A natural compound, aristoyagonine, is identified as a potent bromodomain inhibitor by mid-throughput screening.

Bromodomain-containing protein 4 (Brd4) is known to play a key role in tumorigenesis. It binds acetylated histones to regulate the expression of numerous genes. Because of the importance of brd4 in tumorigenesis, much research has been undertaken to develop brd4 inhibitors with therapeutic potential. As a result, various scaffolds for bromodomain inhibitors have been identified. To discover new scaffolds, we performed mid-throughput screening using two different enzyme assays, alpha-screen and ELISA. We found a novel bromodomain inhibitor with a unique scaffold, aristoyagonine. This natural compound showed inhibitory activity in vitro and tumor growth inhibition in a Ty82-xenograft mouse model. In addition, we tested Brd4 inhibitors in gastric cancer cell lines, and found that aristoyagonine exerted cytotoxicity not only in I-BET-762-sensitive cancer cells, but also in I-BET-762-resistant cancer cells. This is the first paper to describe a natural compound as a Brd4 bromodomain inhibitor.

Enzymatic prenylation and oxime ligation for the synthesis of stable and homogeneous protein-drug conjugates for targeted therapy.

Targeted therapy based on protein-drug conjugates has attracted significant attention owing to its high efficacy and low side effects. However, efficient and stable drug conjugation to a protein binder remains a challenge. Herein, a chemoenzymatic method to generate highly stable and homogenous drug conjugates with high efficiency is presented. The approach comprises the insertion of the CaaX sequence at the C-terminal end of the protein binder, prenylation using farnesyltransferase, and drug conjugation through an oxime ligation reaction. MMAF and an EGFR-specific repebody are used as the antitumor agent and protein binder, respectively. The method enables the precisely controlled synthesis of repebody-drug conjugates with high yield and homogeneity. The utility of this approach is illustrated by the notable stability of the repebody-drug conjugates in human plasma, negligible off-target effects, and a remarkable antitumor activity in vivo. The present method can be widely used for generating highly homogeneous and stable PDCs for targeted therapy.

IRE1 plays an essential role in ER stress-mediated aggregation of mutant huntingtin via the inhibition of autophagy flux.

Huntington's disease (HD), an inherited neurodegenerative disorder, is caused by an expansion of cytosine-adenine-guanine repeats in the huntingtin gene. The aggregation of mutant huntingtin (mtHTT) and striatal cell loss are representative features to cause uncontrolled movement and cognitive defect in HD. However, underlying mechanism of mtHTT aggregation and cell toxicity remains still elusive. Here, to find new genes modulating mtHTT aggregation, we performed cell-based functional screening using the cDNA expression library and isolated IRE1 gene, one of endoplasmic reticulum (ER) stress sensors. Ectopic expression of IRE1 led to its self-activation and accumulated detergent-resistant mtHTT aggregates. Treatment of neuronal cells with ER stress insults, tunicamycin and thapsigargin, increased mtHTT aggregation via IRE1 activation. The kinase activity of IRE1, but not the endoribonuclease activity, was necessary to stimulate mtHTT aggregation and increased death of neuronal cells, including SH-SY5Y and STHdhQ111/111 huntingtin knock-in striatal cells. Interestingly, ER stress impaired autophagy flux via IRE1-TRAF2 pathway, thus enhancing cellular accumulation of mtHTT. Atg5 deficiency in M5-7 cells increased mtHTT aggregation but blocked ER stress-induced mtHTT aggregation. Further, ER stress markers including p-IRE1 and autophagy markers such as p62 were up-regulated exclusively in the striatal tissues of HD mouse models and in HD patients. Moreover, down-regulation of IRE1 expression rescues the rough-eye phenotype by mtHTT in a HD fly model. These results suggest that IRE1 plays an essential role in ER stress-mediated aggregation of mtHTT via the inhibition of autophagy flux and thus neuronal toxicity of mtHTT aggregates in HD.

Intracellular cleavage of osteopontin by caspase-8 modulates hypoxia/reoxygenation cell death through p53.

Osteopontin (OPN) is highly expressed in cancer patients and plays important roles in many stages of tumor progression, such as anti-apoptosis, proliferation, and metastasis. From functional screening of human cDNA library, we isolated OPN as a caspase-8 substrate that regulates cell death during hypoxia/reoxygenation (Hyp/RO). In vitro cleavage assays demonstrate that OPN is cleaved at Asp-135 and Asp-157 by caspase-8. Cellular cleavage of OPN is observed in apoptotic cells exposed to Hyp/RO among various apoptotic stimuli and its cleavage is blocked by zVAD or IETD caspase inhibitor. Further, over-expression of OPN, the form with secretion signal, inhibits Hyp/RO-induced cell death. Caspase cleavage-defective OPN mutant (OPN D135A/D157A) is more efficient to suppress Hyp/RO-induced cell death than wild-type OPN. OPN D135A/D157A sustains AKT activity to increase cell viability through inhibition of caspase-9 during Hyp/RO. In addition, OPN is highly induced in some tumor cells during Hyp/RO, such as HeLa and Huh-7 cells, which is associated with their resistance to Hyp/RO by sustaining AKT activity. Notably, OPN C-terminal cleavage fragment produced by caspase-8 is detected in the nucleus. Plasmid-encoded expression of OPN C-terminal cleavage fragment increases p53 protein level and induces apoptosis of wild-type mouse embryonic fibroblast cells, but not p53(-/-) mouse embryonic fibroblast cells. These observations suggest that the protective function of OPN during Hyp/RO is inactivated via the proteolytic cleavage by caspase-8 and its cleavage product subsequently induces cell death via p53, postulating caspase-8 as a negative regulator of tumorigenic activity of OPN.

An Elaborate New Linker System Significantly Enhances the Efficacy of an HER2-Antibody-Drug Conjugate against Refractory HER2-Positive Cancers.

Human epidermal growth factor receptor 2 (HER2) is overexpressed in breast and gastric cancers and this causes poor clinical outcomes. Although both T-DM1 and Enhertu are approved as an HER2-targeting antibody-drug conjugate (ADC), the effects of these drugs are still not satisfactory to eradicate diverse tumors expressing HER2. To address this shortfall in HER2-targeted therapeutics, an elaborate cleavable linker is created and a novel HER2-targeting ADC composed with trastuzumab and monomethyl auristatin F, which is being investigated in a phase 1 clinical trial and is referred to as LegoChem Bisciences-ADC (LCB-ADC). LCB-ADC displays a higher cytotoxic potency than T-DM1 and it also has a higher G2/M arrest ratio. In animal studies, LCB-ADC produces noticeable tumor growth inhibition compared with trastuzumab or T-DM1 in an HER2 high-expressing N87 xenograft tumor. Especially, LCB-ADC shows good efficacy in terms of suppressing tumor growth in a patient-derived xenograft (PDX) model of HER2-positive gastric cancer as well as in T-DM1-resistant models such as HER2 low-expressing HER2 low expressing JIMT-1 xenograft tumor and PDX. Collectively, the results demonstrate that LCB-ADC with the elaborate linker has a higher efficacy and greater biostability than its ADC counterparts and may successfully treat cancers that are nonresponsive to previous therapeutics.

Role of FLASH in caspase-8-mediated activation of NF-kappaB: dominant-negative function of FLASH mutant in NF-kappaB signaling pathway.

Caspase-8 is the most receptor-proximal, upstream caspase in the caspase cascade and plays a key role in cell death triggered by various death receptors. Here, we addressed the role of endogenous caspase-8 in tumor necrosis factor (TNF)-alpha-induced activation of NF-kappaB. Direct targeting of caspase-8 with siRNA and antisense (AS) approaches abolished TNF-alpha-induced activation of NF-kappaB in NIH3T3, HeLa, and HEK293 cells as determined with luciferase reporter gene and cell fractionation assays. Reconstitution of caspase-8-deficient C33A cells with processing-defective (P/D) mutant of caspase-8 sensitized the cells to TNF-alpha for NF-kappaB activation. In contrast to wild-type caspase-8, death effector domain mutant replacing Asp73 with Ala (caspase-8 (D73A)) failed to activate NF-kappaB and to bind FLICE-associated huge protein (FLASH) in vitro and in vivo. Instead, caspase-8 (D73A) mutant bound to caspase-8 and blocked NF-kappaB activation triggered by TNF-alpha and caspase-8. In addition, expression of an NF-kappaB-activating domain-deletion mutant of FLASH or transfection of FLASH AS oligonucleotides abolished TNF-alpha and caspase-8, but not phorbol 12-myristate 13-acetate, -induced activation of NF-kappaB. Further, immunoprecipitation assays showed that caspase-8 formed triple complex with TRAF2 and FLASH. Taken together, these results suggest that endogenous caspase-8 mediates TNF-alpha-induced activation of NF-kappaB via FLASH.

Inhibition of Bcl10-mediated activation of NF-kappa B by BinCARD, a Bcl10-interacting CARD protein.

We have identified a novel CARD-containing protein from EST database. BinCARD (Bcl10-interacting protein with CARD). BinCARD was ubiquitously expressed. Co-immunoprecipitation, In vitro binding, mammalian two-hybrid, and immunostaining assays revealed that BinCARD interacted with Bcl10 through CARD. BinCARD potently suppressed NF-kappa B activation induced by Bcl10 and decreased the amounts of phosphorylated Bcl10. Mutations at the residue Leu17 or Leu65, which is highly conserved in CARD, abolished the inhibitory effects of BinCARD on both Bcl10-induced activation of NF-kappa B and phosphorylation of Bcl10. Further, expression of BinCARD inhibited Bcl10 phosphorylation induced by T cell activation signal. These results suggest that BinCARD interacts with Bcl10 to inhibit Bcl10-mediated activation of NF-kappa B and to suppress Bcl10 phosphorylation.

NecroX as a novel class of mitochondrial reactive oxygen species and ONOO⁻ scavenger.

Mitochondrial reactive oxygen species and reactive nitrogen species are proven to be major sources of oxidative stress in the cell; they play a prominent role in a wide range of human disorders resulting from nonapoptotic cell death. The aim of this study is to examine the cytoprotective effect of the NecroX series against harmful stresses, including pro-oxidant (tertiarybutylhydroperoxide), doxorubicin, CCl4, and hypoxic injury. In this study, these novel chemical molecules inhibited caspase-independent cell death with necrotic morphology, which is distinctly different from apoptosis, autophagy, and necroptosis. In addition, they displayed strong mitochondrial reactive oxygen species and ONOO⁻ scavenging activity. Further, oral administration of these molecules in C57BL/6 mice attenuated streptozotocin-induced pancreatic islet ß-cell destruction as well as CCl4-induced hepatotoxicity in vivo. Taken together, these results demonstrate that the NecroX series are involved in the blockade of nonapoptotic cell death against mitochondrial oxidative stresses. Thus, these chemical molecules are potential therapeutic agents in mitochondria-related human diseases involving necrotic tissue injury.

SCAMP5 links endoplasmic reticulum stress to the accumulation of expanded polyglutamine protein aggregates via endocytosis inhibition.

Accumulation of expanded polyglutamine proteins is considered to be a major pathogenic biomarker of Huntington disease. We isolated SCAMP5 as a novel regulator of cellular accumulation of expanded polyglutamine track protein using cell-based aggregation assays. Ectopic expression of SCAMP5 augments the formation of ubiquitin-positive and detergent-resistant aggregates of mutant huntingtin (mtHTT). Expression of SCAMP5 is markedly increased in the striatum of Huntington disease patients and is induced in cultured striatal neurons by endoplasmic reticulum (ER) stress or by mtHTT. The increase of SCAMP5 impairs endocytosis, which in turn enhances mtHTT aggregation. On the contrary, down-regulation of SCAMP5 alleviates ER stress-induced mtHTT aggregation and endocytosis inhibition. Moreover, stereotactic injection into the striatum and intraperitoneal injection of tunicamycin significantly increase mtHTT aggregation in the striatum of R6/2 mice and in the cortex of N171-82Q mice, respectively. Taken together, these results suggest that exposure to ER stress increases SCAMP5 in the striatum, which positively regulates mtHTT aggregation via the endocytosis pathway.

E2-25K/Hip-2 regulates caspase-12 in ER stress-mediated Abeta neurotoxicity.

Amyloid-beta (Abeta) neurotoxicity is believed to contribute to the pathogenesis of Alzheimer's disease (AD). Previously we found that E2-25K/Hip-2, an E2 ubiquitin-conjugating enzyme, mediates Abeta neurotoxicity. Here, we report that E2-25K/Hip-2 modulates caspase-12 activity via the ubiquitin/proteasome system. Levels of endoplasmic reticulum (ER)-resident caspase-12 are strongly up-regulated in the brains of AD model mice, where the enzyme colocalizes with E2-25K/Hip-2. Abeta increases expression of E2-25K/Hip-2, which then stabilizes caspase-12 protein by inhibiting proteasome activity. This increase in E2-25K/Hip-2 also induces proteolytic activation of caspase-12 through its ability to induce calpainlike activity. Knockdown of E2-25K/Hip-2 expression suppresses neuronal cell death triggered by ER stress, and thus caspase-12 is required for the E2-25K/Hip-2-mediated cell death. Finally, we find that E2-25K/Hip-2-deficient cortical neurons are resistant to Abeta toxicity and to the induction of ER stress and caspase-12 expression by Abeta. E2-25K/Hip-2 is thus an essential upstream regulator of the expression and activation of caspase-12 in ER stress-mediated Abeta neurotoxicity.

Potentiation of Fas- and TRAIL-mediated apoptosis by IFN-gamma in A549 lung epithelial cells: enhancement of caspase-8 expression through IFN-response element.

Epithelial cell apoptosis triggered cooperatively by multiple cytokines contributes to the injury induced by inflammatory responses in the lung and elsewhere. Here we show that interferon-gamma (IFN-gamma) sensitizes A549 cells, human lung epithelial cells, to cytokine-mediated apoptosis by upregulating caspase-8 expression. Pretreating the cells with IFN-gamma potentiated Fas- and TNF-related apoptosis inducing ligand (TRAIL)-induced cell death, but other forms of apoptosis, not mediated via receptors, were unaffected. Western blotting and inhibitor assays showed that IFN-gamma selectively increased expression of caspases-7 and -8, but not caspases-2, -3, -9, or -10, as a necessary step leading to apoptosis. Assaying promoter activity using a luciferase reporter gene indicated that an IFN-gamma response element was located in the 5'-flanking region of the caspase-8 gene, spanning positions -227 to -219. Taken together, these findings suggest that IFN-gamma potentiates Fas- and TRAIL-mediated apoptosis by increasing caspase-8 expression via an IFN-gamma response element in A549 cells.

Atypical role of proximal caspase-8 in truncated Tau-induced neurite regression and neuronal cell death.

Abnormal Tau protein is known to be closely associated with several neurodegenerative diseases. Previously, we showed that Tau was cleaved by caspase-3 to generate the cleavage product lacking the C-terminus (DeltaTau-1) during neuronal cell death. Here we characterized caspase-8-dependent neurotoxicity of the truncated Tau. Introduction of DeltaTau-1 into primary hippocampal neurons induced loss of neurites in a caspase-dependent manner. Caspase-8 and -6 were proteolytically activated during DeltaTau-1-triggered neuronal cell death, which was suppressed by IETD-fmk, caspase-8 inhibitor. Direct targeting of caspase-8 and its associated FADD with antisense approaches and transient expression of their dominant-negative mutants reduced DeltaTau-1-induced apopotosis. Cells deficient in caspase-8, but not caspase-3, became sensitized to DeltaTau-1-mediated toxicity upon reconstitution with caspase-8. In addition, ectopic expression of mitochondrial antiapoptotic Bcl-2, Bcl-X(L), or inactive caspase-9 short form suppressed DeltaTau-1 toxicity. These results suggest that the truncated Tau protein activates proximal caspase-8 through FADD as a necessary step leading to neuronal cell death and neurite regression, contributing to the progression of abnormal Tau-associated neurodegeneracy.

Caspase cleavage product lacking amino-terminus of IkappaBalpha sensitizes resistant cells to TNF-alpha and TRAIL-induced apoptosis.

In response to a diverse array of signals, IkappaBalpha is targeted for phosphorylation-dependent degradation by the proteasome, thereby activating NF-kappaB. Here we demonstrate a role of the cleavage product of IkappaBalpha in various death signals. During apoptosis of NIH3T3, Jurkat, Rat-1, and L929 cells exposed to tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), Fas, serum deprivation, or TNF-alpha, respectively, IkappaBalpha was cleaved in a caspase-dependent manner. In vitro and in vivo cleavage assays and site-directed mutagenesis showed that caspase-3 cleaved IkappaBalpha between Asp31 and Ser32. Expression of the cleavage product lacking amino-terminus (1-31), DeltaIkappaBalpha, sensitized otherwise resistant NIH3T3 fibroblast cells to apoptosis induced by TNF-alpha or TRAIL, and HeLa tumor cells to TNF-alpha. DeltaIkappaBalpha was more pro-apoptotic compared to wild type or cleavage-resistant (D31E)IkappaBalpha mutant and the sensitization elicited by DeltaIkappaBalpha was as effective as that by the dominant negative mutant, (S32,36A)IkappaBalpha, in NIH3T3 cells. DeltaIkappaBalpha suppressed the transactivation of NF-kappaB induced by TNF-alpha or TRAIL, as reflected by luciferase-reporter activity. Conversely, expression of the p65 subunit of NF-kappaB suppressed TNF-alpha-, TRAIL-, and serum deprivation-induced cell death. On the contrary, DeltaIkappaBalpha was less effective at increasing the death rate of HeLa cells that were already sensitive to death signals including TRAIL, etoposide, or taxol. These results suggest that DeltaIkappaBalpha generated by various death signals sensitizes cells to apoptosis by suppressing NF-kappaB activity.

Essential role of E2-25K/Hip-2 in mediating amyloid-beta neurotoxicity.

The ubiquitin/proteasome system has been proposed to play an important role in Alzheimer's disease (AD) pathogenesis. However, the critical factor(s) modulating both amyloid-beta peptide (Abeta) neurotoxicity and ubiquitin/proteasome system in AD are not known. We report the isolation of an unusual ubiquitin-conjugating enzyme, E2-25K/Hip-2, as a mediator of Abeta toxicity. The expression of E2-25K/Hip-2 was upregulated in the neurons exposed to Abeta(1-42) in vivo and in culture. Enzymatic activity of E2-25K/Hip-2 was required for both Abeta(1-42) neurotoxicity and inhibition of proteasome activity. E2-25K/Hip-2 functioned upstream of apoptosis signal-regulating kinase 1 (ASK1) and c-Jun N-terminal kinase (JNK) in Abeta(1-42) toxicity. Further, the ubiquitin mutant, UBB+1, a potent inhibitor of the proteasome which is found in Alzheimer's brains, was colocalized and functionally interacted with E2-25K/Hip-2 in mediating neurotoxicity. These results suggest that E2-25K/Hip-2 is a crucial factor in regulating Abeta neurotoxicity and could play a role in the pathogenesis of Alzheimer's disease.