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Goal: The long-term goal of our research is to develop safe and effective soluble epoxide hydrolase (sEH) inhibitors. The objective of this study is to evaluate the potency and selectivity of six natural isothiocyanates (ITCs) as sEH inhibitors., Methods: Molecular docking was used to model likely interactions between the ligands and receptors. The sEH inhibitory activity was tested using a validated fluorescence-based assay and PHOME as a substrate. To evaluate their selectivity as sEH inhibitors, the inhibitory potential of the ITCs was determined on microsomal epoxide hydrolase (mEH) and cytochrome P450 (CYP) enzymes in human liver microsomes. Probe substrates such as styrene oxide (mEH substrate) and established substrates for CYP2A6, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4 were used in this study. The metabolites of these substrates were analyzed using validated LC-MS/MS and HPLC-UV assays., Results: Molecular Docking revealed significant differences in binding site preference among the ITCs in silico and pointed to important interactions between the ligands and the catalytic residues of the sEH enzyme. In vitro, the ITCs showed varying degrees of sEH inhibition, but sulforaphane (SFN) and phenyl isothiocyanate (PITC) were the most potent inhibitors with IC 50 values of 3.65 and 7.5 μM, respectively. mEH was not significantly inhibited by any of the ITCs. Erucin and iberin were the only ITCs that did not inhibit the activity of any of the tested CYP enzymes., Conclusion: Our results demonstrate that natural ITCs have the potential to offer safe, selective, and potent sEH inhibition., Competing Interests: Declaration of competing interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Tetrahydrobiopterin (BH4) is an essential cofactor for dopamine and serotonin synthesis in monoaminergic neurons, phenylalanine metabolism in hepatocytes, and nitric oxide synthesis in endothelial and immune cells. BH4 is consumed as a cofactor or is readily oxidized by autooxidation. Quinonoid dihydropteridine reductase (QDPR) is an enzyme that reduces quinonoid dihydrobiopterin (qBH2) back to BH4, and we have previously demonstrated the significance of QDPR in maintaining BH4 in vivo using Qdpr-KO mice. In addition to the levels of BH4 in the cells, the ratios of oxidized to reduced forms of BH4 are supposed to be important for regulating nitric oxide synthase (NOS) via the so-called uncoupling of NOS. However, previous studies were limited due to the absence of specific and high-affinity inhibitors against QDPR. Here, we performed a high-throughput screening for a QDPR inhibitor and identified Compound 9b with an IC 50 of 0.72 μM. To understand the inhibition mechanism, we performed kinetic analyses and molecular dynamics simulations. Treatment with 9b combined with methotrexate (MTX), an inhibitor of another BH4-reducing enzyme, dihydrofolate reductase (DHFR), significantly oxidized intracellular redox states in HepG2, Jurkat, SH-SY5Y, and PC12D cells. Collectively, these findings suggest that 9b may enhance the anticancer and anti-autoimmune effects of MTX., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Methionine adenosyltransferase 2 A (MAT2A) mediates the synthesis of methyl donor S-Adenosylmethionine (SAM), providing raw materials for methylation reactions in cells. MAT2A inhibitors are currently used for the treatment of tumors with methylthioadenosine phosphorylase (MTAP) deficiency in clinical research. Methyltransferase like 3 (METTL3) catalyzes N6-methyladenosine (m 6 A) modification of mRNA in mammalian cells using SAM as the substrate which has been shown to affect the tumorigenesis of non-small cell lung cancer (NSCLC) from multiple perspectives. MAT2A-induced SAM depletion may have the potential to inhibit the methyl transfer function of METTL3. Therefore, in order to expand the applicability of inhibitors, improve anti-tumor effects and reduce toxicity, the combinational effect of MAT2A inhibitor AG-270 and METTL3 inhibitor STM2457 was evaluated in NSCLC. The results showed that this combination induced cell apoptosis rather than cell cycle arrest, which was non-tissue-specific and was independent of MTAP expression status, resulting in a significant synergistic anti-tumor effect. We further elucidated that the combination-induced enhanced apoptosis was associated with the decreased m 6 A level, leading to downregulation of PI3K/AKT protein, ultimately activating the apoptosis-related proteins. Unexpectedly, although combination therapy resulted in metabolic recombination, no significant change in methionine metabolic metabolites was found. More importantly, the combination also exerted synergistic effects in vivo. In summary, the combination of MAT2A inhibitor and METTL3 inhibitor showed synergistic effects both in vivo and in vitro, which laid a theoretical foundation for expanding the clinical application research of the two types of drugs., Competing Interests: Declaration of competing interest The authors declare that there is no conflict of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

4-hydroxyphenylpyruvate dioxygenase (HPPD) Inhibitor Sensitive 1 (HIS1) is an endogenous gene of rice, conferring broad-spectrum resistance to β-triketone herbicides. Similar genes, known as HIS1-like genes (HSLs), exhibit analogous functions and can complement the herbicide-resistant characteristics endowed by HIS1. The identification of HIS1 and HSLs represents a valuable asset, as the intentional pairing of herbicides with resistance genes emerges as an effective strategy for crop breeding. Encoded by HIS1 is a Fe(II)/2-oxoglutarate-dependent oxygenase responsible for detoxifying β-triketone herbicides through hydroxylation. However, the precise structure supporting this function remains unclear. This work, which determined the crystal structure of HIS1, reveals a conserved core motif of Fe(II)/2-oxoglutarate-dependent oxygenase and pinpoints the crucial residue dictating substrate preference between HIS1 and HSL., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

One of common characteristics of solid tumors is low O 2 level, so-called hypoxia, which plays a critical role in chemoresistance. Epigenetic mechanism such as DNA methylation and histone modification is involved in cancer development and progression. There is ample evidence that epigenetic drugs reversed acquired chemoresistance in cancer cells under normal O 2 level, normoxia. However, it remains unknown whether epigenetic drugs improve acquired chemoresistance under hypoxia. The aim of our study was to investigate whether epigenetic drugs can improve the chemoresistance induced under hypoxia in cancer cells. In murine melanoma B16-BL6 (B16) cells, the culture under hypoxia, 1%O 2 caused the elevated expression of hypoxia-inducible factor-1α (HIF-1α) and its target genes. The chemoresistance to 7-ethyl-10-hydroxycamptothecin (SN-38, the active metabolite of irinotecan) was also acquired under hypoxia in B16 cells. In addition, as epigenetic mechanisms, the protein expression of the enhancer of zeste homolog 2 (EZH2), histone methyltransferase and its target histone H3 trimethylation at lysine 27 (H3K27Me3) level increased under hypoxia. The induction of H3K27Me3 under hypoxia was suppressed by EZH2 siRNA and 3-deazaneplanocin A (DZNep), an EZH2 inhibitor. Furthermore, both EZH2 siRNA and DZNep significantly reduced the cell viability after SN-38 treatment and improved the chemoresistance to SN-38 under hypoxia. These results indicated that the chemoresistance to SN-38 under hypoxia would arise from epigenetic mechanism, H3K27Me3 elevation due to EZH2 induction. In conclusion, a histone methyltransferase EZH2 inhibitor, DZNep was capable of tackling acquired chemoresistance via the suppression of histone methylation induced under hypoxic tumor microenvironment., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Endoplasmic reticulum (ER) stress is enhanced in non-alcoholic steatohepatitis (NASH). Among three signalling pathways, the IRE1α/XBP1 signalling pathway is strongly implicated in the pathogenesis of NASH but its significance is still largely uncharacterised. In this report, we constructed a hepatocyte-specific XBP1-Luciferase knock-in mouse model that allows in vivo monitoring of the IRE1α/XBP1 activity in hepatocytes. Using this mouse model, we found that IRE1α/XBP1 was activated within hepatocytes during the pathogenesis of NASH. Significantly, a specific IRE1α kinase-inhibiting RNase attenuator, KIRA8, attenuated NASH in mice. In conclusion, our hepatocyte-specific XBP1 splicing reporter mouse represents a valid model for research and drug development of NASH, which showed that the IRE1α-induced XBP splicing is potentiated in hepatocytes during pathogenesis of NASH. Furthermore, we carried out the proof-of-concept study to demonstrate that the allosteric IRE1α RNase inhibitor serves as a promising therapeutic agent for the treatment of NASH., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Flavivirus, such as Dengue Virus (DENV) and Zika virus (ZIKV), infects millions of people and cause the death of thousands of people every year. Despite many efforts, there is no approved anti-flaviviral treatment available. In particular, some antiflavivirus compounds were investigated the cellular activities of DENV and ZIKV, but lacking the exploration of specific target enzyme, thereby resulting in the hindrance of structure-based drug design. One example is Montlukast, which was found to inhibit the replicon replication in DENV and ZIKV infected cells, with EC 50 values as 1.03 μM (DENV) and 1.14 μM (ZIKV), while the underlying mechanism remains unclear. In our study, the inhibitory mechanisms of Montelukast against the replicon replication of DENV and ZIKV infected cells were studied by using in silico approaches including inverse virtual screening (IVS), molecular dynamics (MD) simulations and binding free energy calculation, and validated through in vitro protease assay, confirming Montelukast could bind to NS2B-NS3 proteases of DENV and ZIKV as a competitive inhibitor (IC 50 for DENV: 25.65 μM, for ZIKV: 15.57 μM). Moreover, Montelukast has no potential off-target effect on NS2B-NS3 protease from thrombin and trypsin inhibitory assay. Overall, Montelukast may be used as a potential candidate to block NS2B-NS3 protease as well as lead for structural modification., Competing Interests: Declaration of competing interest There are no conflicts to declare., (Copyright © 2022. Published by Elsevier Inc.)

The progression of chronic kidney disease (CKD) increases the risks of cardiovascular morbidity and end-stage kidney disease. Indoxyl sulfate (IS), which is derived from dietary l-tryptophan by the action of bacterial l-tryptophan indole-lyase (TIL) in the gut, serves as a uremic toxin that exacerbates CKD-related kidney disorder. A mouse model previously showed that inhibition of TIL by 2-aza-l-tyrosine effectively reduced the plasma IS level, causing the recovery of renal damage. In this study, we found that (+)-sesamin and related lignans, which occur abundantly in sesame seeds, inhibit intestinal bacteria TILs. Kinetic studies revealed that (+)-sesamin and sesamol competitively inhibited Escherichia coli TIL (EcTIL) with K i values of 7 μM and 14 μM, respectively. These K i values were smaller than that of 2-aza-l-tyrosine (143 μM). Molecular docking simulation of (+)-sesamin- (or sesamol-)binding to EcTIL predicted that these inhibitors potentially bind near the active site of EcTIL, where the cofactor pyridoxal 5'-phosphate is bound, consistent with the kinetic results. (+)-Sesamin is a phytochemical with a long history of consumption and is generally regarded as safe. Hence, dietary supplementation of (+)-sesamin encapsulated in enteric capsules could be a promising mechanism-based strategy to prevent CKD progression. Moreover, the present findings would provide a new structural basis for designing more potent TIL inhibitors for the development of mechanism-based therapeutic drugs to treat CKD., (Copyright © 2021 Elsevier Inc. All rights reserved.)

In Brazil, the major vector of arboviruses is Aedes aegypti, which can transmit several alpha and flaviviruses. In this work, a pacifastin protease inhibitor library was constructed and used to select mutants for Ae. aegypti larvae digestive enzymes. The library contained a total of 3.25 × 10 5  cfu with random mutations in the reactive site (P2-P2'). The most successfully selected mutant, TiPI6, a versatile inhibitor, was able to inhibit all three Ae. aegypti larvae proteolytic activities, trypsin-like, chymotrypsin-like and elastase-like activities, with IC 50 values of 0.212 nM, 0.107 nM and 0.109 nM, respectively. In conclusion, the TiPI mutated phage display library was shown to be a useful tool for the selection of an inhibitor of proteolytic activities combined in a mix. TiPI6 is capable of controlling all three digestive enzyme activities present in the larval midgut extract. To our knowledge, this is the first time that one inhibitor containing a Gln at the P1 position showed inhibitory activity against trypsin, chymotrypsin, and elastase-like activities. TiPI6 can be a candidate for further larvicidal studies., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Pyruvate dehydrogenase kinase 1 (PDK1) is a Ser/Thr kinase that inactivates mitochondrial pyruvate dehydrogenase (PDH), leading to switch of glucose metabolism from mitochondrial oxidation to aerobic glycolysis. We previously reported that PDK1 inhibition is a potent therapeutic strategy in multiple myeloma (MM). However, availability of PDK1 inhibitors, which are effective at low concentrations, are limited at present, making PDK1 inhibition difficult to apply in the clinic. In the present study, we examined the efficacy and mechanism of action of JX06, a novel PDK1 inhibitor, against MM cells. We confirmed that PDK1 is highly expressed in normal plasma cells and MM cells using publicly available gene expression datasets. JX06 suppressed cell growth and induced apoptosis against MM cells from approximately 0.5 μM JX06 treatment reduced PDH phosphorylation, suggesting that JX06 is indeed inhibiting PDK1. Intracellular metabolite analysis revealed that JX06 treatment reduced metabolites associated with glucose metabolism of MM cells. Additionally, JX06 in combination with a well-known proteasome inhibitor, bortezomib, significantly increased MM cell death, which raises the possibility of combination use of JX06 with proteasome inhibitors in the clinic. These findings demonstrate that PDK1 can be potentially targeted by JX06 in MM through glycolysis inhibition, leading to a novel therapeutic strategy in MM., Competing Interests: Declaration of competing interest Yawara Kawano reports a relationship with Janssen Pharmaceuticals Inc that includes: speaking and lecture fees. Yawara Kawano reports a relationship with Takeda Pharmaceutical Co Ltd that includes: speaking and lecture fees. Yawara Kawano reports a relationship with Sanofi that includes: speaking and lecture fees. Yawara Kawano reports a relationship with Bristol Myers Squibb Co that includes: speaking and lecture fees. Yawara Kawano reports a relationship with Ono Pharmaceutical Co Ltd that includes: speaking and lecture fees., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The protozoan Plasmodium falciparum is the main aetiological agent of tropical malaria. Characteristic of the phylum is the presence of a plastid-like organelle which hosts several homologs of plant proteins, including a ferredoxin (PfFd) and its NADPH-dependent reductase (PfFNR). The PfFNR/PfFd redox system is essential for the parasite, while mammals share no homologous proteins, making the enzyme an attractive target for novel and much needed antimalarial drugs. Based on previous findings, three chemically reactive residues important for PfFNR activity were identified: namely, the active-site Cys99, responsible for hydride transfer; Cys284, whose oxidation leads to an inactive dimeric form of the protein; and His286, which is involved in NADPH binding. These amino acid residues were probed by several residue-specific reagents and the two cysteines were shown to be promising targets for covalent inhibition. The quantitative and qualitative description of the reactivity of few compounds, including a repurposed drug, set the bases for the development of more potent and specific antimalarial leads., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Previously, we reported that glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a binding partner of prolyl oligopeptidase (POP) in neuroblastoma NB-1 cells and that the POP inhibitor, SUAM-14746, inhibits cytosine arabinoside (Ara-C)-induced nuclear translocation of GAPDH and protects against Ara-C cytotoxicity. To carry out a more in-depth analysis of the interaction between POP and GAPDH, we generated POP-KO NB-1 cells and compared the nuclear translocation of GAPDH after Ara-C with or without SUAM-14746 treatment to wild-type NB-1 cells by western blotting and fluorescence immunostaining. Ara-C did not induce the nuclear translocation of GAPDH and SUAM-14746 did not protect against Ara-C cytotoxicity in POP-KO cells. These results indicate that the anticancer effects of Ara-C not only include the commonly known antimetabolic effects, but also the induction of cell death by nuclear transfer of GAPDH through interaction with POP., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Ferroptosis was recently defined as a novel type of programmed cell death depending on iron and lipid peroxidation. It is biologically different from other types of cell death such as apoptosis. While the involvement of ferroptosis in cancer, patient and animal model have been intensely studied, ferroptosis in human motor neuron model is still clearly unknown. Here we carefully assessed ferroptosis using human iPS cell-derived motor neuron (hiMNs). We found that almost all hiMNs died by the treatment of glutathione peroxidase 4 (GPX4) inhibitors. Importantly, the cell death was rescued by one antioxidant, vitamin E acetate, iron chelators and lipid peroxidase inhibitors with high dynamic ranges. Finally, these data clearly indicated that ferroptosis constitutively occurs in hiMNs, suggesting the possibility that it might play a biologically and pathologically important roles in motor neuron death such as motor neuron disease (MND)/Amyotrophic lateral sclerosis (ALS)., Competing Interests: Declaration of competing interest H.O. is a paid member of the Scientific Advisory Board of San Bio Co., Ltd and K Pharma, Inc. M.Y. is a scientific advisor of K Pharma, Inc. The other authors declare that they have no conflict of interest., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The mitogen-activated protein kinase (MAPK) pathway plays an important role in the colorectal cancer (CRC) progression, being supposed to be activated by the gene mutations, such as BRAF or KRAS. Although the inhibitors of extracellular signal-regulated kinase (ERK) have demonstrated efficacy in the cells with the BRAF or KRAS mutations, a clinical response is not always associated with the molecular signature. The patient-derived organoids (PDO) have emerged as a powerful in vitro model system to study cancer, and it has been widely applied for the drug screening. The present study aims to analyze the association between the molecular characteristics which analyzed by next-generation sequencing (NGS) and sensitivity to the ERK inhibitor (i.e., SCH772984) in PDO derived from CRC specimens. A drug sensitivity test for the SCH772984 was conducted using 14 CRC cell lines, and the results demonstrated that the sensitivity was in agreement with the BRAF mutation, but was not completely consistent with the KRAS status. In the drug sensitivity test for PDO, 6 out of 7 cases with either BRAF or KRAS mutations showed sensitivity to the SCH772984, while 5 out of 6 cases of both BRAF and KRAS wild-types were resistant. The results of this study suggested that the molecular status of the clinical specimens are likely to represent the sensitivity in the PDOs but is not necessarily absolutely overlapping. PDO might be able to complement the limitations of the gene panel and have the potential to provide a novel precision medicine., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Long-term potentiation (LTP) and long-term depression (LTD) are key forms of synaptic plasticity in the hippocampus. LTP and LTD are believed to underlie the processes occurring during learning and memory. Search of mechanisms responsible for switching from LTP to LTD and vice versa is an important fundamental task. Protein synthesis blockers (PSB) are widely used in models of memory impairment and LTP suppression. Here, we found that blockade of serine/threonine phosphatases 1 (PP1) and 2A (PP2A) with the specific blockers, calyculin A (CalyA) or okadaic acid (OA), and simultaneous blockade of the protein translation by anisomycin or cycloheximide leads to a switch from PSB-impaired LTP to LTD. PP1/PP2A-dependent LTD was extremely sensitive to the intensity of the test stimuli, whose increase restored the field excitatory postsynaptic potentials (fEPSP) to the values corresponding to control LTP in the non-treated slices. PP1/PP2A blockade affected the basal synaptic transmission, increasing the paired-pulse facilitation (PPF) ratio, and restored the PSB-impaired PPF 3 h after tetanus. Prolonged exposure to anisomycin led to the NO synthesis increase (measured using fluorescent dye) both in the dendrites and somata of CA1, CA3, dentate gyrus (DG) hippocampal layers. OA partially prevented the NO production in the CA1 dendrites, as well in the CA3 and DG somas. Direct measurements of changes in serine/threonine phosphatase (STPP) activity revealed importance of the PP1/PP2A-dependent component in the late LTP phase (L-LTP) in anisomycin-treated slices. Thus, serine/threonine phosphatases PP1/PP2A influence both basal synaptic transmission and stimulation-induced synaptic plasticity., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Histone lysine demethylase 4D (KDM4D), also known as JMJD2D, plays an important role in cell proliferation and survival and has been associated with several tumor types. KDM4D has emerged as a potential target for the treatment of human cancer. Here, we reported crystal complex structures for two KDM4D inhibitors, OWS [2-(1H-pyrazol-3-yl)isonicotinic acid] and 10r (5-hydroxy-2-methylpyrazolo[1,5-a]pyrido[3,2-e]pyrimidine-3-carbonitrile), which were both determined to 2.0 Å. OWS is a newly discovered KDM4D inhibitor (IC 50  = 4.28 μM) and the critical pharmacophores of this compound are confirmed by the complex structure. Compound 10r is a KDM4D inhibitor reported by us previously. To clarify the binding mode in more detail, the crystal structure was determined and the comparison analysis revealed unique interactions that had never been observed before. Overall, our data provide new structural insights for rational design and offer an opportunity for optimization of KDM4D inhibitors., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021. Published by Elsevier Inc.)

Alpha-1 acid glycoprotein (AGP) is a major acute-phase protein that is involved in drug/ligand binding and regulation of immune response. In response to inflammation, AGP secretion from the liver increases, resulting in elevated concentration of plasma AGP. AGP exhibits multiple N-glycosylation sites, and thus, is highly glycosylated. Although AGP glycosylation is considered to affect its functions, the significance of AGP glycosylation for its secretion is unclear. In this study, we investigated the effects of AGP glycosylation using glycosylation-deficient mouse AGP mutants lacking one, four, or all five N-glycosylation sites. Furthermore, we examined the effects of endoplasmic reticulum (ER) stress-inducing reagents, including tunicamycin and thapsigargin, which induce ER stress in an N-glycosylation-dependent and -independent manner, respectively. Here, we found that glycosylation deficiency and ER stress induce a little or no effect on AGP secretion. Conversely, thapsigargin significantly suppressed AGP secretion in glycosylation-independent manner. These findings indicate that AGP secretion is regulated via thapsigargin-sensitive pathway that might be further controlled by the intracellular calcium concentrations., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest with the content of this manuscript., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Fully understanding the regulatory network under the pluripotency of embryonic stem cells (ESC) is a prerequisite for their safe application. Here, we addressed the characteristics of metastasis-associated (MTA) family members in human ESCs and found that knockdown of the expression of MTA2 and MTA3, but not MTA1, would induce differentiation. High-throughput sequence and quantitative real-time PCR showed that the decreased MTA2 or MTA3 gene transcript mainly led to the emergence of mesendoderm associated markers. Finally, based on the chemical small molecule library screening, we observed that addition of ID8, a specific inhibitor of the dual-specificity tyrosine phosphorylation-regulated kinases (DYRKs), was able to impair the differentiation phenotype induced by MTA2 and MTA3 reduction. Functional assay showed that ID8 could mediate differentiation caused by MTA2 or MTA3 knockdown mainly through inhibition of DYRK4 activity. Therefore, our finding provides the evidence that the functions of MTA family genes in human ESCs are different. Revealing the function of MTA in ESCs with different pluripotency states will help us better understand and apply stem cells., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Inc. All rights reserved.)

γ-Glutamylcyclotransferase (GGCT) is involved in glutathione homeostasis, in which it catalyzes the reaction that generates 5-oxoproline and free amino acids from γ-glutamyl peptides. Increasing evidence shows that GGCT has oncogenic functions and is overexpressed in various cancer tissues, and that inhibition of GGCT activity exerts anticancer effects in vitro and in vivo. Here, we demonstrate that U83836E ((2R)-2-[[4-(2,6-dipyrrolidin-1-ylpyrimidin-4-yl)piperazin-1-yl]methyl]-3,4-dihydro-2,5,7,8,-tetramethyl-2H-1-benzopyran-6-ol, dihydrochloride), a lazaroid that inhibits lipid peroxidation, inhibits GGCT enzymatic activity. U83836E was identified from a high-throughput screen of low molecular weight compounds using a fluorochrome-conjugated GGCT probe. We directly quantified that U83836E specifically inhibited GGCT by measuring the product of a fluorochrome-conjugated GGCT substrate assay, and showed that U83836E inhibited GGCT activity in extracts of NIH3T3 cells overexpressing GGCT. Moreover, U83836E significantly inhibited tumor growth in a xenograft model that used immunodeficient mice orthotopically inoculated with MCF7 human breast cancer cells. These results indicate that U83836E may be a useful GGCT inhibitor for the development of potential cancer therapeutics., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Autotaxin (ATX) and its product lysophosphatidic acid (LPA) have been implicated in lung fibrosis and cancer. We have studied their roles in DNA damage induced by carcinogenic crystalline silica particles (CSi). In an earlier study on bronchial epithelia, we concluded that ATX, via paracrine signaling, amplifies DNA damage. This effect was seen at 6-16 h. A succeeding study showed that CSi induced NLRP3 phosphorylation, mitochondrial depolarization, double strand breaks (DSBs), and NHEJ repair enzymes within minutes. In the current study we hypothesized a role for the ATX-LPA axis also in this rapid DNA damage. Using 16HBE human bronchial epithelial cells, we show ATX secretion at 3 min, and that ATX inhibitors (HA130 and PF8380) prevented both CSi-induced mitochondrial depolarization and DNA damage (detected by γH2AX and Comet assay analysis). Experiments with added LPA gave similar rapid effects as CSi. Furthermore, Rac1 was activated at 3 min, and a Rac1 inhibitor (NSC23766) prevented mitochondrial depolarization and genotoxicity. In mice the bronchial epithelia exhibited histological signs of ATX activation and signs of DSBs (53BP1 positive nuclei) minutes after a single inhalation of CSi. Our data indicate that CSi rapidly activate the ATX-LPA axis and within minutes this leads to DNA damage in bronchial epithelial cells. Thus, ATX mediates very rapid DNA damaging effects of inhaled particles., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Although acute myeloid leukemia (AML) is a highly heterogeneous disease with diverse genetic subsets, one hallmark of AML blasts is myeloid differentiation blockade. Extensive evidence has indicated that differentiation induction therapy represents a promising treatment strategy. Here, we identified that the pharmacological inhibition of the mitochondrial electron transport chain (ETC) complex III by antimycin A inhibits proliferation and promotes cellular differentiation of AML cells. Mechanistically, we showed that the inhibition of dihydroorotate dehydrogenase (DHODH), a rate-limiting enzyme in de novo pyrimidine biosynthesis, is involved in antimycin A-induced differentiation. The activity of antimycin A could be reversed by supplement of excessive amounts of exogenous uridine as well as orotic acid, the product of DHODH. Furthermore, we also found that complex III inhibition exerts a synergistic effect in differentiation induction combined with DHODH inhibitor brequinar as well as with the pyrimidine salvage pathway inhibitor dipyridamole. Collectively, our study uncovered the link between mitochondrial complex III and AML differentiation and may provide further insight into the potential application of mitochondrial complex III inhibitor as a mono or combination treatment in differentiation therapy of AML., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021. Published by Elsevier Inc.)

The coronavirus disease 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), poses an unprecedented global health crisis. It is particularly urgent to develop clinically effective therapies to contain the pandemic. The main protease (M pro ) and the RNA-dependent RNA polymerase (RdRP), which are responsible for the viral polyprotein proteolytic process and viral genome replication and transcription, respectively, are two attractive drug targets for SARS-CoV-2. This review summarizes up-to-date progress in the structural and pharmacological aspects of those two key targets above. Different classes of inhibitors individually targeting M pro and RdRP are discussed, which could promote drug development to treat SARS-CoV-2 infection., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Transactive response DNA-binding protein of 43 kDa (TDP-43) abnormally forms aggregates in certain subtypes of frontotemporal lobar degeneration (FTLD) and in amyotrophic lateral sclerosis (ALS). The pathological forms of TDP-43 have reported to be associated with poly(ADP-ribose) (PAR), which regulates the properties of these aggregates. A recent study has indicated that tankyrase, a member of the PAR polymerase (PARP) family, regulates pathological TDP-43 formation under conditions of stress, and tankyrase inhibitors suppress TDP-43 aggregate formation and cytotoxicity. Since we reported the development of tankyrase inhibitors that are more specific than conventional inhibitors, in this study, we examined their effects on the formation of TDP-43 aggregates in cultured cells. Time-lapse imaging showed that TDP-43 aggregates appeared in the nucleus within 30 min of treatment with sodium arsenite. Several tankyrase inhibitors suppressed the formation of aggregates and decreased the levels of the tankyrase protein. Immunohistochemical studies demonstrated that tankyrase was localized to neuronal cytoplasmic inclusions in the spinal cords of patients with ALS. Moreover, the tankyrase protein levels were significantly higher in the brains of patients with FTLD than in the brains of control subjects. These findings suggest that the inhibition of tankyrase activity protects against TDP-43 toxicity. Tankyrase inhibitors may be a potential treatment to suppress the progression of TDP-43 proteinopathies., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

To overcome dermatological concerns causing abnormally excessive melanin synthesis, highly effective and safe skin depigmentation compounds have been identified in the cosmetic and pharmaceutical industries. Among several methods used to achieve skin depigmentation, inhibition of tyrosinase is one of the most effective, since tyrosinase is a crucial enzyme in melanogenesis. Herein, isolindleyin, a novel inhibitor of human tyrosinase, was introduced and evaluated for its anti-melanogenic effects in human epidermal melanocytes. The results revealed that isolindleyin was directly bound to tyrosinase and it suppressed melanin synthesis. The binding mode between isolindleyin and the active sites of human tyrosinase was investigated using computational molecular docking at the atomic level. Isolindleyin binding was found to be stabilized by hydrophobic interactions between His 367 and Val 377 and by hydrogen bonds between Ser 380 and Asn 364. The results of this study revealed the anti-melanogenic effects of isolindleyin that could contribute toward overcoming dermatological concerns that cause abnormally excessive melanin synthesis., Competing Interests: Declaration of competing interest The authors state no conflict of interest., (Copyright © 2020. Published by Elsevier Inc.)

Emodin, a major component of Chinese herbal rhubarb, delays the progression of chronic renal failure. However, the effect and working mechanisms of Emodin on renal tubulointerstitial fibrosis remains elusive. We hypothesized that emodin inhibits renal tubulointerstitial fibrosis through EZH2, a histone methyltransferase. Our in vivo and in vitro studies demonstrate that emodin reduced extracellular collagen deposition and inhibited Smad3 and CTGF pro-fibrotic signaling pathways, which were correlated with the down-regulation of EZH2 and reduced trimethylation of histone H3 on lysine 27 (H3k27me3) in NRK-49F fibrotic cells and UUO kidneys. Inhibition of EZH2 by 3-DZNeP blocked or attenuated the anti-fibrotic effect of emodin in UUO kidneys and NRK-49F cells. These data indicate that emodin inhibits renal tubulointerstitial fibrosis in obstructed kidneys and this effect is mediated through EZH2., Competing Interests: Declaration of competing interest The authors have no conflicts of interest to declare., (Copyright © 2020 Elsevier Inc. All rights reserved.)

During its intra-erythrocytic growth phase, the malaria parasite Plasmodium falciparum relies heavily on glycolysis for its energy requirements. Pyruvate kinase (PYK) is essential for regulating glycolytic flux and for ATP production, yet the allosteric mechanism of P. falciparum PYK (PfPYK) remains poorly understood. Here we report the first crystal structure of PfPYK in complex with substrate analogues oxalate and the ATP product. Comparisons of PfPYK structures in the active R-state and inactive T-state reveal a 'rock-and-lock' allosteric mechanism regulated by rigid-body rotations of each subunit in the tetramer. Kinetic data and structural analysis indicate glucose 6-phosphate is an activator by increasing the apparent maximal velocity of the enzyme. Intriguingly, the trypanosome drug suramin inhibits PfPYK, which points to glycolysis as a set of potential therapeutic targets against malaria., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Following renal ischemia-reperfusion injury (RIRI), because of the decrease in oxygen supply to the kidney, a large amount of oxygen-free radicals is generated, and in severe cases, tissue cells will undergo apoptosis or even die. Normobaric hyperoxia (NBHO) is a very common clinical adjuvant treatment. It restores the oxygen supply after renal ischemia and combats oxidative stress in tissues, thus playing a protective role. In this study, our aim is to elucidate the protective mechanism of NBHO inhalation in a rat RIRI model. We performed a surgical excision of the left kidney of the rat and established a right kidney solitary kidney model. Later, the right renal pedicle of the rat was clamped using a non-invasive vascular clamp for 45 min. After the vascular clamp was released and reperfused for 24 h, the rat was placed in a closed oxygen chamber. It was subjected to inhalation of high-concentration oxygen (50%-55%), 2 h daily, for 7 days.RIRI induces postoperative weight loss, impaired renal function, increased oxygen free radicals, reduced antioxidant substances, increased histopathological damage, and increased levels of apoptosis. These effects were significantly improved after treatment with NBHO. At the same time, NBHO significantly increased the expression levels of Nrf2 and HO-1 in the tissues after RIRI. To verify whether HO-1 induced by Nrf2 is involved in the resistance to oxidative stress, after the rat RIRI and before inhaling NBHO, we intraperitoneally injected HO-1 specific inhibitor zinc protoporphyrin (ZnPP) (45 μmol/Kg). However, we found that ZnPP reversed the protective effect of NBHO on RIRI in rats. Combining all the results, we have demonstrated the protective effect of NBHO on RIRI, which can be at least partially attributed to the activation of the Nrf2/HO-1 antioxidative stress pathway., Competing Interests: Declaration of competing interest We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service or company that could be construed as influencing the position presented in, or the review of, the manuscript entitled, “Normobaric hyperoxia plays a protective role against renal ischemia-reperfusion injury by activating the Nrf2/HO-1 signaling pathway”o, (Copyright © 2020 Elsevier Inc. All rights reserved.)

Myocardial fibrosis is the pathological consequence of injury-induced fibroblastto-myofibroblast transition, resulting in increased stiffness and diminished cardiac function. Histone modification has been shown to play an important role in the pathogenesis of cardiac fibrosis. Here, we identified H3K27me3 demethylase JMJD3/KDM6B promotes cardiac fibrosis via regulation of fibrogenic pathways. Using neonatal rat cardiac fibroblasts (NRCF), we show that the expression of endogenous JMJD3 is induced by angiotensin II (Ang II), while the principle extracellular matrix (ECM) such as fibronectin, CTGF, collagen I and III are increased. We find that JMJD3 inhibition markedly enhances the suppressive mark (H3K27me3) at the beta (β)-catenin promoter in activated cardiac fibroblasts, and then substantially decreases expression of fibrogenic gene. Both inhibition of β-catenin-mediated transcription with ICG-001 and genetic loss of β-catenin can prevent Ang II-induced ECM deposition. Most importantly, in vivo inhibition of JMJD3 rescues myocardial ischemia-induced cardiac fibrosis and cardiac dysfunction. Collectively, our findings are the first to report a novel role of histone demethylase JMJD3 in the pro-fibrotic cardiac fibroblast phenotype, pharmacological targeting of JMJD3 might represent a promising therapeutic approach for the treatment of human cardiac fibrosis and other fibrotic diseases., Competing Interests: Declaration of competing interest None of the authors have any conflict of interests to report., (Copyright © 2020 Elsevier Inc. All rights reserved.)

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., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Aspartic proteases are important biomarkers of human disease and interesting targets for modulation of immune response via MHC class II antigen processing inhibition. The lack of inhibitors with sufficient selectivity hampers precise analysis of the role of cathepsin E and napsin A in samples containing the ubiquitous and highly abundant homolog cathepsin D. Grassystatins from marine cyanobacteria show promising selectivity for cathepsin E but contain several ester bonds that make their synthesis cumbersome and thus limit availability of the inhibitors. Herewith, we present grassystatin-derived cathepsin E inhibitors with greatly facilitated synthesis but retained selectivity profile. We demonstrate their affinity and selectivity with both enzyme kinetic assays and streptavidin-based pull-down from cells and mouse organs. Our findings suggest that grassystatin-like inhibitors are useful tools for targeted inhibition of cathepsin E and thus provide a novel approach for cancer and immunology research., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Protein tyrosine phosphatase 1B (PTP1B) is a widely expressed 50 kDa enzyme and the first intracellular PTP to be purified from human placental tissue. It has been proved that protein tyrosine phosphatase 1B played a significant role in the negative regulation of insulin signaling pathway and overexpression of PTP1B could lead to the decrease of insulin resistance. Therefore PTP1B has emerged as a novel promising therapeutic target for the treatment of type-2 diabetes mellitus. Computer aided drug design (CADD), chemical synthesis and biological activity assay resulted in the identification of a novel potent PTP1B inhibitor, compound 1a, which shared an IC 50 value of 4.46 μM. Finally, the analysis of molecular dynamics simulation provided the theoretical basis for favorable activity of compound 1a., Competing Interests: Declaration of competing interest There is no conflict of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Coral calcification is intricately linked to the chemical composition of the fluid in the extracellular calcifying medium (ECM), which is situated between the calcifying cells and the skeleton. Here we demonstrate that the acid-base sensing enzyme soluble adenylyl cyclase (sAC) is expressed in calcifying cells of the coral Stylophora pistillata. Furthermore, pharmacological inhibition of sAC in coral microcolonies resulted in acidification of the ECM as estimated by the pH-sensitive ratiometric indicator SNARF, and decreased calcification rates, as estimated by calcein labeling of crystal growth. These results indicate that sAC activity modulates some of the molecular machinery involved in producing the coral skeleton, which could include ion-transporting proteins and vesicular transport. To our knowledge this is the first study to directly demonstrate biological regulation of the alkaline pH of the coral ECM and its correlation with calcification., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Colon cancer is one of the leading causes of cancer-related deaths and its five-year survival rate remains low in locally advanced or metastatic stages of colon cancer. Overexpression of high mobility group protein AT-hook2 (HMGA2) is associated with cancer progression, metastasis, and poor prognosis in many malignancies. Oxidative stress regulates cellular mechanisms and provides an environment that favors the cancer cells to survive and progress, yet, at the same time, oxidative stress can also be utilized as a cancer-damaging strategy. The molecular regulatory roles of HMGA2 in oxidative stress and their involvement in cancer progression are largely unknown. In this study, we investigated the involvement of HMGA2 in regulation of oxidative stress responses by luciferase reporter assays. Moreover, we utilized dicoumarol (DIC), a derivative of coumarin which has been suggested to be involved in oxidation regulation with anticancer effects, and demonstrated that DIC could induce apoptosis and inhibit cell migration of HMGA2 overexpressing colon cancer cells. Further investigation also evidenced that DIC can enhance the cancer inhibition effect of 5-FU in colony formation assays. Taken together, our data revealed novel insights into the molecular mechanisms underlying HMGA2 and highlighted the possibility of targeting the cellular antioxidant system for treating patients and preventing from cancer progression in HMGA2 overexpressing colon cancer cells., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

3-isopropylmalate dehydrogenases (LeuB) belong to the leucine biosynthetic pathway and catalyze the irreversible oxidative decarboxylation of 3IPM to 2-ketoisocaproate that is finally converted into leucine by a branched-chain aminotransferase. Since leucine is an essential amino acid for humans, and it is also vital for the growth of many pathogenic bacteria, the enzymes belonging to this pathway can be considered as potential target sites for designing of a new class of antibacterial agents. We have determined the crystal structure of the Haemophilus influenzae LeuB in complex with the cofactor NAD + and the inhibitor O-IbOHA, at 2.1 Å resolution; moreover, we have investigated the inhibitor mechanism of action by analyzing the enzyme kinetics. The structure of H. influenzae LeuB in complex with the intermediate analog inhibitor displays a fully closed conformation, resembling the previously observed, closed form of the equivalent enzyme of Thiobacillus ferrooxidans in complex with the 3IPM substrate. O-IbOHA was found to bind the active site by adopting the same conformation of 3IPM, and to induce an unreported repositioning of the side chain of the amino acids that participate in the coordination of the ligand. Indeed, the experimentally observed binding mode of O-IbOHA to the H. influenzae LeuB enzyme, reveals aspects of novelty compared to the computational binding prediction performed on M. tuberculosis LeuB. Overall, our data provide new insights for the structure-based rational design of a new class of antibiotics targeting the biosynthesis of leucine in pathogenic bacteria., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The ubiquitin-proteasome pathway is essential for skeletal muscle growth and development. Proteasomes generate oligopeptides in the cytoplasm, and these peptides are considered to be rapidly degraded to amino acids by several intracellular aminopeptidases. However, the role of intracellular aminopeptidases in muscle growth remains unknown. In this study, therefore, we investigated the role of intracellular aminopeptidases in C2C12 myoblast proliferation and differentiation. Inhibition of intracellular aminopeptidases by Bestatin methyl ester (Bes-ME) decreased leucine and alanine aminopeptidase activity, and impaired proliferation and differentiation of C2C12 myoblasts. Furthermore, we observed that the inhibition of intracellular aminopeptidases reduced intracellular levels of amino acid and ATP level, and suppressed the phosphorylation of the mTOR pathway. These results suggested that intracellular aminopeptidases affect C2C12 myoblast proliferation and differentiation via mTOR pathway; however, further studies are required to clarify the role of aminopeptidase in skeletal muscle., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

The tumor suppressor CYLD negatively regulates polyubiquitination-dependent cellular signaling such as nuclear factor (NF)-κB signaling. In addition to CYLD, multiple deubiquitinating enzymes (DUBs) are also involved in the regulation of this signaling pathway, and distinct role of CYLD is yet to be clarified. Here, we identified a small chemical named Subquinocin that inhibited the DUB activity of recombinant CYLD using a wheat cell-free protein synthesis and an AlphaScreen technology. In cells, Subquinocin increased the polyubiquitination of NEMO and RIP1 and enhanced NF-κB activation. Modeling and mutation analyses indicated that Subquinocin interacted with Y940 in CYLD, which locates close to catalytic center of CYLD, and is conserved among the USP-family DUBs. Further biochemical evaluation revealed that Subquinocin inhibited USP-family DUBs, but not other family DUBs including OTU. Although Subquinocin showed a broad specificity toward USP-family DUBs, the inhibitory effect of Subquinocin on NF-κB signaling was negligible in CYLD-KO cells, indicating that CYLD is a major target of Subquinocin on the suppression of NF-κB signaling. In conclusion, Subquinocin identified here is a useful tool to analyze the signal transduction mediated by USP-family DUBs., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Tuberculosis has attracted increased attention worldwide due to its high morality and its resistance to treatment with traditional antibacterial drugs. The l,d-transpeptidase Ldt Mt2 confers resistance to traditional β-lactams and is considered a target for anti-Tuberculosis treatment. Carbapenems are proposed to inhibit Mycobacterium tuberculosis by repressing the activity of Ldt Mt2 . The interaction mechanisms between Ldt Mt2 and carbapenems have been revealed by Ldt Mt2 -carbapenem adduct structures along with various biochemical assays. Interestingly, the lack of the 1-β-methyl group in imipenem may be related to its high binding ability to Ldt Mt2 . However, there is limited evidence on the interaction mode of Ldt Mt2 and panipenem, another carbapenem lacking the 1-β-methyl group. Herein, we identified the biochemical features of panipenem binding to Ldt Mt2 . We further suggest that the presence of the 1-β-methyl group in carbapenems is indeed related to the ligand affinity of Ldt Mt2 and that the presence of the Y308 and Y318 residues in Ldt Mt2 stabilized the conformation of the Ldt Mt2 -carbepenem adduct. Our research provides a structural basis for the development of novel carbapenems against L,D-transpeptidases., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Canarium album (Lour.) Raeusch (C. album) as a normally medicinal and edible plant has been used widely in Asian countries and is considered a source of phytochemicals that are beneficial to human health. Here, we showed at the first time isocorilagin, a polyphenolic compound isolated from C. album, displayed antiviral activity against diverse strains of influenza A virus (IAV), including A/Puerto Rico/8/34 (H1N1), A/Aichi/2/68 (H3N2) and NA-H274Y (H1N1) with IC 50 value of 9.19 ± 1.99, 23.72 ± 2.51 and 4.64 ± 3.01 μM, respectively. Further mechanistic studies revealed that it clearly inhibited neuraminidase activity of IAV and directly influenced the virus release. The molecular docking studies presented isocorilagin could bind to the highly conserved residues in the active sites of NA, implying that isocorilagin may be effective against various influenza strains and not susceptible to produce drug resistance. Taken together, the results strongly suggest that isocorilagin has potential to be an effective, safe and affordable neuraminidase inhibitor against a diverse panel of IAV strains. More importantly, our work expands the biological activities of C. album extracts and provide a new option for the development of anti-influenza drug., Competing Interests: Declaration of competing interest There are no conflicts to declare., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Metabolic programs are rewired in cancer cells to support survival and tumor growth. Among these, recent studies have demonstrated that glutamate-oxaloacetate transaminase 1 (GOT1) plays key roles in maintaining redox homeostasis and proliferation of pancreatic ductal adenocarcinomas (PDA). This suggests that small molecule inhibitors of GOT1 could have utility for the treatment of PDA. However, the development of GOT1 inhibitors has been challenging, and no compound has yet demonstrated selectivity for GOT1-dependent cell metabolism or selective growth inhibition of PDA cell lines. In contrast, potent inhibitors that covalently bind to the transaminase cofactor pyridoxal-5'-phosphate (PLP), within the active site of the enzyme, have been reported for kynurenine aminotransferase (KAT) and gamma-aminobutyric acid aminotransferase (GABA-AT). Given the drug discovery successes with these transaminases, we aimed to identify PLP-dependent suicide substrate-type GOT1 inhibitors. Here, we demonstrate that PF-04859989, a known KAT2 inhibitor, has PLP-dependent inhibitory activity against GOT1 and shows selective growth inhibition of PDA cell lines., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Uveal melanoma (UM) is the most common primary intraocular tumor in adults. Despite of important progress in the local therapy, high radioresistance in primary tumor and chemoresistance in metastatic disease are the major obstacles for UM therapy. Therefore, strategies to overcome resistance to radiation or chemotherapy in UM are urgently needed. In this study, we found that phosphorylation of DNA-PKcs, which is the key factor of non-homologous end joining (NHEJ) pathway, was remarkably overexpressed in ionizing radiation (IR)- and Selumetinib resistant UM cells. Increased amount of NHEJ events were also observed in resistant UM cells. Inhibition of DNA-PKcs by NU7441 significantly impaired DNA repair and re-sensitized resistant UM cells to radiation and Selumetinib both in vitro and in vivo. The results demonstrate increased DNA double strand break repair as a mechanism of resistance to ionizing radiation and Selumetinib, and identify DNA-PKcs as a promising target for radio-and chemotherapy in UM patients., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Lipoxygenases (LOXs) are dioxygenases that catalyze the oxygenation of polyunsaturated fatty acids to hydroperoxyl derivates. These products are precursors for different lipid mediators which are associated with pathogenesis of various diseases such as asthma, atherosclerosis and cancer. Several LOXs suffer from substrate inhibition, a potential regulatory mechanism, yet it is unclear what is the cause of this phenomenon. One such enzyme is the coral 11R-LOX which displays a significant decrease in turnover rate at arachidonic acid concentrations above 30 μM. In this report, site-directed mutagenesis and inhibition assays were employed to shed light on the mechanism of substrate inhibition in 11R-LOX. We found that introduction of a positive charge to the active site entrance with Gly188Arg substitution completely eliminates the slow-down at higher substrate concentrations. Inhibition of 11R-LOX by its catalysis product, 11(R)-hydroperoxyeicosatetraenoic acid, suggests an uncompetitive mechanism. We reason that substrate inhibition in 11R-LOX is due to additional fatty acid binding by the enzyme:substrate complex at an allosteric site situated in the very vicinity of the active site entrance., (Copyright © 2019 Elsevier Inc. All rights reserved.)

RSL3 is a type of small molecular compound which can inactivate glutathione peroxidase 4 (GPX4) and induce ferroptosis, but its role in glioma cell death remains unclear. In this study, we found RSL3 inhibited the viabilities of glioma cells and induced glioma cell death in a dose-dependent manner. In vitro studies revealed that RSL3-induced cell death was accompanied with the changes of autophagy-associated protein levels and was alleviated by pretreatment of 3-Methyladenine, bafilomycin A1 and knockdown of ATG5 with siRNA. The ATP and pyruvate content as well as the protein levels of HKII, PFKP, PKM2 were decreased in cells treated by RSL3, indicating that RSL3 induced glycolysis dysfunction in glioma cells. Moreover, supplement of exterior sodium pyruvate, which was a final product of glycolysis, not only inhibited the changes of autophagy-associated protein levels caused by RSL3, but also prevented RSL3-induced cell death. In vivo data suggested that the inhibitory effect of RSL3 on the growth of glioma cells was associated with glycolysis dysfunction and autophagy activation. Taken together, RSL3 induced autophagic cell death in glioma cells via causing glycolysis dysfunction., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Regulator of chromosome condensation 2 (RCC2) is a regulator of cell-cycle progression linked in multiple cancers to pro-tumorigenic phenomena including promotion of tumor growth, tumor metastases and poorer patient prognoses. However, the role of RCC2 in GBM remains under-investigated. Here, we sought to determine the relevance of RCC2 in GBM, as well as its roles in GBM development, progression and prognosis. Initial clinical evaluation determined significant RCC2 enrichment in GBM when compared to normal brain tissue, and elevated expression was closely associated with a poorer prognosis in glioma patients. Via shRNA inhibition, we determined that RCC2 is essential to tumor proliferation and tumorigenicity in vitro and in vivo. Additionally, RCC2 was determined to promote radioresistance of GBM tumor cells. Investigation of the underlying mechanisms implicated DNA mismatch repair, JAK-STAT pathway and activated transcription of DNA methyltransferase 1 (DNMT1). For validation, pharmacologic inhibition via administration of a DNMT1 inhibitor demonstrated attenuated GBM tumor growth both in vitro and in vivo. Collectively, this study determined a novel therapeutic target for GBM in the form of RCC2, which plays a pivotal role in GBM proliferation and radio-resistance via regulation of DNMT1 expression in a p-STAT3 dependent manner., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The soluble epoxide hydrolase (sEH) is a bifunctional enzyme implicated in the regulation of inflammation. The N-terminal domain harbors a phosphatase activity (N-phos) with an affinity to lipid phosphomonoesters, and the C-terminal domain has an activity to hydrolyze anti-inflammatory lipid epoxides (C-EH). Although many potent inhibitors of C-EH have been discovered, little is known about inhibitors of N-phos. Here, we identify N-substituted amino acids as selective inhibitors of N-phos. Many of the N-substituted amino acids inhibited differently mouse and human N-phos; phenylalanine derivatives are relatively selective for mouse N-phos, whereas tyrosine derivatives are more selective for human N-phos. The best inhibitors, Fmoc-l-Phe(4-CN) (67) and Boc-l-Tyr(Bzl) (23), inhibited mouse and human N-phos competitively with K I in the low micromolar range. These compounds inhibit the N-phos activity 37- (67) and 137-folds (23) more potently than the C-EH. The differences in inhibitor structure activity suggest different active site structure between species, and thus, probably a divergent substrate preference between mouse and human N-phos., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Invasive aspergillosis (IA) is a life-threatening disease impacting immunocompromised individuals. Standard treatments of IA, including polyenes and azoles, suffer from high toxicity and emerging resistance, leading to the need to develop new antifungal agents with novel mechanisms of action. Ergosterol biosynthesis is a classic target for antifungals, and squalene synthase (SQS) catalyzes the first committed step in ergosterol biosynthesis in Aspergillus spp. making SQS of interest in the context of antifungal development. Here, we cloned, expressed, purified and characterized SQS from the pathogen Aspergillus flavus (AfSQS), confirming that it produced squalene. To identify potential leads targeting AfSQS, we tested known squalene synthase inhibitors, zaragozic acid and the phosphonosulfonate BPH-652, finding that they were potent inhibitors. We then screened a library of 744 compounds from the National Cancer Institute (NCI) Diversity Set V for inhibition activity. 20 hits were identified and IC 50 values were determined using dose-response curves. 14 compounds that interfered with the assay were excluded and the remaining 6 compounds were analyzed for drug-likeness, resulting in one compound, celastrol, which had an AfSQS IC 50 value of 830 nM. Enzyme inhibition kinetics revealed that celastrol binds to AfSQS in a noncompetitive manner, but did not bind covalently. Since celastrol is also known to inhibit growth of the highly virulent Aspergillus fumigatus by inhibiting flavin-dependent monooxygenase siderophore A (SidA, under iron starvation conditions), it may be a promising multi-target lead for antifungal development., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Osteoclast lysosomes secrete lytic enzymes into bone resorption lacunae, and sort the lysosomal proton pumping vacuolar-type ATPase (V-ATPase) to the plasma membrane to form the acidic environment required for bone digestion. The a3 isoform of V-ATPase is essential for outward trafficking of the secretory lysosomes and interacts physically with Rab7, a small GTPase that regulates trafficking of late endosomes and lysosomes, to recruit it to lysosomes. However, it is unclear whether organelle acidification by V-ATPase is required for the lysosome trafficking. Here, we showed that incubation of osteoclasts with the V-ATPase inhibitor bafilomycin A1 abolished the osteoclast-characteristic peripheral localization of secretory lysosomes, Rab7, and α-tubulin. Although bafilomycin A1 had little or no effect on Rab7 activation and its interaction with a3, treatment with the inhibitor significantly reduced the lysosomal localization of Rab7. Even constitutively active Rab7 did not localize to lysosomes in the presence of the inhibitor. These results suggest that organelle acidification by V-ATPase is required for localization of activated Rab7 to lysosomes., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Melanin plays an important role in the protection of the skin from ultraviolet irradiation. However, excessive melanin deposition leads to hyperpigmentation and freckles, which are recognized as skin problems, and signs of aging. Tyrosinase, a copper-containing protein, is the rate-limiting enzyme in melanin biosynthesis and first catalyzes the hydroxylation of l-tyrosine to 3,4-dihydroxyphenylalanine (DOPA) and the further oxidization to dopaquinone. To assist the proper regulation of melanin production, we screened compounds and found that 5,6,7,8-tetrahydro-4H-furo[3,2-c]azepine-4-thione (T4FAT), a thioamide derivative, inhibited melanogenesis in B16F10 mouse melanoma cells. T4FAT was not toxic to cells and was stable in water; in addition, it inhibited the activity of tyrosinase derived from mushroom and B16F10 cells in a non-competitive manner. T4FAT downregulated tyrosinase protein expression in B16F10 cells without affecting mRNA expression. As copper binding to the tyrosinase protein is required for both enzymatic activity, correct folding, and maturation, we examined the metal-chelating activities of T4FAT. Equimolar amount of T4FAT resulted in almost complete chelation of copper ions. The thioamide group of T4FAT is essential for copper chelation and tyrosinase inhibition, which subsequently resulted in melanogenesis inhibition in B16F10 cells. Although T4FAT has similar in vitro properties to kojic acid, which is also a copper chelator and approved as a component of cosmetic formulations, T4FAT inhibited melanogenesis in B16F10 cells 30 times more efficiently than kojic acid. These results suggested that T4FAT, a novel copper chelator, may be helpful for the development of new cosmetics for skin whitening., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Little is known about an oncogenic signal transducer β-1,4-galactosyltransferase-V (β-1,4-GalT-V), in human colorectal cancer. Using quantitative RT-PCR, immunohistochemical staining and ELISA assays, we determined that β-1,4-GalT-V gene/protein expression is specifically increased in human colorectal cancer (CRC) tumors, compared to visibly normal tissue. Furthermore, we observed a marked increase in its enzymatic activity, and its product lactosylceramide. Moreover, we found increased dihydrosphingolipid metabolites, in particular dihydrosphingomyelin in cancer tissue compared to normal. Further, inhibition of glycosphingolipid synthesis by the synthetic ceramide analog, D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP), concurrently inhibited colorectal cancer cell (HCT-116) proliferation, as well as β-1,4-GalT-V mass and several glycosphingolipid levels. We conclude that β-1,4-GalT-V may serve as a diagnostic and therapeutic biomarker for the progression of human colorectal cancer, and consequently, inhibition of GSL synthesis may be a novel approach for the treatment of this life-threatening disease., (Copyright © 2018. Published by Elsevier Inc.)

Sirtuins (SIRT1-7), a class of deacetylases, play major roles in DNA damage repair, aging, and metabolism in yeast and in mammals. SIRT7 is localized in the nucleolus. It regulates cellular processes, including genomic stability, rDNA transcription, and cell proliferation, and plays a role in tumorigenesis. SIRT7 deacetylates its substrates histone H3 (at lysine 18) and p53. p53, a tumor suppressor, induces apoptosis or cell cycle arrest and is stabilized by acetylation. p53 deacetylation at K382 by SIRT7 suppressed cancer cell growth by attenuating p53 activity. Therefore, identification of novel SIRT7 enzyme inhibitors is important. In this study, we found a novel inhibitor of SIRT7 (ID: 97491) that decreased SIRT7 activity in a dose-dependent manner. ID: 97491 induced expression of p53 and its acetylation by inhibited SIRT7. Moreover, ID: 97491 upregulated apoptotic effects through the caspase related proteins and inhibited cancer growth in vivo. The study results suggest that ID: 97491 can be a potential candidate to inhibit the deacetylase activity of SIRT7 and prevent tumor progression by increasing p53 stability through acetylation at K373/382., (Copyright © 2018. Published by Elsevier Inc.)

The NSD family (NSD1, NSD2/MMSET/WHSC1, and NSD3/WHSC1L1) are histone lysine methyltransferases (HMTases) essential for chromatin regulation. The NSDs are oncoproteins, drivers of a number of tumors and are considered important drug-targets but the lack of potent and selective inhibitors hampers further therapeutic development and limits exploration of their biology. In particular, MMSET/NSD2 selective inhibition is being pursued for therapeutic interventions against multiple myeloma (MM) cases, especially in multiple myeloma t(4;14)(p16.3;q32) translocation that is associated with a significantly worse prognosis than other MM subgroups. Multiple myeloma is the second most common hematological malignancy, after non-Hodgkin lymphoma and remains an incurable malignancy. Here we report the discovery of LEM-14, an NSD2 specific inhibitor with an in vitro IC 50 of 132 μM and that is inactive against the closely related NSD1 and NSD3. LEM-14-1189, a LEM-14 derivative, differentially inhibits the NSDs with in vitro IC 50 of 418 μM (NSD1), IC 50 of 111 μM (NSD2) and IC 50 of 60 μM (NSD3). We propose LEM-14 and derivative LEM-14-1189 as tools for studying the biology of the NSDs and constitute meaningful steps toward potent NSDs therapeutic inhibitors., (Copyright © 2018 Elsevier Inc. All rights reserved.)