Oxidation of methionine residues to methionine sulfoxide (MetSO) may cause changes in protein structure and function, and may eventually lead to cell damage. Methionine sulfoxide reductases (Msrs) are the only known enzymes that catalyze the reduction of MetSO back to methionine by taking reducing equivalents from the thioredoxin system, and thus protect cells from oxidative damage. Nonetheless, a lack of convenient assays for the enzymes hampers the exploration of their functions. We report the discovery of Msr-blue, the first turn-on fluorescent probe for Msr with a >100-fold fluorescence increment from screening a rationally-designed small library. Intensive studies demonstrated the specific reduction of Msr-blue by the enzymes. Msr-blue is ready to determine Msr activity in biological samples and live cells. Importantly, we disclosed a decline of Msr activity in a Parkinson's model, thus providing a mechanistic linkage between the loss of function of Msrs and the development of neurodegeneration. The strategy for the discovery of Msr-blue would also provide guidance for developing novel probes with longer excitation/emission wavelengths and specific probes for Msr isoforms.

Oxidative stress represents a disorder of the redox equilibrium between the production of free radicals and the capability of cells to eliminate them. As subversion of this redox balance is thought to initiate various diseases, living cells maintain a redox equilibrium diligently. More and more pieces of evidence show that oxidative stress has already become a common risk factor in the pathogenesis of neurodegenerative disorders. So, considerable importance has been given to the prevention of oxidative stress as a potential therapeutic strategy. It is well known that the Nrf2-ARE pathway represents one of the most important cellular endogenous defense mechanisms against oxidative stress. Activation of Nrf2 signaling induces the transcriptional regulation of multiple ARE-dependent antioxidant defense genes. Here, we showed that cardamonin (CD), a chalcone isolated from Alpinia katsumadai, attenuated cell death induced by hydrogen peroxide (H2O2) and 6-hydroxydopamine (6-OHDA) in PC12 cells. Pretreatment of PC12 cells with CD dose-dependently upregulated the expression of phase II antioxidant molecules governed by Nrf2. In contrast, CD failed to provide neuroprotection after silencing Nrf2 expression, indicating that this cytoprotection may be mediated by the activation of transcription factor Nrf2. Our results demonstrate that CD is a novel small molecule activator of Nrf2 in PC12 cells, and suggest that CD may be a potential candidate for the prevention of oxidative stress-mediated neurodegenerative disorders.

•A panel of naphthazarin derivatives were synthesized.•Compound 3 (2-methylnaphthazarin) shows highest cytotoxicity to HL-60 cells.•Compound 3 inhibits thioredoxin reductase (TrxR).•Overexpression of TrxR confers protection, while knockdown of TrxR increases toxicity of 3.•Compound 3 induces oxidative stress-mediated apoptosis of HL-60 cells.Mammalian thioredoxin reductase (TrxR) enzymes play a crucial role in regulating multiple redox-based signaling pathways and have attracted increasing attention as promising anticancer drug targets. We report here the synthesis of a panel of naphthazarin derivatives and discovery of 2-methyl-5,8-dihydroxy-1,4-naphthoquinone (3, 2-methylnaphthazarin) as a potent cytotoxic agent with a submicromolar half maximal inhibitory concentration to the human promyelocytic leukemia HL-60 cells. Mechanism studies reveal that the compound selectively inhibits TrxR to induce oxidative stress-mediated apoptosis of HL-60 cells. Knockdown of TrxR sensitizes the cells to 3 insults, while overexpression of the functional enzyme confers resistance to the compound treatment, underpinning the physiological significance of targeting TrxR by 3. Clarification of the interaction of compound 3 with TrxR unveils a mechanism underlying the cellular action of the compound, and sheds light in considering development of the compound as a potential cancer chemotherapeutic agent.Download high-res image (341KB)Download full-size image

Thioredoxin (Trx) and thioredoxin reductase (TrxR) are essential components of the Trx system which plays pivotal roles in regulating multiple cellular redox signaling pathways. In recent years TrxR/Trx have been increasingly recognized as an important modulator of tumor development, and hence targeting TrxR/Trx is a promising strategy for cancer treatment. In this review we first discuss the structural details of TrxR, the functions of the Trx system, and the rational of targeting TrxR/Trx for cancer treatment. We also highlight small-molecule TrxR/Trx inhibitors that have potential anticancer activity and review their mechanisms of action. Finally, we examine the challenges of developing TrxR/Trx inhibitors as anticancer agents and perspectives for selectively targeting TrxR/Trx.

The first off–on probe, Mito-TRFS, for imaging the mitochondrial thioredoxin reductase (TrxR2) in live cells was reported. In a cellular model of Parkinson's disease (PD), Mito-TRFS staining discloses a drastic decline of the TrxR2 activity, providing a mechanistic link of TrxR2 dysfunction to the etiology of PD.

Reversible thiol modifications are fundamental of cellular redox regulation. Specific thiol detection, including thiol sensing and protein thiols labeling, is critical to study such modifications. We reported the discovery of 4-methylsulfonyl-N-n-butyl-1,8-naphthalimide (MSBN), a highly selective fluorogenic probe for thiols based on the 1,8-naphthalimide scaffold. Thiols react with MSBN nearly quantitatively via nucleophilic aromatic substitution to replace the methylsulfonyl group and restore the quenched fluorescence (>100-fold increase). MSBN was employed to selectively image thiols in live cells and specifically label protein thiols with a turn-on signal to determine diverse reversible protein thiol modifications. In addition, we introduced a bulky group into the MSBN as a mass tag to create a probe MSBN-TPP, which readily discriminates the reduced thioredoxin from the oxidized one. The specific reaction of MSBN with thiols and the easy manipulation of the naphthalimide unit enable MSBN a versatile scaffold in developing novel probes for thiol-based protein bioconjugation and studying various thiol modifications.

A novel turn-on type of ultrafast biothiol fluorescent probe, Naph-EA-mal, was designed, synthesized and evaluated. The probe contains a naphthalimide moiety as a fluorophore, a maleimide unit as a thiol acceptor, and 1,2-ethylenediamine as a linker. Naph-EA-mal displays high selectivity and a fast response toward thiols in aqueous solution. The reaction mechanism of the probe with thiols was confirmed by 1H NMR and HRMS. Test strips were fabricated and a sharp color change was observed by the naked-eye. Furthermore, Naph-EA-mal was successfully applied to label protein thiols, image thiols in living cells, quantify thiol content in cells lysate, and determine the reversible protein thiols oxidation in fixed cells.

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.

The first thiol-specific turn-on probe, BODIPY-TS, utilizing a thiosulfonate scaffold as the thiol recognition unit was reported. BODIPY-TS displays low toxicity, and features high sensitivity, fast response and quantitative reaction towards thiols. The structural novelty of BODIPY-TS would guide the development of novel thiol probes.

The cellular antioxidant system plays key roles in blocking or retarding the pathogenesis of adult neurodegenerative disorders as elevated oxidative stress has been implicated in the pathophysiology of such diseases. Molecules with the ability in enhancing the antioxidant defense thus are promising candidates as neuroprotective agents. We reported herein the synthesis of piperlongumine analogues and evaluation of their cytoprotection against hydrogen peroxide- and 6-hydroxydopamine-induced neuronal cell oxidative damage in the neuron-like PC12 cells. The structure–activity relationship was delineated after the cytotoxicity and protection screening. Two compounds (4 and 5) displayed low cytotoxicity and confer potent protection of PC12 cells from the oxidative injury via upregulation of a panel of cellular antioxidant molecules. Genetically silencing the transcription factor Nrf2, a master regulator of the cellular stress responses, suppresses the cytoprotection, indicating the critical involvement of Nrf2 for the cellular action of compounds 4 and 5 in PC12 cells.

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.

Hydroxytyrosol (3,4-dihydroxyphenylethanol, HT), a major polyphenol in olive oils, has received increasing attention due to its multiple pharmacological activities. However, it is not well understood how HT works on the neuronal system. We report herein that HT efficiently scavenges free radicals in vitro and displays cytoprotection against oxidative stress-induced damage in PC12 cells. HT completely protects the cells from hydrogen peroxide-induced death and rescues the cells from 6-hydroxydopamine-induced damage. Mechanistic studies reveal that Nrf2 is a prerequisite for the neuroprotection of HT as knocking down Nrf2 eliminated this action. HT, via activation of the Keap1-Nrf2 pathway, elevates a panel of cytoprotective enzymes, including glutamate–cysteine ligase, HO-1, NQO1 and thioredoxin reductase. Our study reveals that HT provides dual neuroprotection and cellular antioxidant defense as both a free radical scavenger and Nrf2 activator, suggesting the potential pharmaceutical usage of HT for the treatment of neurodegenerative disorders.

Natural compounds containing phenoxyl groups and/or Michael acceptor units appear to possess antioxidant and cytoprotective properties. The ginger principal constituent 6-shogaol (6-S) represents one of such compounds. In this study, we reported that 6-S efficiently scavenges various free radicals in vitro, and displays remarkable cytoprotection against oxidative stress-induced cell damage in the neuron-like rat pheochromocytoma cell line, PC12 cells. Pretreatment of PC12 cells with 6-S significantly upregulates a series of phase II antioxidant molecules, such as glutathione, heme oxygenase 1, NAD(P)H: quinone oxidoreductase 1, thioredoxin reductase 1, and thioredoxin 1. A mechanistic study revealed that 6-S enhanced the translocation of Nrf2 from the cytosol to the nucleus and knockdown of Nrf2 abolished such protection, indicating that this cytoprotection is mediated by the activation of the transcription factor Nrf2. Another ginger constituent 6-gingerol (6-G), having a similar structure of 6-S but lacking the alpha,beta-unsaturated ketone structure (Michael acceptor moiety), failed to shelter PC12 cells from oxidative stress. Our results demonstrate that 6-S is a novel small molecule activator of Nrf2 in PC12 cells, and suggest that 6-S might be a potential candidate for the prevention of oxidative stress-mediated neurodegenerative disorders.

Xanthohumol (2′,4′,4-trihydroxy-6′-methoxy-3′-prenylchalcone, Xn), a polyphenol chalcone from hops (Humulus lupulus), has received increasing attention due to its multiple pharmacological activities. As an active component in beers, its presence has been suggested to be linked to the epidemiological observation of the beneficial effect of regular beer drinking. In this work, we synthesized Xn with a total yield of 5.0% in seven steps and studied its neuroprotective function against oxidative-stress-induced neuronal cell damage in the neuronlike rat pheochromocytoma cell line PC12. Xn displays moderate free-radical-scavenging capacity in vitro. More importantly, pretreatment of PC12 cells with Xn at submicromolar concentrations significantly upregulates a panel of phase II cytoprotective genes as well as the corresponding gene products, such as glutathione, heme oxygenase, NAD(P)H:quinone oxidoreductase, thioredoxin, and thioredoxin reductase. A mechanistic study indicates that the α,β-unsaturated ketone structure in Xn and activation of the transcription factor Nrf2 are key determinants for the cytoprotection of Xn. Targeting the Nrf2 by Xn discloses a previously unrecognized mechanism underlying the biological action of Xn. Our results demonstrate that Xn is a novel small-molecule activator of Nrf2 in neuronal cells and suggest that Xn might be a potential candidate for the prevention of neurodegenerative disorders.

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.

The cellular endogenous antioxidant system plays pivotal roles in counteracting or retarding the pathogenesis of many neurodegenerative diseases. Molecules with the ability to enhance the antioxidant defense thus are promising candidates for neuroprotective drugs. 6-Dehydrogingerdione (6-DG), one of the major components of dietary ginger, has received increasing attention due to its multiple pharmacological activities. However, how this pleiotropic molecule works on the neuronal system has not been studied. This paper reports that 6-DG efficiently scavenges various free radicals in vitro and displays remarkable cytoprotection against oxidative stress-induced neuronal cell damage in the neuron-like rat pheochromocytoma cell line, PC12 cells. Pretreatment of PC12 cells with 6-DG significantly up-regulates a panel of phase II genes as well as the corresponding gene products, such as glutathione, heme oxygenase, NAD(P)H:quinone oxidoreductase, and thioredoxin reductase. Mechanistic study indicates that activation of the Keap1-Nrf2-ARE pathway is the molecular basis for the cytoprotection of 6-DG. This is the first revelation of this novel mechanism of 6-DG as an Nrf2 activator against oxidative injury, providing the potential therapeutic use of 6-DG as neuroprotective agent.

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.

PMQA, an 8-aminoquinoline-based ratiometric fluorescent sensor, demonstrates the Zn2+-induced red-shift of emission (85 nm), and was successfully applied to image zinc in living cells. Compared to 2:1 stoichiometry in PMQA–Zn2+, PMQA–Cu2+ shows 1:1 composition. Both nitrogen atoms from the aminoquinoline are missing in binding of zinc, while they are critically involved in Cu2+ chelation. The structure difference between PMQA–Zn2+ and PMQA–Cu2+ might shed light in designing novel zinc probes without suffering from copper interference.

•The probe DOQED selectively binds the Cu2+ to quench the intrinsic fluorescence.•The X-ray crystallography of DOQED-Cu2+ complex confirms the binding mode.•The S2– replaces the Cu2+ from DOQED-Cu2+ and recovers the quenched fluorescence.•DOQED has potential applications in detecting Cu2+ and S2– in biological samples.Diethyl 2,2′-(2-oxo-2-(quinolin-8-ylamino)ethylazanediyl) diacetate (DOQED), an aminoquinoline-based fluorescent probe, was synthesized, and successfully applied in detection of Cu2 + and S2 − in an aqueous buffered solution. The emission signal of DOQED was selectively quenched by Cu2 +, and was exclusively recovered by subsequent addition of S2 −. The formation of a [Cu(DOQED)]2 + complex was confirmed by X-ray crystallography.The aminoquinoline-based probe DOQED displays good selectivity and sensitivity in detection of Cu2+ and S2– in an aqueous solution. The emission signal of DOQED was selectively quenched by Cu2+, and exclusively recovered by the addition of S2–. The formation of a Cu-DOQED complex was confirmed by X-ray crystallography.

•Alantolactone (ATL) inhibits thioredoxin reductase (TrxR).•ATL induces generation of reactive oxygen species in HeLa cells.•ATL causes accumulation of the oxidized thioredoxin in cells.•Knockdown of TrxR sensitizes cells to ATL treatment.•ATL elicits oxidative stress-mediated apoptosis in HeLa cells.The mammalian thioredoxin reductase (TrxR) isoenzymes, TrxR1 in cytosol or nucleus, TrxR2 in mitochondria, and TrxR3 in testis, are essential seleno-flavoenzymes with a conserved penultimate selenocysteine (Sec) residue at the C-terminus, and have attracted increasing interests as potential targets for development of cancer chemotherapeutic agents. The sesquiterpene lactone alantolactone (ATL), an active component from the traditional folk medicine Inula helenium, has been documented possessing multiple pharmacological functions, especially the anticancer activity. However, the underlying mechanism has not been well defined. We reported that ATL inhibits both the recombinant TrxR and the enzyme in the cellular environment. The alpha-methylene-gamma-lactone moiety in ATL and the Sec residue in TrxR are critical for targeting TrxR by ATL. By employing our newly developed pull down assay, we demonstrated the remarkable elevation of the oxidized thioredoxin in HeLa cells after ATL treatment. In addition, ATL elicits accumulation of reactive oxygen species, and eventually induces apoptosis of HeLa cells. Importantly, overexpression of the functional TrxR attenuates the cytotoxicity of ATL, while knockdown of the enzyme sensitizes the cells to ATL treatment. Targeting TrxR thus discloses a novel molecular mechanism underlying the cellular action of ATL, and sheds light in considering the usage of ATL as a potential cancer chemotherapeutic agent.

•DHC inhibited thioredoxin reductase (TrxR1).•DHC treatment elicited accumulation of ROS.•DHC induced oxidative stress-mediated apoptosis in HeLa cells.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.Download high-res image (188KB)Download full-size image

•Plumbagin (PLB) selectively kills human promyelocytic leukemia HL-60 cells.•Inhibition of thioredoxin reductase (TrxR) by PLB contributes to its cytotoxicity.•PLB causes elevation of ROS and disruption of cellular redox balance.•PLB induces oxidative stress-mediated apoptosis of HL-60 cells.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.Download high-res image (351KB)Download full-size image

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.

Oxidation of methionine residues to methionine sulfoxide (MetSO) may cause changes in protein structure and function, and may eventually lead to cell damage. Methionine sulfoxide reductases (Msrs) are the only known enzymes that catalyze the reduction of MetSO back to methionine by taking reducing equivalents from the thioredoxin system, and thus protect cells from oxidative damage. Nonetheless, a lack of convenient assays for the enzymes hampers the exploration of their functions. We report the discovery of Msr-blue, the first turn-on fluorescent probe for Msr with a >100-fold fluorescence increment from screening a rationally-designed small library. Intensive studies demonstrated the specific reduction of Msr-blue by the enzymes. Msr-blue is ready to determine Msr activity in biological samples and live cells. Importantly, we disclosed a decline of Msr activity in a Parkinson's model, thus providing a mechanistic linkage between the loss of function of Msrs and the development of neurodegeneration. The strategy for the discovery of Msr-blue would also provide guidance for developing novel probes with longer excitation/emission wavelengths and specific probes for Msr isoforms.

The first thiol-specific turn-on probe, BODIPY-TS, utilizing a thiosulfonate scaffold as the thiol recognition unit was reported. BODIPY-TS displays low toxicity, and features high sensitivity, fast response and quantitative reaction towards thiols. The structural novelty of BODIPY-TS would guide the development of novel thiol probes.

The first off–on probe, Mito-TRFS, for imaging the mitochondrial thioredoxin reductase (TrxR2) in live cells was reported. In a cellular model of Parkinson's disease (PD), Mito-TRFS staining discloses a drastic decline of the TrxR2 activity, providing a mechanistic link of TrxR2 dysfunction to the etiology of PD.