Marine-Fungi-Derived Gliotoxin Promotes Autophagy to Suppress Mycobacteria tuberculosis Infection in Macrophage.

The Mycobacterium tuberculosis (MTB) infection causes tuberculosis (TB) and has been a long-standing public-health threat. It is urgent that we discover novel antitubercular agents to manage the increased incidence of multidrug-resistant (MDR) or extensively drug-resistant (XDR) strains of MTB and tackle the adverse effects of the first- and second-line antitubercular drugs. We previously found that gliotoxin (1), 12, 13-dihydroxy-fumitremorgin C (2), and helvolic acid (3) from the cultures of a deep-sea-derived fungus, Aspergillus sp. SCSIO Ind09F01, showed direct anti-TB effects. As macrophages represent the first line of the host defense system against a mycobacteria infection, here we showed that the gliotoxin exerted potent anti-tuberculosis effects in human THP-1-derived macrophages and mouse-macrophage-leukemia cell line RAW 264.7, using CFU assay and laser confocal scanning microscope analysis. Mechanistically, gliotoxin apparently increased the ratio of LC3-II/LC3-I and Atg5 expression, but did not influence macrophage polarization, IL-1ß, TNF-a, IL-10 production upon MTB infection, or ROS generation. Further study revealed that 3-MA could suppress gliotoxin-promoted autophagy and restore gliotoxin-inhibited MTB infection, indicating that gliotoxin-inhibited MTB infection can be treated through autophagy in macrophages. Therefore, we propose that marine fungi-derived gliotoxin holds the promise for the development of novel drugs for TB therapy.

Quercetin alleviates gliotoxin-induced duckling tissue injury by inhibiting oxidative stress, inflammation and increasing heterophil extracellular traps release.

Aspergillus fumigatus causes aspergillosis with high morbidity and mortality in the duck industry. As a vital virulence factor produced by A. fumigatus, gliotoxin (GT) is widely present in food and feed, threatening duck industry and human health. Quercetin is a polyphenol flavonoid compound from natural plants with anti-inflammatory and antioxidant functions. However, the effects of quercetin on ducklings with GT poisoning are unknown. The model of ducklings with GT poisoning was established, and the protective effects and molecular mechanisms of quercetin on ducklings with GT poisoning were investigated. Ducklings were divided into control, GT, and quercetin groups. A model of GT (2.5 mg/kg) poisoning in ducklings was successfully established. Quercetin protected GT-induced liver and kidney functions and alleviated GT-induced alveolar wall thickening in lungs, cell fragmentation, and inflammatory cell infiltration in liver and kidney. Quercetin decreased malondialdehyde (MDA) and increased superoxide dismutase (SOD) and catalase (CAT) after GT treatment. Quercetin significantly reduced GT-induced mRNA expression levels of inflammatory factors. Furthermore, quercetin increased GT-reduced heterophil extracellular traps (HETs) in serum. These results indicated that quercetin protected ducklings against GT poisoning by inhibiting oxidative stress, inflammation and increasing HETs release, which confirms the potential applicability of quercetin in treating GT-induced duckling poisoning.

Gliotoxin, a natural product with ferroptosis inducing properties.

As one of the mycotoxins produced by Aspergillus fumigatus, gliotoxin has a variety of pharmacological effects, such as anti-tumor, antibacterial, immunosuppressive. Antitumor drugs induce tumor cell death in several forms, including apoptosis, autophagy, necrosis and ferroptosis. Ferroptosis is a recently identified unique form of programmed cell death characterized by iron-dependent accumulation of lethal lipid peroxides, which induces cell death. A large amount of preclinical evidence suggests that ferroptosis inducers may enhance the sensitivity of chemotherapy and the induction of ferroptosis may be an effective therapeutic strategy to prevent acquired drug resistance. In our study, gliotoxin was characterized as a ferroptosis inducer and showed strong anti-tumor activity with IC50 of 0.24 µM and 0.45 µM in H1975 and MCF-7 cells at 72 h, respectively. Gliotoxin may provide a new natural template for the designing of ferroptosis inducers.

6-Heterocyclic carboxylic ester derivatives of gliotoxin lead to LSD1 inhibitors in gastric cancer cells.

Gliotoxin is a representative compound of the epipolythiodioxopiperazine (ETP) class of fungal metabolites. Histone Lysine Specific Demethylase 1 (LSD1) is highly expressed in a variety of cancers. Herein, a series of 6-heterocyclic carboxylic ester derivatives of gliotoxin was designed and synthesized as new LSD1 inhibitors and their biological evaluations in human gastric MGC-803 and HGC-27 cells were carried out. All of the derivatives effectively suppressed the enzymatic activities of LSD1. In particular, compound 4e exhibited excellent LSD1 inhibition with IC50 = 62.40 nM, as well as anti-proliferation against MGC-803 and HGC-27 cells with IC50 values of 0.31 µM and 0.29 µM, respectively. 4e also had a remarkable capacity to inhibit the colony formation, suppress migration and induce the apoptosis of these two cancer cell lines. In sum, our findings identified and characterized the 6-heterocyclic carboxylic ester derivatives of gliotoxin as potent and cellular active LSD1 inhibitors, which may provide a novel chemotype of LSD1 inhibitors for gastric cancer treatment.

Gliotoxin Induced Ferroptosis by Downregulating SUV39H1 Expression in Esophageal Cancer Cells.

BACKGROUND: Gliotoxin, a secondary metabolite isolated from marine-derived Aspergillus fumigatus, has demonstrated anti-tumor properties in several cancers. Ferroptosis, a recently discovered type of programmed cell death that depends on the accumulation of iron and lipid peroxides, participates in the occurrence and development of various diseases, including cancer. A recent patent, US20200383943, has suggested that the promotion of ferroptosis is a method of cancer treatment. Therefore, the development of drugs that induce ferroptosis in cancer cells would constitute a novel therapeutic approach. OBJECTIVE: Gliotoxin is a natural compound which has exhibited anti-tumor properties in multiple cancers, however, studies of the effect of gliotoxin on esophageal cancer are lacking. Although cancer treatment has shown great progress, including traditional surgery, chemotherapy, radiotherapy, and immunotherapy, the prognosis of esophageal cancer is still poor. Therefore, the development of new treatment approaches for esophageal cancer is necessary. METHODS: The effects of gliotoxin on esophageal cancer cells were determined by functional assays, such as CCK-8, wound healing and transwell assays. We used online tools to predict the target genes of gliotoxin, followed by further verification using Western blotting assays. To assess the role of gliotxin in inducing ferroptosis in esophageal cancer, we detected characteristics associated with ferroptosis including ROS, MDA, GSH and Fe2+. RESULTS: Using online tools SEA and SwissTargetPrediction, we predicted that SUV39H1 was the gliotoxin target gene. Furthermore, in esophageal cancer tissues, SUV39H1 was expressed at higher levels than in normal tissues, while in patients with Esophageal Squamous Cell Carcinoma (ESCC), high expression levels of SUV39H1 indicated a poor prognosis. In vitro, we observed that gliotoxin increased ESCC cell death and inhibited cell migration. We treated ESCC cells with pan-caspase inhibitor Z-VAD-FMK or ferroptosis inhibitors, including Fer-1 and DFO. Our results showed that Fer-1 and DFO reduced the toxic effects of gliotoxin, while Z-VAD-FMK did not. Furthermore, gliotoxin treatment reduced tumor weight and volume in the xenograft tumor mouse model. CONCLUSION: In summary, our findings indicate that gliotoxin downregulated SUV39H1 expression in ESCC cells and induced ferroptosis, suggesting a novel natural therapy for ESSC.

Evaluation of kinetic effects of Gliotoxin in different breast cancer cell lines.

In the present research, the antiproliferative properties of Gliotoxin, which is obtained from marine fungus and thought to be a promising metabolite, on MCF-7 and MDA-MB-231 breast cancer cells, which have different molecular subtypes, were evaluated. Different cell kinetic parameters were employed for this aim. In experiments, cell viability, cell index, mitotic index, BrdU labeling index, and apoptotic index were assessed. Gliotoxin concentrations of 1.5625 µM, 3.125 µM, and 6.25 µM were used in studies for both cell lines. As a result of the values obtained from cell viability and xCELLigence Real-Time Cell Analysis (RTCA) System, 1.5625 µM concentration was determined as IC50 dose. This concentration was applied to all other parameters and anticancer activities were observed.

Julichrome derivatives and gliotoxin from a soil derived Streptomyces sp.

Six julichrome derivatives including a new monomeric julichrome named as julichrome Q10 (1), and previous reported julichrome Q6 (2), julichrome Q6.6 (4), julichrome Q3.5 (5), julichrome Q5.6 (6), julichrome Q2.3 (7), along with a diketopiperazine gliotoxin (3) were isolated from a soil derived strain Streptomyces sp. The structures of these compounds were identified by HR-ESI-MS, UV, IR and NMR methods. The isolated compounds were tested for their in vitro cytotoxicity against human hepatocarcinoma HepG-2 and SMMC-7721 cell lines, human breast cancer MCF-7 and MDA-MB-231 cell lines, and human normal heptical LO2 cell line. Gliotoxin (3) showed the most cytotoxic activity against the tested tumor cell lines, with IC50 values ranging from 0.11 to 1.45 µM. Julichrome Q6.6 (4) displayed selective cytotoxic activity against SMMC-7721, MCF-7 and MDA-MB-231 cell lines.

Identification of Gliotoxin isolated from marine fungus as a new pyruvate kinase M2 inhibitor.

Pyruvate kinase M2 (PKM2) functions as an important rate-limiting enzyme of aerobic glycolysis that is involved in tumor initiation and progression. However, there are few studies on effective PKM2 inhibitors. Gliotoxin is a marine-derived fungal secondary metabolite with multiple biological activities, including immunosuppression, cytotoxicity, and et al. In this study, we found that Gliotoxin directly bound to PKM2 and inhibited its glycolytic activity in a dose-dependent manner accompanied by the decreases in glucose consumption and lactate production in the human glioma cell line U87. Moreover, Gliotoxin suppressed tyrosine kinase activity of PKM2, leading to a dramatic reduction in Stat3 phosphorylation in U87 cells. Furthermore, Gliotoxin suppressed cell viability in U87 cells, and cytotoxicity of Gliotoxin on U87 cells was obviously augmented under hypoxia condition compared to normal condition. Finally, Gliotoxin was demonstrated to induce cell apoptosis of U87 cells and synergize with temozolomide. Our findings identify Gliotoxin as a new PKM2 inhibitor with anti-tumor activity, which lays the foundation for the development of Gliotoxin as a promising anti-tumor drug in the future.

Targeting Nuclear NOTCH2 by Gliotoxin Recovers a Tumor-Suppressor NOTCH3 Activity in CLL.

NOTCH signaling represents a promising therapeutic target in chronic lymphocytic leukemia (CLL). We compared the anti-neoplastic effects of the nuclear NOTCH2 inhibitor gliotoxin and the pan-NOTCH γ-secretase inhibitor RO4929097 in primary CLL cells with special emphasis on the individual roles of the different NOTCH receptors. Gliotoxin rapidly induced apoptosis in all CLL cases tested, whereas RO4929097 exerted a variable and delayed effect on CLL cell viability. Gliotoxin-induced apoptosis was associated with inhibition of the NOTCH2/FCER2 (CD23) axis together with concomitant upregulation of the NOTCH3/NR4A1 axis. In contrast, RO4929097 downregulated the NOTCH3/NR4A1 axis and counteracted the spontaneous and gliotoxin-induced apoptosis. On the cell surface, NOTCH3 and CD23 expression were mutually exclusive, suggesting that downregulation of NOTCH2 signaling is a prerequisite for NOTCH3 expression in CLL cells. ATAC-seq confirmed that gliotoxin targeted the canonical NOTCH signaling, as indicated by the loss of chromatin accessibility at the potential NOTCH/CSL site containing the gene regulatory elements. This was accompanied by a gain in accessibility at the NR4A1, NFκB, and ATF3 motifs close to the genes involved in B-cell activation, differentiation, and apoptosis. In summary, these data show that gliotoxin recovers a non-canonical tumor-suppressing NOTCH3 activity, indicating that nuclear NOTCH2 inhibitors might be beneficial compared to pan-NOTCH inhibitors in the treatment of CLL.

Dechdigliotoxins A-C, Three Novel Disulfide-Bridged Gliotoxin Dimers from Deep-Sea Sediment Derived Fungus Dichotomomyces cejpii.

Dechdigliotoxins A-C (1-3), which represented the first examples of gliotoxin dimers with an unprecedented exocyclic disulfide linkage, were obtained from a deep-sea derived fungus Dichotomomyces cejpii FS110. The structures of these compounds were elucidated on the basis of spectroscopic analysis and the absolute configurations were unambiguously determined through quantum chemical calculations, as well as DP4+ probability simulations. The proposed biosynthetic pathway suggested 1-3 were generated from unusual L-Phe and D-Ser. All the isolates were evaluated for their cytotoxicity against four tumor cell lines.

Gliotoxin Enhances Autophagic Cell Death via the DAPK1-TAp63 Signaling Pathway in Paclitaxel-Resistant Ovarian Cancer Cells.

Death-associated protein kinase 1 (DAPK1) expression induced by diverse death stimuli mediates apoptotic activity in various cancers, including ovarian cancer. In addition, mutual interaction between the tumor suppressor p53 and DAPK1 influences survival and death in several cancer cell lines. However, the exact role and connection of DAPK1 and p53 family proteins (p53, p63, and p73) in drug-resistant ovarian cancer cells have not been studied previously. In this study, we investigated whether DAPK1 induction by gliotoxin derived from marine fungus regulates the level of transcriptionally active p63 (TAp63) to promote apoptosis in an autophagy-dependent manner. Pre-exposure of paclitaxel-resistant ovarian cancer cells to gliotoxin inhibited the expression of multidrug resistant-associated proteins (MDR1 and MRP1-3), disrupted the mitochondrial membrane potential, and induced caspase-dependent apoptosis through autophagy induction after subsequent treatment with paclitaxel. Gene silencing of DAPK1 prevented TAp63-mediated downregulation of MDR1 and MRP1-3 and autophagic cell death after sequential treatment with gliotoxin and then paclitaxel. However, pretreatment with 3-methyladenine (3-MA), an autophagy inhibitor, had no effect on the levels of DAPK1 and TAp63 or on the inhibition of MDR1 and MRP1-3. These results suggest that DAPK1-mediated TAp63 upregulation is one of the critical pathways that induce apoptosis in chemoresistant cancer cells.

Effects of the Combination of Gliotoxin and Adriamycin on the Adriamycin-Resistant Non-Small-Cell Lung Cancer A549 Cell Line.

Acquired drug resistance constitutes an enormous hurdle in cancer treatment, and the search for effective compounds against resistant cancer is still advancing. Marine organisms are a promising natural resource for the discovery and development of anticancer agents. In this study, we examined whether gliotoxin (GTX), a secondary metabolite isolated from marine-derived Aspergillus fumigatus, inhibits the growth of adriamycin (ADR)-resistant non-small-cell lung cancer (NSCLC) cell lines A549/ADR. We investigated the effects of GTX on A549/ADR cell viability with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and the induction of apoptosis in A549/ADR cells treated with GTX via fluorescence-activated cell sorting analysis, Hoechst staining, annexin V/propidium iodide staining, tetraethylbenzimidazolylcarbocyanine iodide (JC-1) staining, and western blotting. We found that GTX induced apoptosis in A549/ADR cells through the mitochondria-dependent pathway by disrupting mitochondrial membrane potential and activating p53, thereby increasing the expression levels of p21, p53 upregulated modulator of apoptosis (PUMA), Bax, cleaved poly (ADP-ribose) polymerase (PARP), and cleaved caspase-9. More importantly, we discovered that GTX works in conjunction with ADR to exert combinational effects on A549/ADR cells. In conclusion, our results suggest that GTX may have promising effects on ADR-resistant NSCLC cells by inducing mitochondria-dependent apoptosis and through the combined effects of sequential treatment with ADR.

Reduced-gliotoxin induces ROS-mediated anoikis in human colorectal cancer cells.

Reduced-gliotoxin is a small molecule derived from the secondary metabolites of marine fungi; compared to other gliotoxin analogues, it exhibits potent anticancer effects. However, the molecular basis of the death of colorectal cancer (CRC) cells induced by reduced-gliotoxin is unclear. Thus, the aim of this study was to investigate the potency of reduced-gliotoxin against CRC cells and to elucidate the underlying mechanisms. Cell morphology, flow cytometric analysis and western bolt analysis were performed to examine the functions and mechanisms of cell death induced by reduced-gliotoxin. Our findings demonstrated that reduced-gliotoxin triggered rapid cell detachment and induced anoikis in CRC cells. Mechanistically, our data indicated that the anoikis induced by reduced-gliotoxin was associated with the disruption of integrin-associated cell detachment and multiple signaling pathways. Furthermore, reduced-gliotoxin induced the excessive production of reactive oxygen species (ROS) and the disruption of mitochondrial membrane potential (MMP), resulting in the activation of both endogenous and exogenous apoptotic pathways and eventually, in the apoptosis of CRC cells. The blockage of ROS generation with N-acetylcysteine (NAC) attenuated the anoikis induced by reduced-gliotoxin. Taken together, these results suggest that reduced-gliotoxin may prove to be a potential candidate in the treatment of CRC.

KIF1Bß increases ROS to mediate apoptosis and reinforces its protein expression through O 2- in a positive feedback mechanism in neuroblastoma.

Relapse-prone, poor prognosis neuroblastoma is frequently characterized by deletion of chr1p36 where tumor suppressor gene KIF1Bß resides. Interestingly, many 1p36-positive patients failed to express KIF1Bß protein. Since altered cellular redox status has been reported to be involved in cell death and protein modification, we investigated the relationship between reactive oxygen species (ROS) and KIF1Bß. Here, we showed that wild-type KIF1Bß protein expression positively correlates with superoxide (O2-) and total ROS levels in neuroblastoma cells, unlike apoptotic loss-of-function KIF1Bß mutants. Overexpression of KIF1Bß apoptotic domain variants increases total ROS and, specifically O2-, whereas knockdown of endogenous KIF1Bß decreases ROS and O2-. Interestingly, O2- increases KIF1Bß protein expression, independent of the proteasomal degradation pathway. Scavenging O2- or ROS decreases KIF1Bß protein expression and subsequent apoptosis. Moreover, treatment with investigational redox compound Gliotoxin increases O2-, KIF1Bß protein expression, apoptosis and colony formation inhibition. Overall, our findings suggest that ROS and O2- may be important downstream effectors of KIF1Bß-mediated apoptosis. Subsequently, O2- produced may increase KIF1Bß protein expression in a positive feedback mechanism. Therefore, ROS and, specifically O2-, may be critical regulators of KIF1Bß-mediated apoptosis and its protein expression in neuroblastoma.

Gliotoxin--bane or boon?

Gliotoxin (GT) is the most important epidithiodioxopiperazine (ETP)-type fungal toxin. GT was originally isolated from Trichoderma species as an antibiotic substance involved in biological control of plant pathogenic fungi. A few isolates of GT-producing Trichoderma virens are commercially marketed for biological control and widely used in agriculture. Furthermore, GT is long known as an immunosuppressive agent and also reported to have anti-tumour properties. However, recent publications suggest that GT is a virulence determinant of the human pathogen Aspergillus fumigatus. This compound is thus important on several counts - it has medicinal properties, is a pathogenicity determinant, is a potential diagnostic marker and is important in biological crop protection. The present article addresses this paradox and the ecological role of GT. We discuss the function of GT as defence molecule, the role in aspergillosis and suggest solutions for safe application of Trichoderma-based biofungicides.

Gliotoxin Inhibits Proliferation and Induces Apoptosis in Colorectal Cancer Cells.

The discovery of new bioactive compounds from marine natural sources is very important in pharmacological research. Here we developed a Wnt responsive luciferase reporter assay to screen small molecule inhibitors of cancer associated constitutive Wnt signaling pathway. We identified that gliotoxin (GTX) and some of its analogues, the secondary metabolites from marine fungus Neosartorya pseufofischeri, acted as inhibitors of the Wnt signaling pathway. In addition, we found that GTX downregulated the ß-catenin levels in colorectal cancer cells with inactivating mutations of adenomatous polyposis coli (APC) or activating mutations of ß-catenin. Furthermore, we demonstrated that GTX induced growth inhibition and apoptosis in multiple colorectal cancer cell lines with mutations of the Wnt signaling pathway. Together, we illustrated a practical approach to identify small-molecule inhibitors of the Wnt signaling pathway and our study indicated that GTX has therapeutic potential for the prevention or treatment of Wnt dependent cancers and other Wnt related diseases.

Epipolythiodiketopiperazines from the Marine Derived Fungus Dichotomomyces cejpii with NF-κB Inhibitory Potential.

The Ascomycota Dichotomomyces cejpii was isolated from the marine sponge Callyspongia cf. C. flammea. A new gliotoxin derivative, 6-acetylmonodethiogliotoxin (1) was obtained from fungal extracts. Compounds 2 and 3, methylthio-gliotoxin derivatives were formerly only known as semi-synthetic compounds and are here described as natural products. Additionally the polyketide heveadride (4) was isolated. Compounds 1, 2 and 4 dose-dependently down-regulated TNFα-induced NF-κB activity in human chronic myeloid leukemia cells with IC50s of 38.5 ± 1.2 µM, 65.7 ± 2.0 µM and 82.7 ± 11.3 µM, respectively. The molecular mechanism was studied with the most potent compound 1 and results indicate downstream inhibitory effects targeting binding of NF-κB to DNA. Compound 1 thus demonstrates potential of epimonothiodiketopiperazine-derived compounds for the development of NF-κB inhibitors.

Gliotoxin potentiates osteoblast differentiation by inhibiting nuclear factor-κB signaling.

The differentiation of pluripotent mesenchymal stem cells to mature osteoblasts is crucial for the maintenance of the adult skeleton. In rheumatic arthritis, osteoblast differentiation is impaired by the overproduction of cytokine tumor necrosis factor (TNF)­α. It has been demonstrated that TNF-α is able to inhibit osteoblast differentiation through the activation of nuclear factor (NF)-κB signaling. As a result of the critical role of TNF-α and NF-κB in the pathogenesis of bone-loss associated diseases, these factors are regarded as key targets for the development of therapeutic agents. In the current study, the role of the NF-κB inhibitor gliotoxin (GTX) in the regulation of osteoblast differentiation was evaluated. The non-toxic GTX doses were determined to be ≤ 3 µg/ml. It was revealed that GTX was able to block TNF-α-induced inhibition of osteoblast differentiation, as indicated by alkaline phosphatase (ALP) activity and ALP staining assays, as well as the expression levels of osteoblast-associated genes Col I, Ocn, Bsp, Runx2, Osx and ATF4. Additionally, it was identified that gliotoxin directly promoted bone morphogenetic protein-2-induced osteoblast differentiation. GTX was found to inhibit the accumulation of NF-κB protein p65 in the nucleus and reduce NF-κB transcriptional activity, suggesting that GTX potentiated osteoblast differentiation via the suppression of NF-κB signaling.

Bioprospection of cytotoxic compounds in fungal strains recovered from sediments of the Brazilian coast.

The cytotoxic activities of extracts (50 µg/ml) from 48 fungal strains, recovered from sediments of Pecém's offshore port terminal (Northeast coast of Brazil), against HCT-116 colon cancer cell lines were investigated. The most promising extract was obtained from strain BRF082, identified as Dichotomomyces cejpii by phylogenetic analyses of partial RPB2 gene sequence. Thus, it was selected for bioassay-guided isolation of the cytotoxic compounds. Large-scale fermentation of BRF082 in potato dextrose broth, followed by chromatographic purification of the bioactive fractions from the liquid medium, yielded gliotoxin (4) and its derivatives acetylgliotoxin G (3), bis(dethio)bis(methylsulfanyl)gliotoxin (1), acetylgliotoxin (5), 6-acetylbis(dethio)bis(methylsulfanyl)gliotoxin (2), besides the quinazolinone alkaloid fiscalin B. All isolated compounds were tested for their cytotoxicities against the tumor cell lines HCT-116, revealing 4 and 3 as the most cytotoxic ones (IC50 0.41 and 1.06 µg/ml, resp.).

Gliotoxin suppresses NF-κB activation by selectively inhibiting linear ubiquitin chain assembly complex (LUBAC).

A linear ubiquitin chain, which consists of ubiquitin molecules linked via their N- and C-termini, is formed by a linear ubiquitin chain assembly complex (LUBAC) composed of HOIP, HOIL-1L, and SHARPIN, and conjugation of a linear ubiquitin chain on the NF-κB essential modulator (NEMO) is deeply involved in NF-κB activation induced by various signals. Since abnormal activation of NF-κB is associated with inflammatory disease and malignancy, we searched for an inhibitor of LUBAC by high-throughput screening (HTS) with a Tb(3+)-fluorescein FRET system. As a result, we found that the fungal metabolite gliotoxin inhibits LUBAC selectively by binding to the RING-IBR-RING domain of HOIP, the catalytic center of LUBAC. Gliotoxin has been well-known as an inhibitor of NF-κB activation, though its action mechanism has remained elusive. Here, we show that gliotoxin inhibits signal-induced NF-κB activation by selectively inhibiting LUBAC-mediated linear ubiquitin chain formation.