Phytohabitols A-C, δ-Lactone-Terminated Polyketides from an Actinomycete of the Genus Phytohabitans.

Phytohabitols A-C (1-3), new terminally δ-lactonized linear polyketides, were isolated from the culture extract of a rare actinomycete of the genus Phytohabitans. The structures of 1-3, substituted with multiple methyl and hydroxy groups on a conjugated and a skipped diene-containing backbone, were elucidated by NMR and MS spectroscopic analyses. The absolute configuration of 1 was determined by chemical derivatization and chiral anisotropic analysis, coupled with ROESY and J-based configuration analysis. In addition, closely similar 1H and 13C NMR data and optical rotations among 1-3 supported the same stereochemistry of these polyketides. The related streptomycetes metabolites lagunapyrones B, C, and D have α-pyrone rings on the linear part in place of the δ-lactone, but their chirality at the C19-C21 stereocenters were opposite from those described here, posing a question on the previous assignment made solely by comparison of the optical rotations of four possible diastereomers. Compounds 1-3 inhibited migration of cancer cells with IC50 values of 15, 11, and 8.3 µM, respectively, at noncytotoxic concentrations. In addition, 1-3 displayed potent antitrypanosomal activity against Trypanosoma cruzi with IC50 values of 12, 6.4, and 18 µM, comparable to a commonly used therapeutic drug, benznidazole (IC50 16 µM).

Cyclopeptides from the Mushroom Pathogen Fungus Cladobotryum varium.

Three new cyclopeptides with serial Phe residues were identified with the aid of HPLC-DAD analysis, from the culture broth of Cladobotryum varium, a fungal pathogen causing mushroom cobweb disease. Cladoamides A (1) and B (2) have two consecutive N-methylphenylalanine units in the destruxin class cyclic depsipentapeptide framework, while cladoamide C (3) has a three consecutive Phe motif in a cyclopentapeptide structure. Of these three cyclopeptides, 1 showed potent autophagy-inducing activity at 10 µg/mL, comparable to a positive control, rapamycin. For the determination of the absolute configurations of the Ile residues in 1 and 3, new conditions for separating Ile and allo-Ile, using a pentafluorophenyl-bonded solid phase and methanolic solvent, were established within the analytical scheme of the advanced Marfey's method, thus offering a convenient alternative to the C3 Marfey's method, which requires elution with a three-solvent mixture. The sequence of two d-Phe and one l-Phe in 3 was determined through NMR chemical shift prediction by DFT-based calculations and chemical synthesis, which demonstrated the significance of noncovalent interactions in the accurate calculation of stable conformers for peptides with multiple aromatic rings.

14-3-3εa directs the pulsatile transport of basal factors toward the apical domain for lumen growth in tubulogenesis.

The Ciona notochord has emerged as a simple and tractable in vivo model for tubulogenesis. Here, using a chemical genetics approach, we identified UTKO1 as a selective small molecule inhibitor of notochord tubulogenesis. We identified 14-3-3εa protein as a direct binding partner of UTKO1 and showed that 14-3-3εa knockdown leads to failure of notochord tubulogenesis. We found that UTKO1 prevents 14-3-3εa from interacting with ezrin/radixin/moesin (ERM), which is required for notochord tubulogenesis, suggesting that interactions between 14-3-3εa and ERM play a key role in regulating the early steps of tubulogenesis. Using live imaging, we found that, as lumens begin to open between neighboring cells, 14-3-3εa and ERM are highly colocalized at the basal cortex where they undergo cycles of accumulation and disappearance. Interestingly, the disappearance of 14-3-3εa and ERM during each cycle is tightly correlated with a transient flow of 14-3-3εa, ERM, myosin II, and other cytoplasmic elements from the basal surface toward the lumen-facing apical domain, which is often accompanied by visible changes in lumen architecture. Both pulsatile flow and lumen formation are abolished in larvae treated with UTKO1, in larvae depleted of either 14-3-3εa or ERM, or in larvae expressing a truncated form of 14-3-3εa that lacks the ability to interact with ERM. These results suggest that 14-3-3εa and ERM interact at the basal cortex to direct pulsatile basal accumulation and basal-apical transport of factors that are essential for lumen formation. We propose that similar mechanisms may underlie or may contribute to lumen formation in tubulogenesis in other systems.

Protein kinase A inhibition facilitates the antitumor activity of xanthohumol, a valosin-containing protein inhibitor.

Xanthohumol (XN), a simple prenylated chalcone, can be isolated from hops and has the potential to be a cancer chemopreventive agent against several human tumor cell lines. We previously identified valosin-containing protein (VCP) as a target of XN; VCP can also play crucial roles in cancer progression and prognosis. Therefore, we investigated the molecular mechanisms governing the contribution of VCP to the antitumor activity of XN. Several human tumor cell lines were treated with XN to investigate which human tumor cell lines are sensitive to XN. Several cell lines exhibited high sensitivity to XN both in vitro and in vivo. shRNA screening and bioinformatics analysis identified that the inhibition of the adenylate cyclase (AC) pathway synergistically facilitated apoptosis induced by VCP inhibition. These results suggest that there is crosstalk between the AC pathway and VCP function, and targeting both VCP and the AC pathway is a potential chemotherapeutic strategy for a subset of tumor cells.

Involvement of the MEK/ERK pathway in EGF-induced E-cadherin down-regulation.

E-cadherin is a major component of the epithelial adherens junction. However, the regulatory mechanism of E-cadherin expression is still poorly understood. In this study, we found that EGF decreased E-cadherin expression at both mRNA and protein levels in colorectal carcinoma LoVo cells. Since E-cadherin down-regulation is a well-known hallmark of the EMT (Epithelial-Mesenchymal Transition), we investigated whether EGF induced E-cadherin down-regulation during the EMT. EGF was unable to affect the expression of mesenchymal markers (such as N-cadherin, vimentin or fibronectin) or EMT-regulating transcription factors (such as SNAIL, SLUG, ZEB1, ZEB2 or TWIST), suggesting that EGF induced E-cadherin down-regulation via an EMT-independent mechanism. On the other hand, the MEK inhibitor U0126 was found to suppress EGF-induced E-cadherin down-regulation at the transcriptional level, suggesting that the MEK/ERK pathway is involved in EGF-induced E-cadherin down-regulation. Moreover, we also found that EGF disrupted cell-cell contact, stimulated cells to form an elongated shape with filamentous protrusions, and induced cell migration in LoVo cells. These effects were suppressed by U0126. Therefore, EGF is suggested to induce E-cadherin down-regulation at the transcriptional level through the MEK/ERK pathway, which might result in, at least in part, the induction of cellular morphological changes and cell migration in LoVo cells.

Screening and target identification of bioactive compounds that modulate cell migration and autophagy.

Cell migration is a fundamental step for embryonic development, wound repair, immune responses, and tumor cell invasion and metastasis. It is well known that protrusive structures, namely filopodia and lamellipodia, can be observed at the leading edge of migrating cells. The formation of these structures is necessary for cell migration; however, the molecular mechanisms behind the formation of these structures remain largely unclear. Therefore, bioactive compounds that modulate protrusive structures are extremely powerful tools for studying the mechanisms behind the formation of these structures and subsequent cell migration. Therefore, we have screened for bioactive compounds that inhibit the formation of filopodia, lamellipodia, or cell migration from natural products, and attempted to identify the target molecules of our isolated compounds. Additionally, autophagy is a bulk, non-specific protein degradation system that is involved in the pathogenesis of cancer and neurodegenerative disorders. Recent extensive studies have revealed the molecular mechanisms of autophagy, however, they also remain largely unclear. Thus, we also have screened for bioactive compounds that modulate autophagy, and identified the target molecules. In the present article, we introduce the phenotypic screening system and target identification of four bioactive compounds.

Chemistry and biology of the compounds that modulate cell migration.

Cell migration is a fundamental step for embryonic development, wound repair, immune responses, and tumor cell invasion and metastasis. Extensive studies have attempted to reveal the molecular mechanisms behind cell migration; however, they remain largely unclear. Bioactive compounds that modulate cell migration show promise as not only extremely powerful tools for studying the mechanisms behind cell migration but also as drug seeds for chemotherapy against tumor metastasis. Therefore, we have screened cell migration inhibitors and analyzed their mechanisms for the inhibition of cell migration. In this mini-review, we introduce our chemical and biological studies of three cell migration inhibitors: moverastin, UTKO1, and BU-4664L.

Evaluation of drug toxicity profiles based on the phenotypes of ascidian Ciona intestinalis.

In vivo toxicity evaluation using model organisms is an important step for the development of new drugs. Here, we report that Ciona intestinalis, a chordate invertebrate, is beneficial to drug toxicity evaluation for the following reasons: rapid embryonic and larval development, resemblance to vertebrates, ease of management, low cost, transparent body, and low risk of ethical issues. The dynamic phenotypic change of Ciona larvae during metamorphosis prompted us to examine the effect of cytotoxic drugs on its development by quantifying six toxicity endpoints: degenerated tail size, ampulla length, rotation of body axis, stomach size, heart rate, and body size. As a result, mitochondrial respiratory inhibitors, tubulin polymerization/depolymerization inhibitors, or DNA/RNA synthesis inhibitors showed distinct toxicity profiles against these six endpoints, but drugs with the same targets showed a similar toxicity profile in Ciona. Our results suggest Ciona is an effective animal model for profiling drug toxicity and exploring the mechanisms of drugs with unknown targets.

Apoptosis induction associated with the ER stress response through up-regulation of JNK in HeLa cells by gambogic acid.

BACKGROUND: Gambogic acid (GA) was extracted from the dried yellow resin of gamboge (Garcinia hanburyi) which is traditionally used as a coloring material for painting and cloth dying. Gamboge has been also used as a folk medicine for an internal purgative and externally infected wound. We focused on the mechanisms of apoptosis induction by GA through the unfold protein response (ER stress) in HeLa cells. METHODS: The cytotoxic effect of GA against HeLa cells was determined by trypan blue exclusion assay. Markers of ER stress such as XBP-1, GRP78, CHOP, GADD34 and ERdj4 were analyzed by RT-PCR and Real-time RT-PCR. Cell morphological changes and apoptotic proteins were performed by Hoechst33342 staining and Western blotting technique. RESULTS: Our results indicated a time- and dose-dependent decrease of cell viability by GA. The ER stress induction is determined by the up-regulation of spliced XBP1 mRNA and activated GRP78, CHOP, GADD34 and ERdj4 expression. GA also induced cell morphological changes such as nuclear condensation, membrane blebbing and apoptotic body in Hela cells. Apoptosis cell death detected by increased DR5, caspase-8, -9, and -3 expression as well as increased cleaved-PARP, while decreased Bcl-2 upon GA treatment. In addition, phosphorylated JNK was up-regulated but phosphorylated ERK was down-regulated after exposure to GA. CONCLUSIONS: These results suggest that GA induce apoptosis associated with the ER stress response through up-regulation of p-JNK and down-regulation of p-ERK in HeLa cells.

[Metabolomics and molecular targeted therapy of cancer].

Metabolomics is defined as the quantitative measurement of the dynamic multiparametric metabolites. Recent technological advances in the quantification of cellular metabolites, such as capillary electrophoresis (CE) -TOFMS, have prompted the comprehensive analysis of the global metabolism. Now, metabolomics has been used in the identification of new biomarker for toxicology and disease diagnosis, and the elucidation of fermentation processes. Moreover, it is considered that metabolomics would be a powerful tool for the target identification of small molecular bioactive compounds. Here we introduce our recent study that metabolomics was applied to identify the molecular target of glucopiericidin A, which was isolated through a screening of natural products for an inhibitor of cellular filopodia protrusion in carcinoma cells.

5-Lipoxygenase and cysteinyl leukotriene receptor 1 regulate epidermal growth factor-induced cell migration through Tiam1 upregulation and Rac1 activation.

Cell migration is an essential step for tumor metastasis. The small GTPase Rac1 plays an important role in cell migration. Previously, we reported that epidermal growth factor (EGF) induced two waves of Rac1 activation; namely, at 5 min and 12 h after stimulation. A second wave of EGF-induced Rac1 activation was required for EGF-induced cell migration, however, the spatiotemporal regulation of the second wave of EGF-induced Rac1 activation remains largely unclear. In this study, we found that 5-lipoxygenase (5-LOX) is activated in the process of EGF-induced cell migration, and that leukotriene C4 (LTC4 ) produced by 5-LOX mediated the second wave of Rac1 activation, as well as cell migration. Furthermore, these effects caused by LTC4 were found to be blocked in the presence of the antagonist of cysteinyl leukotriene receptor 1 (CysLT1). This blockage indicates that LTC4 -mediated CysLT1 signaling regulates the second EGF-induced wave of Rac1 activation. We also found that 5-LOX inhibitors, CysLT1 antagonists and the knockdown of CysLT1 inhibited EGF-induced T cell lymphoma invasion and metastasis-inducing protein 1 (Tiam1) expression. Tiam1 expression is required for the second wave of EGF-induced Rac1 activation in A431 cells. Therefore, our results indicate that the 5-LOX/LTC4 /CysLT1 signaling pathway regulates EGF-induced cell migration by increasing Tiam1 expression, leading to a second wave of Rac1 activation. Thus, CysLT1 may serve as a new molecular target for antimetastatic therapy. In addition, the CysLT1 antagonist, montelukast, which is used clinically for allergy treatment, might have great potential as a novel type of antimetastatic agent.

Boron-based inhibitors of acyl protein thioesterases 1 and 2.

Ras proteins are of importance in cell proliferation, and hence their mutated forms play causative roles in many kinds of cancer in different tissues. Inhibition of the Ras-depalmitoylating enzyme acyl protein thioesterases APT1 and -2 is a new approach to modulating the Ras cycle. Here we present boronic and borinic acid derivatives as a new class of potent and nontoxic APT inhibitors. These compounds were detected by extensive library screening using chemical arrays and turned out to inhibit human APT1 and -2 in a competitive mode. Furthermore, one of the molecules was demonstrated to inhibit Erk1/2 phosphorylation significantly.

Establishment of a new detection system for the dimerization of IRE1α by BiFC assay.

We developed a new detection system for the activation of an endoplasmic reticulum (ER) stress sensor, inositol requiring kinase 1 α (IRE1α), by evaluating dimerization of it by bimolecular fluorescence complementation (BiFC) assay. By detecting the fluorescence derived from the reconstituted cerulean, this assay system enabled us to distinguish the activation behaviors of IRE1α as to ER stress-inducing compounds.

Comparative analysis of the expression patterns of UPR-target genes caused by UPR-inducing compounds.

Endoplasmic reticulum (ER) stress, due to an accumulation of unfolded proteins in the ER, leads to a process known as the unfolded protein response (UPR). Since the several compounds used to induce UPR have different modes of action, their mechanisms of protein accumulation are thought to be different, but it is unclear whether these compounds can upregulate UPR target genes with similar kinetics. Hence, we sought to compare the expression patterns of nine UPR target genes induced by seven UPR-inducing compounds. Hierarchical clustering analysis revealed that the expression patterns of the UPR target genes induced by the seven compounds were classified into two clusters; cluster A (thapsigargin, tunicamycin, 2-deoxyglucose, and dithiothreitol) and cluster B (brefeldin A, monensin, and eeyarestatin I). Thus, this study suggests the existence of at least two types of UPR target gene expression profiles, which depend on the mode of action of the compounds.

Cisplatin-induced activation of TGF-ß signaling contributes to drug resistance.

Growing evidence suggests an association between epithelial-mesenchymal transition (EMT), a hallmark of tumor malignancy, and chemoresistance to a number of anti-cancer drugs. However, the mechanism of EMT induction in the process of acquiring anti-cancer drug resistance remains unclear. To address this issue, we obtained a number of cisplatin-resistant clones from LoVo cells and found that almost all of them lost cell-cell contacts. In these clones, the epithelial marker E-cadherin was downregulated, whereas the mesenchymal marker N-cadherin was upregulated. Moreover, the expression of EMT-related transcription factors, including Slug, was elevated. On the other hand, the upregulation of other mesenchymal marker Vimentin was weak, suggesting that the mesenchymal-like phenotypic changes occurred in these cisplatin-resistant clones. These mesenchymal-like features of cisplatin-resistant clones were partially reversed to parental epithelial-like features by treatment with transforming growth factor-ß (TGF-ß) receptor kinase inhibitors, indicating that TGF-ß signaling is involved in cisplatin-induced the mesenchymal-like phenotypic changes. Moreover, cisplatin was observed to enhance the secretion of TGF-ß into the culture media without influencing TGF-ß gene transcription. These results suggest that cisplatin may induce the mesenchymal-like phenotypic changes by enhancing TGF-ß secretion, ultimately resulting in drug resistance.

Involvement of 14-3-3 proteins in the second epidermal growth factor-induced wave of Rac1 activation in the process of cell migration.

Immense previous efforts have elucidated the core machinery in cell migration, actin remodeling regulated by Rho family small GTPases including RhoA, Cdc42, and Rac1; however, the spatiotemporal regulation of these molecules remains largely unknown. Here, we report that EGF induces biphasic Rac1 activation in the process of cell migration, and UTKO1, a cell migration inhibitor, inhibits the second EGF-induced wave of Rac1 activation but not the first wave. To address the regulation mechanism and role of the second wave of Rac1 activation, we identified 14-3-3ζ as a target protein of UTKO1 and also showed that UTKO1 abrogated the binding of 14-3-3ζ to Tiam1 that was responsible for the second wave of Rac1 activation, suggesting that the interaction of 14-3-3ζ with Tiam1 is involved in this event. To our knowledge, this is the first report to use a chemical genetic approach to demonstrate the mechanism of temporal activation of Rac1.

Antitumor effects of novel highly hydrophilic and non-ATP-competitive MEK1/2 inhibitor, SMK-17.

The mitogen-activated protein kinase (MAPK) signal pathway plays a central role in regulating tumor cell proliferation, survival, and differentiation. The components of this pathway, Ras/Raf/MEK/ERK, are frequently activated in human cancers. Targeting this pathway is considered to be a promising anticancer strategy. In particular, MEK is an attractive drug target because of its high selectivity to ERK. We can expect potent growth inhibitory and proapoptotic effects by inhibiting MEK. Here, we report derivatives of N-[2-(2-chloro-4-iodo-phenylamino)-3,4-difluorophenyl]-methanesulfonamide as novel MEK1/2 inhibitors. Among these compounds, we found SMK-17 to be a potent MEK1/2 inhibitor with high aqueous solubility. The in-silico docking study suggested that SMK-17 is bound to an allosteric pocket of MEK1. The kinetic study and the kinase profiler analysis confirmed the allosteric nature of SMK-17. SMK-17 inhibited MEK1 kinase activity in a non-ATP-competitive manner and it was highly selective to MEK1 and 2. SMK-17 inhibited the growth of tumor cell lines in vitro. Especially, it seemed that cell lines harboring highly phosphorylated MEK1/2 and ERK1/2 were highly sensitive to SMK-17. Moreover, unlike previously reported MEK inhibitors, PD184352 or U0126, SMK-17 did not inhibit the phosphorylation of ERK5. In vivo, SMK-17 exhibited potent antitumor activity in animal models on oral administration. SMK-17 selectively blocked the MAPK pathway signaling without affecting other signal pathways, which resulted in significant antitumor efficacy without notable side effects. These findings suggest that SMK-17, an exquisitely selective, orally available MEK1/2 inhibitor, is a useful chemical biology tool for characterizing the function of MEK/MAPK signaling both in vitro and in vivo.

Target identification of bioactive compounds.

To fully understand the regulation of cellular events, functional analysis of each protein involved in the regulatory systems is required. Among a variety of methods to uncover protein function, chemical genetics is a remarkable approach in which small molecular compounds are used as probes to elucidate protein functions within signaling pathways. However, identifying the target of small molecular bioactive compounds isolated by cell-based assays represents a crucial hurdle that must be overcome before chemical genetic studies can commence. A variety of methods and technologies for identifying target proteins have been reported. This review therefore aims to describe approaches for identifying these molecular targets.

Bisprenyl naphthoquinone and chlorinated calcimycin congener bearing thiazole ring from an actinomycete of the genus Phytohabitans.

A bisprenyl naphthoquinone, phytohabinone (1), and a calcimycin congener with unusual modifications, phytohabimicin (2), were isolated from the culture extract of Phytohabitans sp. RD003013. The structures of 1 and 2 were determined by NMR and MS analyses, and the absolute configuration of 2 was established by using electronic circular dichroism (ECD) calculation. The prenylation pattern of 1 was unprecedented among the known prenylated naphthoquinones. Compound 2 represents a spiroacetal core of polyketide origin substituted with a thiazole carboxylic acid and a dichrolopyrrole moiety, which is an unprecedented modification pattern in the known calcimycin family natural products. Remarkably, 2 showed moderate antimicrobial activity against a Gram-negative bacterium Ralstonia solanacearum while calcimycin was inactive. Additionally, 2 inhibits the migration of EC17 cancer cells at noncytotoxic concentrations.

Isolation of Caldorazole, a Thiazole-Containing Polyketide with Selective Cytotoxicity under Glucose-Restricted Conditions.

Caldorazole (1) was isolated from the marine cyanobacterium Caldora sp. collected on Ishigaki Island, Okinawa, Japan. Its structure was determined to be a new polyketide that contained two thiazole rings and an O-methylenolpyruvamide moiety. Caldorazole (1) showed strong cytotoxicity toward tumor cells that had been seeded at a high density. Cell death induced by 1 in HeLa and A431 cells was also observed only in the presence of the glycolysis blocker 2-deoxy-d-glucose (2DG). Co-treatment with 1 and 2DG remarkably decreased ATP levels in these cells. Furthermore, 1 selectively inhibited complex I in the mitochondrial respiratory chain. Thus, 1 was demonstrated to exert cytotoxicity toward human tumor cells by blocking mitochondrial respiration.