green, we further demonstrated that the cytotoxicity of different selenocompounds is correlated to their ability metabolizing to selenols in cells. To the best of our knowledge, Sel-green is the first selenol probe that works under physiological conditions. The elucidation of the structure-activity relationship for selective recognition of selenols paves the way for further design of novel probes to better understand the pivotal role of Sec as well as selenoproteins in vivo.

A new chemodosimeter for the highly selective sensing and imaging of biothiols was designed and realized in phosphate-buffered saline solution at pH 7.4 through a fluorescence "off-on" response. A unique mechanism featuring a two-step cascade (biothiols -> H2O) sequence for this remarkable recognition is disclosed for the first time.

The selenoprotein thioredoxin reductase (TrxR) plays a pivotal role in regulating cellular redox homeostasis and has attracted increasing attention as a promising anticancer drug target. We report here that 2-(4-aminophenyl)-1,3,2-dithiarsinane (PAO-PDT, 4), a potent and highly selective small molecule inhibitor of TrxR, stoichiometrically binds to the C-terminal selenocysteine/cysteine pair in the enzyme in vitro and induces oxidative stress-mediated apoptosis in HL-60 cells. The molecular action of 4 in cells involves inhibition of TrxR, elevation of reactive oxygen species, depletion of cellular thiols, and activation of caspase-3. Knockdown of TrxR sensitizes the cells to 4 treatment, whereas overexpression of the functional enzyme alleviates the cytotoxicity, providing physiological relevance for targeting TrxR by 4 in cells. The simplicity of the structure and the presence of an easily manipulated amine group will facilitate the further development of 4 as a potential cancer chemotherapeutic agent.

Shikonin, a major active component of the Chinese herbal plant Lithospermum erythrorhizon, has been applied for centuries in traditional Chinese medicine. Although shikonin demonstrates potent anticancer efficacy in numerous types of human cancer cells, the cellular targets of shikonin have not been fully defined. We report here that shikonin may interact with the cytosolic thioredoxin reductase (TrxR1), an important selenocysteine (Sec)-containing antioxidant enzyme with a C-terminal -Gly-Cys-Sec-Gly active site, to induce reactive oxygen species (ROS)-mediated apoptosis in human promyelocytic leukemia HL-60 cells. Shikonin primarily targets the Sec residue in TrxR1 to inhibit its physiological function, but further shifts the enzyme to an NADPH oxidase to generate superoxide anions, which leads to accumulation of ROS and collapse of the intracellular redox balance. Importantly, overexpression of functional TrxR1 attenuates the cytotoxicity of shikonin, whereas knockdown of TrxR1 sensitizes cells to shikonin treatment. Targeting TrxR1 with shikonin thus discloses a previously unrecognized mechanism underlying the biological activity of shikonin and provides an in-depth insight into the action of shikonin in the treatment of cancer.

Mammalian thioredoxin reductases (TrxRs) are selenocysteine-containing homodimeric flavin enzymes that catalyze the NADPH-dependent reduction of oxidized thioredoxins. Increasing evidence indicates that TrxRs are potential targets for anticancer drug development. This review summarizes patented inhibitors of mammalian TrxRs with an emphasis on those having potential applications in treatment of cancer. Areas covered: A background introduction of TrxR as well as the relevance of TrxR and cancer is provided in the first part of this review. Then, a brief discussion of TrxR assays is followed in the second part. The patented TrxRs' inhibitors that were categorized into four classes, i. e., metal complexes, Michael acceptors, sulfur/selenium-containing compounds and others, are summarized in the third part of the review. Expert opinion: There is currently no clinical anticancer drug that specifically targets TrxR. One major hurdle in finding a successful TrxR inhibitor as a therapeutic drug is the specific inhibition of TrxR by an inhibitor. As most inhibitors described in literature and patents target the selenol group in the C-terminus of TrxR enzymes, it is hard to avoid cross interactions of such inhibitors with thiols. Novel strategies are proposed to achieve discovery of highly selective inhibitors of TrxR enzymes.

The first fluorescent probe for mammalian thioredoxin reductase (TrxR), TRFS-green, was designed, synthesized, and fully evaluated. The probe features a 1,2-dithiolane scaffold with a quenched naphthalimide fluorophore. TRFS-green displays a green fluorescence off-on change induced by the TrxR-mediated disulfide cleavage and subsequent intramolecular cyclization to liberate the masked naphthalimide fluorophore. It was demonstrated in vitro that TRFS-green manifests high selectivity toward TrxR over other related enzymes and various small molecule thiols as well as biological reducing molecules. HPLC analyses indicated that TRFS-green was exclusively converted to naphthalimide catalyzed by TrxR. The ability in triggering on the fluorescence signal by cellular protein extracts correlates well with the endogenous TrxR activity in different cells. Furthermore, inhibition of TrxR by 2,4-dinitrochlorobenzene or depletion of TrxR by immunoprecipitation remarkably decreases the reduction of TRFS-green by cellular protein extracts. Finally, TRFS-green was successfully applied in imaging TrxR activity in living cells. The fluorescence signal of TRFS-green in living cells was inhibited by pretreating the cells with TrxR inhibitor in a dose-dependent manner, potentiating the development of living cell-based screening assay for identifying TrxR inhibitors. We expect the novel fluorescent probe TRFS-green would facilitate the discovery of TrxR-targeting small molecules for potential therapeutic agents and provide significant advances in understanding the physiological/pathophysiological functions of TrxR in vivo.

Thioredoxin reductase (TrxR) and thioredoxin (Trx) are two major components of the thioredoxin system, which plays essential roles in regulating cellular redox signaling. Mammalian TrxRs are essential seleno-flavoenzymes with a conserved penultimate selenocysteine (Sec) residue at the C-terminus, and have attracted considerable interests as promising targets for anticancer drugs. Securinine (SCR), a major active alkaloid lactone from the Chinese herbal medicine Securinega suffruticosa, has been established clinical success in treatment of neurological disorders. Recently, increasing evidence demonstrates that SCR has potential cytotoxicity to various types of tumor cells, which enables this old central nervous system drug as a potential cancer therapeutic agent. However, the mechanism underlying the anticancer activity of SCR is not well defined. We reported here that SCR inhibits both the purified TrxR and the enzyme in intact cells. SCR elicits accumulation of reactive oxygen species (ROS), elevation of oxidized glutathione and Trx, disturbs redox homeostasis, and eventually leads to oxidative stress-mediated HeLa cell apoptosis. Importantly, pharmacological inhibition or knockdown of TrxR sensitizes the cells to SCR treatment, underpinning the physiological significance of targeting TrxR by SCR. Our discovery discloses a novel mechanism underlying the anticancer activity of SCR and provides basic data for further development of SCR as a cancer chemotherapeutic drug.

In a screen for mammalian thioredoxin reductases inhibitors, an MeOH extract from the roots of Saussurea lappa C.B. Clarke (Compositae) inhibited the activity of cytosolic thioredoxin reductase (TrxR1). Bioassay-guided separation of the extract led to the isolation of a new TrxR1 inhibitor, dehydrocostus lactone (DHC), a guaiane-type sesquiterpene. The content of DHC in the extract was determined to be 0.4%. DHC inhibited human cervical carcinoma HeLa cells with an IC50 of ∼12.00 μM but displayed less cytotoxicity to human immortalized normal liver cells L02. We observed that DHC killed HeLa cells through induction of apoptosis. DHC inhibited the activity of TrxR1 in HeLa cells, which elicited an accumulation of reactive oxygen species (ROS) in cells and a collapse of the intracellular redox equilibrium and eventually induced apoptosis of HeLa cells.

Parthenolide (PTL), a major active sesquiterpene lactone from the herbal plant Tanacetum parthenium, has been applied in traditional Chinese medicine for centuries. Although PTL demonstrates potent anticancer efficacy in numerous types of malignant cells, the cellular targets of PTL have not been well defined. We reported here that PTL interacts with both cytosolic thioredoxin reductase (TrxR1) and mitochondrial thioredoxin reductase (TrxR2), two ubiquitous selenocysteine-containing antioxidant enzymes, to elicit reactive oxygen species-mediated apoptosis in HeLa cells. PTL selectively targets the selenocysteine residue in TrxR1 to inhibit the enzyme function, and further shifts the enzyme to an NADPH oxidase to generate superoxide anions, leading to reactive oxygen species accumulation and oxidized thioredoxin. Under the conditions of inhibition of TrxRs in cells, PTL does not cause significant alteration of cellular thiol homeostasis, supporting selective target of TrxRs by PTL. Importantly, overexpression of functional TrxR1 or Trx1 confers protection, whereas knockdown of the enzymes sensitizes cells to PTL treatment. Targeting TrxRs by PTL thus discloses an unprecedented mechanism underlying the biological activity of PTL, and provides deep insights to understand the action of PTL in treatment of cancer.