Combined thioredoxin reductase and glutaminase inhibition exerts synergistic anti-tumor activity in MYC-high high-grade serous ovarian carcinoma.

Approximately 50%-55% of high-grade serous ovarian carcinoma (HGSOC) patients have MYC oncogenic pathway activation. Because MYC is not directly targetable, we have analyzed molecular pathways enriched in MYC-high HGSOC tumors to identify potential therapeutic targets. Here, we report that MYC-high HGSOC tumors show enrichment in genes controlled by NRF2, an antioxidant signaling pathway, along with increased thioredoxin redox activity. Treatment of MYC-high HGSOC tumors cells with US Food and Drug Administration (FDA)-approved thioredoxin reductase 1 (TrxR1) inhibitor auranofin resulted in significant growth suppression and apoptosis in MYC-high HGSOC cells in vitro and also significantly reduced tumor growth in an MYC-high HGSOC patient-derived tumor xenograft. We found that auranofin treatment inhibited glycolysis in MYC-high cells via oxidation-induced GAPDH inhibition. Interestingly, in response to auranofin-induced glycolysis inhibition, MYC-high HGSOC cells switched to glutamine metabolism for survival. Depletion of glutamine with either glutamine starvation or glutaminase (GLS1) inhibitor CB-839 exerted synergistic anti-tumor activity with auranofin in HGSOC cells and OVCAR-8 cell line xenograft. These findings suggest that applying a combined therapy of GLS1 inhibitor and TrxR1 inhibitor could effectively treat MYC-high HGSOC patients.

The Thioredoxin-1 Inhibitor, PX-12, Suppresses Local Osteosarcoma Progression.

BACKGROUND/AIM: The thioredoxin-1 (Trx-1) inhibitor, PX-12, is active against several cancer types. This study aimed to evaluate its effects on local osteosarcoma (OS) progression and to describe PX-12-related signal transduction pathways. MATERIALS AND METHODS: Publicly available expression cohort data were analyzed to determine the relationship between the expression levels of TXN, which codes for the Trx protein, and survival in patients with OS. Murine LM8 OS cells were stimulated with PX-12. Apoptosis-related protein levels, cell viability, caspase activity, and wound healing were evaluated. PX-12 efficacy in suppressing tumor progression was evaluated in C3H mice injected with LM8 cells. RESULTS: High TXN expression was a negative prognostic factor for metastasis and overall survival in OS patients. PX-12 induced apoptosis in OS cells via the oxidative stress-MAPK-caspase 3 pathway and suppressed OS cell migration. PX-12 suppressed local OS progression. CONCLUSION: PX-12 is a potential therapeutic agent for use in suppressing local OS progression.

Inhibition of the PERK/TXNIP/NLRP3 Axis by Baicalin Reduces NLRP3 Inflammasome-Mediated Pyroptosis in Macrophages Infected with Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb) remains a significant threat to global health as it induces granuloma and systemic inflammatory responses during active tuberculosis. Mtb can induce macrophage pyroptosis, leading to the release of IL-1ß and tissue damage, promoting its spread. Here, we established an in vitro Mtb-infected macrophage model to seek an effective antipyroptosis agent. Baicalin, isolated from Radix Scutellariae, was found to reduce pyroptosis in Mtb-infected macrophages. Baicalin could inhibit activation of the PERK/eIF2α pathway and thus downregulates TXNIP expression and subsequently reduces activation of the NLRP3 inflammasome, resulting in reduced pyroptosis in Mtb-infected macrophages. In conclusion, baicalin reduced pyroptosis by inhibiting the PERK/TXNIP/NLRP3 axis and might thus be a new adjuvant host-directed therapy (HDT) drug.

Protective effect of dimethyl itaconate against fibroblast-myofibroblast differentiation during pulmonary fibrosis by inhibiting TXNIP.

Fibroblast-myofibroblast differentiation (FMD) is a critical cellular phenotype during the occurrence and deterioration of pulmonary fibrosis (PF). FMD can increase with an elevated level of reactive oxygen species (ROS) on fibroblasts under oxidative stress. Thioredoxin-interacting protein (TXNIP) is an α-arrestin family protein that regulates the level of intracellular ROS. Nuclear factor erythroid 2-related factor 2 (Nrf2) can protect against FMD in PF. However, the relationship between Nrf2 and TXNIP in FMD remains elusive. Therefore, we established TGF-ß1-induced FMD in vitro and bleomycin (BLM)-induced mouse PF model in vivo to explore whether the activation of Nrf2 can inhibit TXNIP-mediated FMD in PF. Dimethyl itaconate (DMI) was selected to activate Nrf2. Our results showed that TXNIP was elevated and FMD was aggravated in mice lung tissues after BLM administration compared with the saline group. Inversely, Nrf2 decreased TXNIP expression and alleviated FMD in PF. In vitro, TXNIP overexpression enhanced FMD and increased the level of ROS. In contrast, TXNIP deficiency by small interfering RNA (siRNA) attenuated TGF-ß1-induced FMD and reduced ROS. An increase in ROS by H2 O2 can upregulate TXNIP expression. Moreover, Nrf2 also inhibited TGF-ß1-induced FMD and the increase of ROS, with reducing expression of TXNIP, and the inhibitory effect was better than TXNIP siRNA. These results suggest that activation of Nrf2 by DMI can protect against PF via inhibiting TXNIP expression. Our study may provide new therapeutic targets and treatment approaches for PF.

Thioredoxin interacting protein regulates age-associated neuroinflammation.

Immune system hypersensitivity is believed to contribute to mental frailty in the elderly. Solid evidence indicates NOD-like receptor pyrin domain containing-3 (NLRP3)-inflammasome activation intimately connects aging-associated chronic inflammation (inflammaging) to senile cognitive decline. Thioredoxin interacting protein (TXNIP), an inducible protein involved in oxidative stress, is essential for NLRP3 inflammasome activity. This study aims to find whether TXNIP/NLRP3 inflammasome pathway is involved in senile dementia. According to our studies on sex-matched mice, TXNIP was significantly upregulated in aged animals, paralleled by the NLRP3-inflammasome over-activity leading to enhanced caspase-1 cleavage and IL-1ß maturation, in both sexes. This was closely associated with depletion of the anti-aging and cognition enhancing protein klotho, in aged males. Txnip knockout reversed age-related NLRP3-hyperactivity and enhanced thioredoxin (TRX) levels. Further, TXNIP inhibition along with verapamil replicated TXNIP/NLRP3-inflammasome downregulation in aged animals, with FOXO-1 and mTOR upregulation. These alterations concurred with substantial improvements in both cognitive and sensorimotor abilities. Together, these findings substantiate the pivotal role of TXNIP to drive inflammaging in parallel with klotho depletion and functional decline, and delineate thioredoxin system as a potential target to decelerate senile dementia.

Luteolin ameliorates LPS-induced acute liver injury by inhibiting TXNIP-NLRP3 inflammasome in mice.

BACKGROUND: Chemical liver injury is one of the main causes of acute liver failure and death. To date, however, treatment strategies for acute liver injury have been limited. Therefore, there is an urgent need to find new therapeutic targets and effective drugs. NOD-like receptor pyrin domain containing 3 (NLRP3) inflammasome is a complex of multiple proteins that has been shown to induce cell death under inflammatory and stress pathologic conditions and is thought to provide new targets for the treatment of a variety of diseases. PURPOSE: The purpose of this study was to investigate whether luteolin has a protective effect on the liver and further elucidate whether it is realized through the thioredoxin interacting protein (TXNIP)-NLRP3 axis. STUDY DESIGN: Acute hepatic injury in mice caused by intraperitoneal injection of lipopolysaccharide (LPS) was treated with or without luteolin. METHODS: Male C57BL/6 mice and mouse primary hepatocytes were selected. TXNIP protein knockdown was achieved by siRNA, qPCR and Western blot were performed to explore the mechanism of luteolin in alleviating acute liver injury. RESULTS: The results indicated that luteolin had a markedly protective effect on acute liver injury induced by LPS in mice by inhibiting the TXNIP-NLRP3 axis. Luteolin inhibits NLRP3 inflammasome activation by suppressing TXNIP, apoptosis associated speck-like protein containing a CARD domain (ASC), caspase-1, interleukin-1ß (IL-1ß) and IL-18 to reduce liver injury. In addition, luteolin inhibits LPS-induced liver inflammation by inhibiting the production of inflammation-related gene tumor necrosis factor-α (TNF-α), IL-10, and IL-6. What's more, luteolin alleviated LPS-induced hepatocyte injury by inhibiting oxidative stress and regulating MDA, SOD, and GSH levels. However, the protective effect of luteolin on acute LPS-induced liver injury in mice was blocked by si-TXNIP in vitro. CONCLUSIONS: These combined data showed that luteolin may alleviate LPS-induced liver injury through the TXNIP-NLPR3 axis, providing new therapeutic targets and therapeutic drugs for subsequent studies.

Verapamil as an Adjunct Therapy to Reduce tPA Toxicity in Hyperglycemic Stroke: Implication of TXNIP/NLRP3 Inflammasome.

Thrombolytic therapy has remained quite challenging in hyperglycemic patients for its association with poor prognosis and increased hemorrhagic conversions. We recently showed that tissue plasminogen activator (tPA)-induced cerebrovascular damage is associated with thioredoxin-interacting protein (TXNIP) upregulation, which has an established role in the detrimental effects of hyperglycemia. In the present work, we investigated whether verapamil, an established TXNIP inhibitor, may provide protection against hyperglycemic stroke and tPA-induced blood-brain barrier (BBB) disruption. Acute hyperglycemia was induced by intraperitoneal administration of 20% glucose, 15 min prior to transient middle cerebral artery occlusion (tMCAO). Verapamil (0.15 mg/kg) or saline was intravenously infused with tPA at hyperglycemic reperfusion, 1 h post tMCAO. After 24 h of ischemia/reperfusion (I/R), mice were assessed for neurobehavioral deficits followed by sacrifice and evaluation of brain infarct volume, edema, and microbleeding. Alterations in TXNIP, inflammatory mediators, and BBB markers were further analyzed using immunoblotting or immunostaining techniques. As adjunctive therapy, verapamil significantly reduced tPA-induced BBB leakage, matrix metalloproteinase 9 (MMP-9) upregulation, and tight junction protein deregulation, which resulted in lesser hemorrhagic conversions. Importantly, verapamil strongly reversed tPA-induced TXNIP/NLRP3 (NOD-like receptor pyrin domain-containing-3) inflammasome activation and reduced infarct volume. This concurred with a remarkable decrease in high-mobility group box protein 1 (HMGB-1) and nuclear factor kappa B (NF-κB) stimulation, leading to less priming of NLRP3 inflammasome. This preclinical study supports verapamil as a safe adjuvant that may complement thrombolytic therapy by inhibiting TXNIP's detrimental role in hyperglycemic stroke.

Synthesis and biological evaluation of disulfides as anticancer agents with thioredoxin inhibition.

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.

Ligustrazine alleviates cyclophosphamide-induced hepatotoxicity via the inhibition of Txnip/Trx/NF-κB pathway.

AIMS: Cyclophosphamide (CP) is a common therapeutic drug for cancer, but exposure to CP can cause acute hepatotoxicity. This study aimed to elucidate the protective effects of Ligustrazine (2, 3, 5, 6-tetramethylpyrazine, TMP) on hepatotoxicity induced by CP or its active metabolite 4-hydroperoxycyclophosphamide (4-HC). MAIN METHODS: We presented a comprehensive investigation about the hepatoprotection of TMP on CP-induced mice and 4-HC-treated HSC-LX2 cells. Liver function was detected via enzyme-linked immunosorbent assay (ELISA). Hepatic histopathology analysis was performed via hematoxylin and eosin (H&E) and Masson staining. Survival of hepatocytes was detected by TUNEL assay. Related proteins in the thioredoxin (Trx)-interacting protein (Txnip)/Trx/Nuclear factor-kappa B (NF-κB) pathway were measured by western blotting. KEY FINDINGS: The results indicated that CP or 4-HC could increase the levels of alanine aminotransferase and aspartate aminotransferase, enhance inflammatory factors and oxidative indicators, and suppress the activity of oxidoreductases. Moreover, significant changes in liver histological structure, fibrosis, and cell death were observed through the activation of Txnip/Trx/NF-κB pathway. In contrast, administration of TMP significantly reversed these above changes. Furthermore, TMP intervention participated in the inhibition of NLRP3 inflammasome accompanied with pyroptosis, as well as upregulating Trx expression and downregulating p-NF-κB, while the protective effect of TMP was limited to the involvement of Txnip overexpression. SIGNIFICANCE: TMP treatment could significantly alleviate the hepatotoxicity process as evidenced by improving the structure and function of the liver, inhibiting oxidative stress and inflammation accompanied with pyroptosis, which was positively correlated with the inhibition of Txnip/Trx/NF-κB pathway.

Libertellenone H, a Natural Pimarane Diterpenoid, Inhibits Thioredoxin System and Induces ROS-Mediated Apoptosis in Human Pancreatic Cancer Cells.

Libertellenone H (LH), a marine-derived pimarane diterpenoid isolated from arctic fungus Eutypella sp. D-1, has shown effective cytotoxicity on a range of cancer cells. The present study is to explore the anticancer effect of LH on human pancreatic cancer cells and to investigate the intracellular molecular target and underlying mechanism. As shown, LH exhibited anticancer activity in human pancreatic cancer cells by promoting cell apoptosis. Mechanistic studies suggested that LH-induced reactive oxygen species (ROS) accumulation was responsible for apoptosis as antioxidant N-acetylcysteine (NAC) and antioxidant enzyme superoxide dismutase (SOD) antagonized the inhibitory effect of LH. Zymologic testing demonstrated that LH inhibited Trx system but had little effect on the glutathione reductase and glutaredoxin. Mass spectrometry (MS) analysis revealed that the mechanism of action was based on the direct conjugation of LH to the Cys32/Cys35 residue of Trx1 and Sec498 of TrxR, leading to a decrease in the cellular level of glutathione (GSH) and activation of downstream ASK1/JNK signaling pathway. Taken together, our findings revealed LH was a marine derived inhibitor of Trx system and an anticancer candidate.

Targeting antioxidant enzymes enhances the therapeutic efficacy of the BCL-XL inhibitor ABT-263 in KRAS-mutant colorectal cancers.

Cancer chemotherapeutic drugs exert cytotoxic effects by modulating intracellular reactive oxygen species (ROS) levels. However, whether ROS modulates the efficacy of targeted therapeutics remains poorly understood. Previously, we reported that upregulation of the anti-apoptotic protein, BCL-XL, by KRAS activating mutations was a potential target for KRAS-mutant colorectal cancer (CRC) treatment. Here, we demonstrated that the BCL-XL targeting agent, ABT-263, increased intracellular ROS levels and targeting antioxidant pathways augmented the therapeutic efficacy of this BH3 mimetic. ABT-263 induced expression of genes associated with ROS response and increased intracellular ROS levels by enhancing mitochondrial superoxide generation. The superoxide dismutase inhibitor, 2-methoxyestradiol (2-ME), exhibited synergism with ABT-263 in KRAS-mutant CRC cell lines. This synergistic effect was attributed to the inhibition of mTOR-dependent translation of the anti-apoptotic MCL-1 protein via caspase 3-mediated cleavage of AKT and S6K. In addition, combination treatment of ABT-263 and 2-ME demonstrated a synergistic effect in in vivo patient-derived xenografts harboring KRAS mutations. Our data suggest a novel role for ROS in BH3 mimetic-based targeted therapy and provide a novel strategy for treatment of CRC patients with KRAS mutations.

The thioredoxin-1 inhibitor Txnip restrains effector T-cell and germinal center B-cell expansion.

Thioredoxin-1 (Trx1) is a vital component for cellular redox homeostasis. In T cells, Trx1 donates electrons for the de novo synthesis of deoxyribonucleotides to allow rapid cell proliferation. The Trx-interacting protein (Txnip) binds to the reduced Trx1 and inhibits its activity. However, the role of Txnip in adaptive immunity in vivo is unknown. Here, we show that absence of Txnip increased proliferation of effector T cells and GC B-cell responses in response to lymphocytic choriomeningitis virus and Qß virus-like particles, respectively, but did not affect development and homeostasis of T and B cells. While downregulation of Txnip and concomitant upregulation of Trx1 is critical for rapid T-cell expansion upon viral infection, re-expression of Txnip and consequently inhibition of Trx1 is important to restrain late T-cell expansion. Importantly, we demonstrated that T-cell receptor (TCR) engagement but not CD28 costimulation is critically required for Txnip downregulation. Thus, this study further uncovers positive and negative control of lymphocyte proliferation by the Trx1 system.

miR-30c-5p Alleviated Pyroptosis During Sepsis-Induced Acute Kidney Injury via Targeting TXNIP.

Sepsis-induced acute kidney injury (SAKI) is a common complication of hospitalized patients, often leading to unacceptable mortality. Limited effective treatment or diagnosis biomarkers are available and the underlying mechanism remains unclear. The miR-30c-5p is considered as a critical mediator of kidney diseases and aberrantly decreased in patients with SAKI, while the mechanism is still unclear. For this purpose, the role of miR-30c-5p in SAKI has been investigated in this study. Here, we first confirmed that miR-30c-5p expression decreased in our septic models and was associated with the activation of NLRP3/caspase-1-mediated pyroptosis. Overexpression of miR-30c-5p alleviated the kidney injury via suppressing HK-2 cell pyroptosis. Furthermore, we identified that TXNIP was a direct target of miR-30c-5p. Upregulation of miR-30c-5p repressed the expression of TXNIP, which inhibited NLRP3, ASC, and caspase-1 expression, as well as secretion of inflammatory cytokines. In conclusion, our data suggested that miR-30c-5p negatively controlled the NLRP3 signal pathway-related pyroptosis and sepsis-induced injury via TXNIP, indicating that this axis might be a positive therapeutic target for the patient with SAKI.

Thioredoxin-dependent system. Application of inhibitors.

One of the systems responsible for maintaining cellular redox homeostasis is the thioredoxin-dependent system. An equally important function of this system is the regulation of the expression of many proteins by the transcription factor NF-κB or the apoptosis regulating kinase (ASK-1). Since it has been shown that the Trx-dependent system can contribute to both the enhancement of tumour angiogenesis and growth as well as apoptosis of neoplastic cells, the search for compounds that inhibit the level/activity of Trx and/or TrxR and thus modulate the course of the neoplastic process is ongoing. It has been shown that many naturally occurring polyphenolic compounds inactivate elements of the thioredoxin system. In addition, the effectiveness of Trx is inhibited by imidazole derivatives, while the activity of TrxR is reduced by transition metal ions complexes, dinitrohalobenzene derivatives, Michael acceptors, nitrosourea and ebselen. In addition, research is ongoing to identify new selective Trx/TrxR inhibitors.

Electrophilic reactivity of the Busulfan metabolite, EdAG, towards cellular thiols and inhibition of human thioredoxin-1.

Busulfan is an alkylating agent used in chemotherapy conditioning regimens prior to hematopoietic stem cell transplantation (HSCT). However, its administration is associated with a great risk of adverse toxicities, which have been historically attributed to busulfan's mechanism of non-specific DNA alkylation. A phase II generated metabolite of busulfan, EdAG (γ-glutamyldehydroalanylglycine), is a dehydroalanine analog of glutathione (GSH) with an electrophilic moiety, suggesting it may bind to proteins and disrupt biological function. However, EdAG's reactions with common cellular thiols such as glutathione (GSH) and l-cysteine are understudied, along with possible inhibition of glutathionylation-dependent enzymes (with active site cysteine residues). We established a physiologically-relevant in vitro model to readily measure thiol loss over time. Using this model, we compared the apparent rates of thiol depletion in the presence of EdAG or arecoline, a toxic constituent of the areca (betel) nut and known GSH depletor. Simulated kinetic modeling revealed that the mean (±SE) alpha (α) second order rate constants describing GSH and l-cysteine depletion in the presence of EdAG were 0.00522 (0.00845) µM-1∙min-1 and 0.0207 (0.00721) µM-1∙min-1, respectively; in the presence of arecoline, the apparent rates of depletion were 0.0619 (0.009) µM-1∙min-1 and 0.2834 (0.0637) µM-1∙min-1 for GSH and l-cysteine, respectively. Under these experimental conditions, we conclude that EdAG was a weaker electrophile than arecoline. Arecoline and EdAG both depleted apparent l-cysteine concentrations to a much greater extent than GSH, approximately 4.58-fold and 3.97-fold change greater, respectively. EdAG modestly inhibited (∼20%) the human thioredoxin-1 (hTrx-1) catalyzed reduction of insulin with a mean IC50 of 93 µM [95% CI: 78.6-110 µM). In summary, EdAG's ability to spontaneously react with endogenous thiols and inhibit hTrx-1 are potentially biochemically relevant in humans. These findings continue to support the growing concept that EdAG, an underrecognized phase II metabolite of busulfan, plays a role in untoward cellular toxicities during busulfan pharmacotherapy.

Fibroblast-enriched endoplasmic reticulum protein TXNDC5 promotes pulmonary fibrosis by augmenting TGFß signaling through TGFBR1 stabilization.

Pulmonary fibrosis (PF) is a major public health problem with limited therapeutic options. There is a clear need to identify novel mediators of PF to develop effective therapeutics. Here we show that an ER protein disulfide isomerase, thioredoxin domain containing 5 (TXNDC5), is highly upregulated in the lung tissues from both patients with idiopathic pulmonary fibrosis and a mouse model of bleomycin (BLM)-induced PF. Global deletion of Txndc5 markedly reduces the extent of PF and preserves lung function in mice following BLM treatment. Mechanistic investigations demonstrate that TXNDC5 promotes fibrogenesis by enhancing TGFß1 signaling through direct binding with and stabilization of TGFBR1 in lung fibroblasts. Moreover, TGFß1 stimulation is shown to upregulate TXNDC5 via ER stress/ATF6-dependent transcriptional control in lung fibroblasts. Inducing fibroblast-specific deletion of Txndc5 mitigates the progression of BLM-induced PF and lung function deterioration. Targeting TXNDC5, therefore, could be a novel therapeutic approach against PF.

6-Dithio-2'-deoxyguanosine analogs induce reactive oxygen species-mediated tumor cell apoptosis via bi-targeting thioredoxin 1 and telomerase.

Thioredoxin 1 (Trx1) and telomerase play key roles in the development and progression process of most tumors, and they both are promising drug therapy targets. We have, for the first time, discovered that Trx1 and telomerase had a dual-target synergistic effect. Based on that results, we designed a series of 6-dithio-2'-deoxyguanosine analogs (named as YLS00X) and verified whether they can inhibit Trx1 and telomerase simultaneously. TrxR1/Trx1 system activity and telomerase expression were significantly inhibited by 6-dithio-2'-deoxyguanosine analogs, especially YLS004. YLS004 can also cause ROS accumulation, and induce tumor cell apoptosis. The vitro antitumor activity of 6-dithio-2'-deoxyguanosine analogs using MTT assay on 11 different human cancer cells and found that human colon cancer cells(HCT116) and melanoma cells (A375) were the most sensitive cells to 6-dithio-2'-deoxyguanosine analogs treatment and vivo xenografts models also confirmed that. The serum biochemical parameters and multiple organs HE staining results of subacute experiments indicated that YLS004 might be mildly toxic to immune organs, including the thymus, spleen, and hematopoietic system. Besides, YLS004 was rapidly metabolized in the rats' blood. Our study revealed that YLS004, a Trx1 and telomerase inhibitor, has strong anti-tumor effects to colon cancer and melanoma cells and is a promising new candidate drug.

A prodrug of epigallocatechin-3-gallate alleviates high glucose-induced pro-angiogenic factor production by inhibiting the ROS/TXNIP/NLRP3 inflammasome axis in retinal Müller cells.

Diabetic retinopathy (DR) is a neurovascular complication of diabetes mellitus that leads to blindness in the working-age population. Retinal Müller cells proliferate and produce pro-angiogenic factors, including vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF), via the reactive oxygen species (ROS)/thioredoxin interacting protein (TXNIP)/NACHT, LRR and PYD domain-containing protein 3 (NLRP3) inflammasome axis to promote proliferative DR. Epigallocatechin-3-gallate (EGCG) plays anti-oxidant, anti-inflammatory, anti-proliferative and anti-angiogenic roles in Müller cells. A prodrug of EGCG (pro-EGCG) enhances the bioavailability of EGCG. In an in vitro model of high glucose-stimulated Müller cells, pro-EGCG inhibited proliferation and pro-angiogenic factor production by down-regulating the activity of the ROS/TXNIP/NLRP3 inflammasome axis. In a mouse DR model, pro-EGCG reduced ROS accumulation, NLRP3 inflammasome activation, Müller cell proliferation, and production of the pro-angiogenic factors VEGF and HGF. In summary, pro-EGCG mitigated hyperglycaemia-challenged Müller cell proliferation and pro-angiogenic factor production by inhibiting ROS/TXNIP/NLRP3 inflammasome signalling, implying a potential therapeutic strategy for DR.

Citarinostat and Momelotinib co-target HDAC6 and JAK2/STAT3 in lymphoid malignant cell lines: a potential new therapeutic combination.

Histone deacetylase (HDAC) inhibitors represent an encouraging class of antitumor drugs. HDAC inhibitors induce a series of molecular and biological responses and minimal toxicity to normal cells. Citarinostat (Acy-241) is a second generation, orally administered, HDAC6-selective inhibitor. Momelotinib (CYT387) is an orally administered inhibitor of Janus kinase/signal transducer of transcription-3 (JAK/STAT3) signaling. Momelotinib showed efficacy in patients with myelofibrosis. We hypothesized that both HDAC and JAK/STAT pathways were important in lymphoproliferative disorders, and that inhibiting JAK/STAT3 and HDAC simultaneously might enhance the efficacy of momelotinib and citarinostat without increasing toxicity. Accordingly, we tested the citarinostat + momelotinib combination in lymphoid cell lines. Citarinostat + momelotinib showed strong cytotoxicity; it significantly reduced mitochondrial membrane potential, down-regulated Bcl-2 and Bcl-xL, and activated caspases 9 and 3. Caspase-8 was upregulated in only two lymphoid cell lines, which indicated activation of the extrinsic apoptotic pathway. We identified a lymphoid cell line that was only slightly sensitive to the combination treatment. We knocked down thioredoxin expression by transfecting with small interfering RNA that targeted thioredoxin. This knockdown increased cell sensitivity to the combination-induced cell death. The combination treatment reduced Bcl-2 expression, activated caspase 3, and significantly inhibited cell viability and clonogenic survival.

Predicting 19 F†NMR Chemical Shifts: A Combined Computational and Experimental Study of a Trypanosomal Oxidoreductase-Inhibitor Complex.

The absence of fluorine from most biomolecules renders it an excellent probe for NMR spectroscopy to monitor inhibitor-protein interactions. However, predicting the binding mode of a fluorinated ligand from a chemical shift (or vice versa) has been challenging due to the high electron density of the fluorine atom. Nonetheless, reliable 19 F chemical-shift predictions to deduce ligand-binding modes hold great potential for in silico drug design. Herein, we present a systematic QM/MM study to predict the 19 F NMR chemical shifts of a covalently bound fluorinated inhibitor to the essential oxidoreductase tryparedoxin (Tpx) from African trypanosomes, the causative agent of African sleeping sickness. We include many protein-inhibitor conformations as well as monomeric and dimeric inhibitor-protein complexes, thus rendering it the largest computational study on chemical shifts of 19 F nuclei in a biological context to date. Our predicted shifts agree well with those obtained experimentally and pave the way for future work in this area.