Caldomycin, a new guanidopolyamine produced by a novel agmatine homocoupling enzyme involved in homospermidine biosynthesis.

An extreme thermophilic bacterium, Thermus thermophilus produces more than 20 unusual polyamines, but their biosynthetic pathways, including homospermidine, are not yet fully understood. Two types of homospermidine synthases have been identified in plants and bacteria, which use spermidine and putrescine or two molecules of putrescine as substrates. However, homospermidine synthases with such substrate specificity have not been identified in T. thermophilus. Here we identified a novel agmatine homocoupling enzyme that is involved in homospermidine biosynthesis in T. thermophilus. The reaction mechanism is different from that of a previously described homospermidine synthase, and involves conjugation of two molecules of agmatine, which produces a diamidino derivative of homospermidine (caldomycin) as an immediate precursor of homospermidine. We conclude that there is a homospermidine biosynthetic pathway from agmatine via caldomycin synthase followed by ureohydrolase in T. thermophilus. Furthermore, it is shown that caldomycin is a novel compound existing in nature.

Synthesis and Biological Evaluation of 2-Azolylmethylene-3-(2H)-benzofuranone Derivatives as Potent Monoamine Oxidases Inhibitors.

A series of 2-azolylmethylene-3-(2H)-benzofuranone derivatives, 2-indolylmethylene-3-(2H)-benzofuranone and 2-pyrrolylmethylene-3-(2H)-benzofuranone derivatives, were synthesized, and their monoamine oxidase (MAO) A and B inhibitory activities were evaluated. Compounds 1b, 3b, 6b, 7b, and 10b showed strong inhibitory activity against MAO-A, and compound 3b showed the highest potency and selectivity, with an IC50 value of 21 nM and a MAO-A selectivity index of 48. Compounds 3c, 4c, 9a, 9c, 10c, 11a, and 11c showed strong inhibitory activity against MAO-B, and compound 4c showed the highest potency and selectivity, with an IC50 value of 16 nM and a MAO-B selectivity index of >1100. Further analysis of these compounds indicated that compound 3b for MAO-A and compound 4c for MAO-B were competitive inhibitors, with Ki values of 10 and 6.1 nM, respectively. Furthermore, computational analyses, such as quantitative structure-activity relationship (QSAR) analysis of the 2-azolylmethylene-3-(2H)-benzofuranone derivatives conducting their pIC50 values with the Molecular Operating Environment (MOE) and Mordred, and molecular docking analysis using MOE-Dock supported that the 2-azolylmethylene-3-(2H)-benzofuranone derivatives are a privileged scaffold for the design and development of novel MAO inhibitors.

A Tricyclic Aromatic Polyketide Isolated from the Marine-Derived Fungus Curvularia aeria.

A novel tricyclic polyketide, curvulanone (1), was isolated from the marine-derived fungus Curvularia aeria. The structure of 1 was determined by NMR and single-crystal X-ray crystallography. 1 had a cyclopentabenzopyranone with 3-acetic acid structure that is rarely found in natural compounds. Monoamine oxidase and sirtuin 1 inhibitory test was exhibited and 1 showed their inhibitory activity.

Synthesis of 2,8-Dioxabicyclo[3.3.1]nonane Derivatives and Their Neuroprotective Activities.

Twenty natural-product-like 2,8-dioxabicyclo[3.3.1]nonane derivatives were synthesized and their neuroprotective activities were tested using human monoamine oxidases (MAO) A and B and acetyl and butyryl cholinesterases (ChE). Compound 1s showed inhibitory activity for MAO-A, MAO-B and acetylcholinesterase (AChE) (IC50 values 34.0, 2.3 and 11.0 µM, respectively). The inhibition mode of (-)-1s for MAO-B was investigated. Chiral HPLC of (±)-1s separated the enantiomers and (-)-1s showed MAO-B inhibitory activity. Molecular docking simulation of (-)-1s and MAO-B revealed the binding mode.

Intracellular polyamine depletion induces N-linked galactosylation of the monoclonal antibody produced by CHO DP-12 cells.

The heterogeneity of the N-linked glycan profile of therapeutic monoclonal antibodies (mAbs) derived from animal cells affects therapeutic efficacy and, therefore, needs to be appropriately controlled during the manufacturing process. In this study, we examined the effects of polyamines on the N-linked glycan profiles of mAbs produced by CHO DP-12 cells. Normal cell growth of CHO DP-12 cells and their growth arrest by α-difluoromethylornithine (DFMO), an inhibitor of the polyamine biosynthetic pathway, was observed when 0.5% fetal bovine serum was added to serum-free medium, despite the presence of cadaverine and aminopropylcadaverine, instead of putrescine and spermidine in cells. Polyamine depletion by DFMO increased IgG galactosylation, accompanied by ß1,4-galactosyl transferase 1 (B4GAT1) mRNA elevation. Additionally, IgG production in polyamine-depleted cells was reduced by 30% compared to that in control cells. Therefore, we examined whether polyamine depletion induces an ER stress response. The results indicated increased expression levels of chaperones for glycoprotein folding in polyamine-depleted cells, suggesting that polyamine depletion causes ER stress related to glycoprotein folding. The effect of tunicamycin, an ER stress inducer that inhibits N-glycosylation, on the expression of B4GALT1 mRNA was examined. Tunicamycin treatment increased B4GALT1 mRNA expression. These results suggest that ER stress caused by polyamine depletion induces B4GALT1 mRNA expression, resulting in increased IgG galactosylation in CHO cells. Thus, introducing polyamines, particularly SPD, to serum-free CHO culture medium for CHO cells may contribute to consistent manufacturing and quality control of antibody production.

Inhibition of Polyamine Catabolism Reduces Cellular Senescence.

The aging of the global population has necessitated the identification of effective anti-aging technologies based on scientific evidence. Polyamines (putrescine, spermidine, and spermine) are essential for cell growth and function. Age-related reductions in polyamine levels have been shown to be associated with reduced cognitive and physical functions. We have previously found that the expression of spermine oxidase (SMOX) increases with age; however, the relationship between SMOX expression and cellular senescence remains unclear. Therefore, we investigated the relationship between increased SMOX expression and cellular senescence using human-liver-derived HepG2 cells. Intracellular spermine levels decreased and spermidine levels increased with the serial passaging of cells (aged cells), and aged cells showed increased expression of SMOX. The levels of acrolein-conjugated protein, which is produced during spermine degradation, also increases. Senescence-associated ß-gal activity was increased in aged cells, and the increase was suppressed by MDL72527, an inhibitor of acetylpolyamine oxidase (AcPAO) and SMOX, both of which are enzymes that catalyze polyamine degradation. DNA damage accumulated in aged cells and MDL72527 reduced DNA damage. These results suggest that the SMOX-mediated degradation of spermine plays an important role in cellular senescence. Our results demonstrate that cellular senescence can be controlled by inhibiting spermine degradation using a polyamine-catabolizing enzyme inhibitor.

Potentiation of Anticancer Activity of G2/M Blockers by Mild Hyperthermia.

BACKGROUND/AIM: Hyperthermia (HT), combined with chemotherapy, has been used to treat various types of cancer. This study aimed to investigate the HT-sensitivity of malignant and non-malignant cells, and then evaluate the combination effect of docetaxel (DTX) and a newly synthesized chromone derivative (compound A) with HT. MATERIALS AND METHODS: The number of viable cells was determined using the MTT method. Cell cycle distribution was analyzed using a cell sorter, and DNA fragmentation pattern was detected using agarose gel electrophoresis. RESULTS: Among 12 cultured cells, oral squamous cell carcinoma (OSCC), especially Ca9-22 cells, and myelogenous leukemia cells showed higher sensitivity to HT than lung carcinoma and glioblastoma cell lines, while normal oral cells were the most resistant. Cytotoxicity of DTX on Ca9-22 cells was maximum at 41-42°C and 45~60 min exposure to HT. DXT, compound A, and HT induced G2/M arrest of Ca-22 cells. Mild HT enhanced the DTX- and compound A-induced subG1 arrest, in a synergistic fashion. CONCLUSION: The combination G2/M blockers and mild-HT can potentially be used for the treatment of OSCC.

A Comparative Study of Tumor-Specificity and Neurotoxicity between 3-Styrylchromones and Anti-Cancer Drugs.

Background. Many anti-cancer drugs used in clinical practice cause adverse events such as oral mucositis, neurotoxicity, and extravascular leakage. We have reported that two 3-styrylchromone derivatives, 7-methoxy-3-[(1E)-2-phenylethenyl]-4H-1-benzopyran-4-one (Compound A) and 3-[(1E)-2-(4-hydroxyphenyl)ethenyl]-7-methoxy-4H-1-benzopyran-4-one (Compound B), showed the highest tumor-specificity against human oral squamous cell carcinoma (OSCC) cell lines among 291 related compounds. After confirming their superiority by comparing their tumor specificity with newly synthesized 65 derivatives, we investigated the neurotoxicity of these compounds in comparison with four popular anti-cancer drugs. Methods: Tumor-specificity (TSM, TSE, TSN) was evaluated as the ratio of mean CC50 for human normal oral mesenchymal (gingival fibroblast, pulp cell), oral epithelial cells (gingival epithelial progenitor), and neuronal cells (PC-12, SH-SY5Y, LY-PPB6, differentiated PC-12) to OSCC cells (Ca9-22, HSC-2), respectively. Results: Compounds A and B showed one order of magnitude higher TSM than newly synthesized derivatives, confirming its prominent tumor-specificity. Docetaxel showed one order of magnitude higher TSM, but two orders of magnitude lower TSE than Compounds A and B. Compounds A and B showed higher TSM, TSE, and TSN values than doxorubicin, 5-FU, and cisplatin, damaging OSCC cells at concentrations that do not affect the viability of normal epithelial and neuronal cells. QSAR prediction based on the Tox21 database suggested that Compounds A and B may inhibit the signaling pathway of estrogen-related receptors.

Assay of Arginyltransferase Activity by a Fluorescent HPLC Method.

Syntheses of fluorescent substrate and product for arginyltransferase, N-aspartyl-4-dansylamidobutylamine (Asp4DNS) and N-arginylaspartyl-4-dansylamidobutylamine (ArgAsp4DNS), respectively, including their precursor 4-dansylamidobutylamine (4DNS), are described. Then, HPLC conditions are summarized for a baseline separation of the three compounds in 10 min. The present method, which permits the simultaneous determination of Asp4DNS, 4DNS, and ArgAsp4DNS (in eluting order), is advantageous in measuring arginyltransferase activity and detecting the unfavorable enzyme(s) in 105,000 × g supernatant of tissues to ensure accurate determination.

Putrescine Biosynthesis from Agmatine by Arginase (TtARG) in Thermus thermophilus.

In the three domains of life, three biosynthetic pathways are known for putrescine. The first route is conversion of ornithine to putrescine by ornithine decarboxylase (ODC: SpeC), the second route is the conversion of arginine to agmatine by arginine decarboxylase (ADC: SpeA), followed by the conversion of agmatine to putrescine by agmatine ureohydrolase (AUH: SpeB), and the third route is the conversion of agmatine to N-carbamoylputrescine by agmatine deiminase (agmatine iminohydrolase, AIH), followed by the conversion of N-carbamoylputrescine to putrescine by N-carbamoylputrescine amidohydrolase (NCPAH). An extreme thermophile, Thermus thermophilus produces putrescine, although this bacterium lacks homologs for putrescine synthesizing pathways, such as ODC, AUH, AIH and NCPAH. To identify genes involved in putrescine biosynthesis in T. thermophilus, putrescine biosynthesis was examined by disruption of a predicted gene for agmatinase (agmatine ureohydrolase), or by using purified enzyme. It was found that arginase (TTHA1496) showed an agmatinase activity utilizing agmatine as a substrate. These results indicate that this bacterium can use arginase for putrescine biosynthesis. Arginase is a major contributor to putrescine biosynthesis under physiological conditions. The presence of an alternative pathway for converting agmatine into putrescine is functionally important for polyamine metabolism supporting survival at extreme environments.

Total Synthesis and Monoamine Oxidase Inhibitory Activities of (±)-Entonalactam A and Its Derivatives.

The first total synthesis of isoindolinone (±)-entonalactam A (6), originally obtained from the fungus Entonaema sp., was achieved in 14 steps from commercially available 5-bromovanillin via benzophenone intermediates. Isoindolinone, phthalide, and benzophenone analogues of natural products were also synthesized. The monoamine oxidase (MAO) A and B inhibitory activities were tested. The isoindolinone derivative 30 exhibited inhibition of both MAO-A and -B (IC50 = 17.8 and 15.8 µM, respectively).

Antifungal activity of dehydrocurvularin for Candida spp. through the inhibition of adhesion to human adenocarcinoma cells.

Cell adhesion plays a crucial role in candidiasis through invasion of the human body and obtaining resistance to drugs by forming biofilms. Cell adhesion thus is a critical target for combating candidiasis by preventing the entry of fungal hyphae into the epithelium. We report here that dehydrocurvularin (1), isolated from the marine-derived fungus Curvularia aeria, exhibited anti-fungal activities for Candida albicans and Candida auris. This compound also prevented the adherence of C. albicans to human adenocarcinoma cells. Real-time RT-PCR analysis showed that exposure to 1 results in decreased expression of HWP1, EFG1, and ECE1, genes involved in Candida adhesion to epithelial cells and hyphal morphogenesis.

Polyamines produced by an extreme thermophile are essential for cell growth at high temperature.

An extreme thermophile, Thermus thermophilus grows at an optimum temperature of around 70°C and produces 16 different polyamines including long-chain and branched-chain polyamines. We found that the composition of polyamines in the thermophile cells changes with culture temperature. Long-chain and branched-chain polyamines (unusual polyamines) were increased in the cells grown at high temperature such as 80°C, but they were minor components in the cells grown at relatively lower temperature such as 60°C. The effects of polyamines on cell growth were studied using T. thermophilus HB8 ΔspeA deficient in arginine decarboxylase. Cell growth of this mutant strain was significantly decreased at 70°C. This mutant strain cannot produce polyamines and grows poorly at 75°C. It was also determined whether polyamines are directly involved in protecting DNA from DNA double-strand breaks (DSBs) induced by heat. Polyamines protected DNA against double-strand breaks. Therefore, polyamines play essential roles in cell growth at extremely high temperature through maintaining a functional conformation of DNA against DSBs and depurination.

A Dual Anti-Inflammatory and Anti-Proliferative 3-Styrylchromone Derivative Synergistically Enhances the Anti-Cancer Effects of DNA-Damaging Agents on Colon Cancer Cells by Targeting HMGB1-RAGE-ERK1/2 Signaling.

The current anti-cancer treatments are not enough to eradicate tumors, and therefore, new modalities and strategies are still needed. Most tumors generate an inflammatory tumor microenvironment (TME) and maintain the niche for their development. Because of the critical role of inflammation via high-mobility group box 1 (HMGB1)-receptor for advanced glycation end-products (RAGE) signaling pathway in the TME, a novel compound possessing both anti-cancer and anti-inflammatory activities by suppressing the HMGB1-RAGE axis provides an effective strategy for cancer treatment. A recent work of our group found that some anti-cancer 3-styrylchromones have weak anti-inflammatory activities via the suppression of this axis. In this direction, we searched such anti-cancer molecules possessing potent anti-inflammatory activities and discovered 7-methoxy-3-hydroxy-styrylchromone (C6) having dual suppressive activities. Mechanism-of-action studies revealed that C6 inhibited the increased phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) under the stimulation of HMGB1-RAGE signaling and thereby suppressed cytokine production in macrophage-like RAW264.7 cells. On the other hand, in colorectal cancer HCT116 cells, C6 inhibited the activation of ERK1/2, cyclin-dependent kinase 1, and AKT, down-regulated the protein level of XIAP, and up-regulated pro-apoptotic Bax and caspase-3/7 expression. These alterations are suggested to be involved in the C6-induced suppression of cell cycle/proliferation and initiation of apoptosis in the cancer cells. More importantly, in cancer cells, the treatment of C6 potentiates the anti-cancer effects of DNA-damaging agents. Thus, C6 may be a promising lead for the generation of a novel class of cancer therapeutics.

A benzaldehyde derivative obtained from Hypoxylon truncatum NBRC 32353 treated with hygromycin B.

The ribosome-targeted antifungal agent hygromycin B (HygB) alters the secondary metabolite profiles of fungi. Hypoxylon truncatum NBRC 32353 fermented in the presence of hygromycin B in barley medium activated secondary metabolite synthesis. A new benzaldehyde derivative truncaaldehyde (1) was obtained, along with thirteen known compounds (2-14). The structures of the new compounds were revealed using NMR and single-crystal X-ray crystallography. The total synthesis of (±)-1 was achieved using a four-step sequence, and chiral separation was accomplished. The isolated compounds were tested for their monoamine oxidase (MAO) -A and -B inhibitory activities, with six compounds ((±)-1, 4, 5, 7, 8, and 10) showing inhibitory activity.

Stimulation of the Runx2 P1 promoter by collagen-derived dipeptide prolyl-hydroxyproline bound to Foxg1 and Foxo1 in osteoblasts.

Collagen-derived dipeptide prolyl-hydroxyproline (Pro-Hyp) directly binds to the forkhead box g1 (Foxg1) protein and causes it to undergo structural alteration. Pro-Hyp also promotes the production of a regulator of osteoblast differentiation, Runt-related transcription factor 2 (Runx2), through Foxg1, inducing osteoblast differentiation. In addition, Pro-Hyp disrupts the interaction between Foxg1 and Runx2, and Foxg1 appears to interact with Runx2 in the absence of Pro-Hyp. To elucidate the mechanism of Pro-Hyp that promotes osteoblast differentiation, we investigated whether Pro-Hyp regulates the Runx2 P1 promoter together with Foxg1. The present study revealed that Pro-Hyp is taken up by osteoblastic cells via the solute carrier family 15 member (Slc15a) 4. In the presence of Pro-Hyp, Runx2 is translocated from the nucleus to the cytoplasm and Foxg1 is translocated from the cytoplasm to the nucleus. We also found that Pro-Hyp promoted the interaction between Forkhead box o1 (Foxo1) and Runx2 and the dissociation of Foxg1 from Runx2. Moreover, we identified the Pro-Hyp response element in the Runx2 distal P1 promoter at nt -375 to -316, including the Runx2 binding sites and Fox core sequence. In the presence of Pro-Hyp, Runx2 is dissociated from the Pro-Hyp response element in the Runx2 distal P1 promoter. Subsequently, Foxg1 and Foxo1 activated the Runx2 promoter by binding to the Pro-Hyp response element. In summary, we delineated the mechanism by which Pro-Hyp stimulates the bone-related Runx2 distal P1 promoter activity in osteoblastic cells through Foxg1, Foxo1, and Runx2.

Synthesis and biological evaluation of 3-styrylchromone derivatives as selective monoamine oxidase B inhibitors.

A series of 3-styrylchromone derivatives was synthesized and evaluated for monoamine oxidase (MAO) A and B inhibitory activities. Most of all derivatives inhibited MAO-B selectively, except compound 21. Compound 19, which had a methoxy group at R2 on the chromone ring and chlorine at R4 on phenyl ring, potently inhibited MAO-B, with an IC50 value of 2.2 nM. Compound 1 showed the highest MAO-B selectivity, with a selectivity index of >3700. Further analysis of these compounds indicated that compounds 1 and 19 were reversible and mixed-type MAO-B inhibitors, suggesting that their mode of action may be through tight-binding inhibition to MAO-B. Quantitative structure-activity relationship (QSAR) analyses of the 3-styrylchromone derivatives were conducted using their pIC50 values, through Molecular Operating Environment (MOE) and Dragon. There were 1796 descriptors of MAO-B inhibitory activity, which showed significant correlations (P < 0.05). Further investigation of the 3-styrylchromone structures as useful scaffolds was performed through three-dimensional-QSAR studies using AutoGPA, which is based on the molecular field analysis algorithm using MOE. The MAO-B inhibitory activity model constructed using pIC50 value index exhibited a determination coefficients (R2) of 0.972 and a Leave-One-Out cross-validated determination coefficients (Q2) of 0.914. These data suggest that the 3-styrylchromone derivatives assessed herein may be suitable for the design and development of novel MAO inhibitors.

A 3-styrylchromone converted from trimebutine 3D pharmacophore possesses dual suppressive effects on RAGE and TLR4 signaling pathways.

Receptor for advanced glycation end-products (RAGE) and Toll-like receptors (TLRs) are potential therapeutic targets in the treatment of acute and chronic inflammatory diseases. We previously reported that trimebutine, a spasmolytic drug, suppresses RAGE pro-inflammatory signaling pathway in macrophages. The aim of this study was to convert trimebutine to a new small molecule using in silico 3D pharmacophore similarity search, and dissect the mechanistic anti-inflammatory basis. Of note, a unique 3-styrylchromone (3SC), 7-methoxy-3-trimethoxy-SC (7M3TMSC), converted from trimebutine 3D pharmacophore potently suppressed both high mobility group box 1-RAGE and lipopolysaccharide-TLR4 signaling pathways in macrophage-like RAW264.7 cells. More importantly, 7M3TMSC inhibited the phosphorylation of extracellular signaling-regulated kinase 1 and 2 (ERK1/2) and downregulated the production of cytokines, such as interleukin-6. Furthermore, 3D pharmacophore-activity relationship analyses revealed that the hydrogen bond acceptors of the trimethoxy groups in a 3-styryl moiety and the 7-methoxy-group in a chromone moiety in this compound are significant in the dual anti-inflammatory activity. Thus, 7M3TMSC may provide an important scaffold for the development of a new type of anti-inflammatory dual effective drugs targeting RAGE/TLR4-ERK1/2 signaling.

A Unique Anti-Cancer 3-Styrylchromone Suppresses Inflammatory Response via HMGB1-RAGE Signaling.

Background: High mobility group box 1 (HMGB1)-receptor for advanced glycation endo-products (RAGE) axis serves as a key player in linking inflammation and carcinogenesis. Recently, papaverine was revealed to suppress the HMGB1-RAGE inflammatory signaling pathway and cancer cell proliferation. Therefore, a dual suppressor targeting this axis is expected to become a new type of therapeutic agent to treat cancer. Methods: Papaverine 3D pharmacophore mimetic compounds were selected by the LigandScout software from our in-house, anti-cancer chemical library and assessed for their anti-inflammatory activities by a HMGB1-RAGE-mediated interleukin-6 production assay using macrophage-like RAW264.7 cells. Molecular-biological analyses, such as Western blotting, were performed to clarify the mechanism of action. Results: A unique 6-methoxy-3-hydroxy-styrylchromone was found to possess potent anti-inflammatory and anti-cancer activities via the suppression of the HMGB1-RAGE-extracellular signal-regulated kinase 1/2 signaling pathway. Furthermore, the 3D pharmacophore-activity relationship analyses revealed that the hydroxyl group at the C4' position of the benzene ring in a 3-styryl moiety was significant in its dual suppressive effects. Conclusions: These findings indicated that this compound may provide a valuable scaffold for the development of a new type of anti-cancer drug possessing anti-inflammatory activity and as a tool for understanding the link between inflammation and carcinogenesis.

Synthesis and antifungal activity of polycyclic pyridone derivatives with anti-hyphal and biofilm formation activity against Candida albicans.

Thirty-five pyridone derivatives were synthesized, with derivatization conducted on polycyclic pyridone scaffolds, including cis- or trans-oxydecalin and other cyclic structures, by domino-Knoevenagel-electrocyclic reactions. The anti-fungal activities of the synthesized compounds were tested against Candida albicans. Ten compounds inhibited hyphal formation without inhibiting growth. Pyridones with anti-hyphal formation activity (4c, 6d, 12a and 12c) were tested for their ability to inhibit biofilm formation. Compound 6d showed both anti-hyphal and biofilm inhibition activity.