Synthesis and biological evaluation of flavonoid-based IP6K2 inhibitors.

Inositol polyphosphates (IPs) are a group of inositol metabolites that act as secondary messengers for external signalling cues. They play various physiological roles such as insulin release, telomere length maintenance, cell metabolism, and aging. Inositol hexakisphosphate kinase 2 (IP6K2) is a key enzyme that produces 5-diphosphoinositol 1,2,3,4,6-pentakisphosphate (5-IP7), which influences the early stages of glucose-induced exocytosis. Therefore, regulation of IP6Ks may serve as a promising strategy for treating diseases such as diabetes and obesity. In this study, we designed, synthesised, and evaluated flavonoid-based compounds as new inhibitors of IP6K2. Structure-activity relationship studies identified compound 20s as the most potent IP6K2 inhibitor with an IC50 value of 0.55 µM, making it 5-fold more potent than quercetin, the reported flavonoid-based IP6K2 inhibitor. Compound 20s showed higher inhibitory potency against IP6K2 than IP6K1 and IP6K3. Compound 20s can be utilised as a hit compound for further structural modifications of IP6K2 inhibitors.

Design and synthesis of a novel BODIPY-labeled PSMA inhibitor.

Prostate-specific membrane antigen (PSMA) is a zinc-bound metalloprotease which is highly expressed in metastatic prostate cancer. It has been considered an excellent target protein for prostate cancer imaging and targeted therapy because it is a membrane protein and its active site is located in the extracellular region. We successfully synthesized and evaluated a novel PSMA ligand conjugated with BODIPY650/665. Compound 1 showed strong PSMA-inhibitory activity and selective uptake into PSMA-expressing tumors. Compound 1 has the potential to be utilized as a near infrared (NIR) optical imaging probe targeting PSMA-expressing cancers.

Structure-activity relationship studies of prostate-specific membrane antigen (PSMA) inhibitors derived from α-amino acid with (S)- or (R)-configuration at P1' region.

Prostate-specific membrane antigen (PSMA), a type II membrane glycoprotein, is considered an excellent target for the diagnosis or treatment of prostate cancer. We previously investigated the effect of ß- and γ-amino acids with (S)- or (R)-configuration in the S1 pocket on the binding affinity for PSMA. However, comprehensive studies on the effect of α-amino acid with (R)-configuration in the S1' pocket has not been reported yet. We selected ZJ-43 (1) and DCIBzL (5) as templates and synthesized their analogues with (S)- or (R)-configuration in the P1 and P1' regions. The PSMA-inhibitory activities of compounds with altered chirality in the P1' region were dropped dramatically, with their IC50 values changing from nM to µM ranges. The compounds with (S)-configuration at both P1 and P1' regions were more potent than the others. The findings of this study may provide insights regarding the structural modification of PSMA inhibitor in the S1' binding pocket.

Design and synthesis of dye-conjugated hepsin inhibitors.

Hepsin is a type II serine protease that is highly expressed in neoplastic prostate. It is an attractive biomarker for imaging metastatic prostate cancer because of its overexpression in advanced prostate cancer and the location of its active site on the cell surface. We designed and synthesized novel hepsin-targeted imaging probes by conjugating the hepsin-binding ligand with near-infrared (NIR) optical dyes. The Leu-Arg dipeptides, attached to BODIPY or SulfoCy7, exhibited strong hepsin-inhibitory activities with Ki values of 21 and 22 nM, respectively. Compound 2 showed selective uptake and retention in hepsin-overexpressing cells. This is the first report of hepsin-targeted optical probes with strong binding affinities and high selectivity over matriptase. Compound 2 has the potential to be used for developing hepsin-based imaging probes and be as a prototype molecule in the design of new hepsin inhibitors.

Discovery of gastric cancer specific biomarkers by the application of serum proteomics.

Current diagnostic markers for gastric cancer are not sufficiently specific or sensitive for use in clinical practice. The aims of this study are to compare the proteomes of serum samples from patients with gastric cancers and normal controls, and to develop useful tumor markers of gastric cancer by quantitative proteomic analysis. We identified a total of 388 proteins with a ≤1% FDR and with at least two unique peptides from the sera of each group. Among them, 215, 251, and 260 proteins were identified in serum samples of patients in an advanced cancer group, early cancer group, and normal control group, respectively. We selected differentially expressed proteins in cancer patients compared with those of normal controls via semiquantitative analyses comparing the spectral counts of identified proteins. These differentially expressed proteins were successfully verified using an MS-based quantitative assay, multiple reactions monitoring analysis. Four proteins (vitronectin, clusterin isoform 1, thrombospondin 1, and tyrosine-protein kinase SRMS) were shown to have significant changes between the cancer groups and the normal control group. These four serum proteins were able to discriminate gastric cancer patients from normal controls with sufficient specificity and selectivity.

A key lysine residue in the AXH domain of ataxin-1 is essential for its ubiquitylation.

Spinocerebellar ataxia type 1 (SCA1), an autosomal-dominant neurodegenerative disorder, is caused by expansion of the polyglutamine tract within ataxin-1 (ATXN1). The AXH domain of ATXN1 can mediate neurodegeneration through its interaction with other proteins. We have previously showed that the ubiquitin-conjugating enzyme UbcH6 modulates the transcriptional repression activity of ATXN1 through ubiquitylation. In the present study, we sought to identify sites in the AXH domain that are ubiquitylated by UbcH6. Systematic replacement of each lysine residue in the AXH domain revealed that the lysine at 589 (K589) of ATXN1 is essential for its ubiquitylation by UbcH6. Mass spectrometry studies further confirmed the ubiquitylation site. Interestingly, protein aggregation was significantly enhanced in mutant AXH K589R, implying that the aggregation is strongly associated with the level of ATXN1 expression. Our study may suggest a therapeutic potential of UbcH6 in the treatment of SCA1.

Analysis of nitrated proteins in Saccharomyces cerevisiae involved in mating signal transduction.

Protein tyrosine nitration (PTN) is a PTM that regulates signal transduction and inflammatory responses, and is related to neurodegenerative and cardiovascular diseases. The cellular function of PTN remains unclear because the low stoichiometry of PTN limits the identification and quantification of nitrated peptides. Effective enrichment is an important aspect of PTN analysis. In this study, we analyzed the in vivo nitroproteome elicited by mating signal transduction in Saccharomyces cerevisiae using a novel chemical enrichment method followed by LC-MS/MS. Nitroproteome profiling successfully identified changes in the nitration states of 14 proteins during mating signal transduction in S. cerevisiae, making this the first reported in vivo nitroproteome in yeast. We investigated the biological functions of these nitroproteins and their relationships to mating signal transduction in S. cerevisiae using a protein-protein interaction network. Our results suggest that PTN and denitration may be involved in nonreactive nitrogen species-mediated signal transduction and can provide clues for understanding the functional roles of PTN in vivo.

Phospholipids of tumor extracellular vesicles stratify gefitinib-resistant nonsmall cell lung cancer cells from gefitinib-sensitive cells.

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) such as gefitinib are one of gold standard treatment options for nonsmall-cell lung cancer (NSCLC) patients, which eventually fail due to the acquired resistance and relapse because of the development of secondary activating mutations such as T790M in EGFR. Predicting chemo-responsiveness of cancer patients provides a major challenge in chemotherapy. The goal of the present study is to determine whether phospholipid signatures of tumor extracellular vesicles (EV) are associated with gefitinib-resistance of NSCLC. A sophisticated MS-based shotgun lipidomic assays were performed for in-depth analysis of the lipidomes of gefitinib-resistant (PC9R) and responsive (PC9) NSCLC cells and their shed EV from these cell lines (PC9EV or PC9REV). Lipid MALDI-MS analysis showed that EV phospholipid composition was significantly distinct in PC9R, compared to PC9 cells. Following statistical analyses has identified 35 (20 positive and 15 negative ion mode) differentially regulated lipids, which are significantly over- or underexpressed in PC9R EV, compared to PC9 EV (p value < 0.01, fold change > 1.5). Our phospholipid signatures suggest that EV associates with drug sensitivity, which is worthy of additional investigation to assess chemoresistance in patients with NSCLC treated with anti-EGFR TKIs.

Characteristics of Plasma Treated Electrospun Polycaprolactone (PCL) Nanofiber Scaffold for Bone Tissue Engineering.

Polycaprolactone (PCL) nanofibers (PCL-NF) with uniform fibrous structure were fabricated by electrospinning. However, PCL-NF has hydrophobic surface, lacks functional groups and hence it is not a good substrate for cell adhesion. To improve the cell adhesion, PCL-NF surfaces were modified by low pressure RF discharge plasma treatment using monomer such as acrylic acid or oxygen gas. The plasma treated PCL-NFs improved the wettability and cell proliferation.

Preparation of O2 Plasma Treated Polycaprolactone/Nano TiO2 Composites and In Vitro Bioactivity.

Polycaprolactone (PCL)/TiO2 composite films (PTCFs) were prepared by a solvent casting method at various concentrations of TiO2 (1, 3, 5, and 10 wt%) and then treated using oxygen plasma. The hydrophilicity of the oxygen plasma treated PTCFs increased as the treatment time was increased, due to the oxygen induced production of polar species at the surface of the PTCFs. In vitro bioactivities of the composite films were examined by immersion in simulated body fluid for up to 7 days. It was found that the oxygen plasma treatment significantly influenced the in vitro bioactivity of the PTCFs.

The standardized BHH10 extract, a combination of Astragalus membranaceus, Cinnamomum cassia, and Phellodendron amurense, reverses bone mass and metabolism in a rat model of postmenopausal osteoporosis.

Jasin-hwan-gagambang (BHH10), a modified prescription of Jasin-hwan, contains Astragalus membranaceus, Cinnamomum cassia, and Phellodendron amurense, and it has been traditionally used to treat osteoporosis and other inflammatory diseases. In this study, we systematically investigated the protective effects of BHH10 in ovariectomy (OVX)-induced rats. Sprague-Dawley rats were randomly divided into sham and OVX subgroups. The rats in the OVX group were treated with vehicle, BHH10, alendronate (ALN), and 17ß-estradiol (E2). BHH10 treatment significantly inhibited OVX-induced increases in body weight and uterus atrophy. In addition, it significantly increased the bone mineral density (BMD) and prevented a decrease in trabecular bone volume, connectivity density, trabecular number, thickness, and separation at the total femur and femur neck. The OVX rats showed significant decreases in the serum levels of calcium and phosphorous and significant increases in the serum levels of cholesterol, low-density lipoprotein cholesterol, alkaline phosphatase, osteocalcin, C-telopeptide type 1 collagen, and bone morphogenetic protein-2. These changes were significantly reduced to near sham levels by administration of BHH10 to OVX rats. BHH10-treated rats had a greater bone mass, a better structural architecture of the bone, and higher levels of biochemical markers of the bone than did the ALN-treated or E2-treated rats. These results suggest that BHH10 reverses osteoporosis in OVX rats by stimulating bone formation or regulating bone resorption and is not associated with toxicity.

Extracellular vesicles shed from gefitinib-resistant nonsmall cell lung cancer regulate the tumor microenvironment.

Epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs), including gefitinib, are the first-line treatment of choice for nonsmall cell lung cancer patients who harbor activating EGFR mutations, however, acquired resistance to EGFR-TKIs is inevitable. The main objective of this study was to identify informative protein signatures of extracellular vesicles (EV) derived from gefitinib-resistant nonsmall cell lung cancer cells using proteomics analysis. Nano-LC-MS/MS analysis identified with high confidence (false discovery rate < 0.05, fold change ≥2) 664 EV proteins enriched in PC9R cells, which are resistant to gefitinib due to EGFR T790M mutation. Computational analyses suggested components of several signal transduction mechanisms including the AKT (also PKB, protein kinase B)/mTOR (mechanistic target of rapamycin) pathway are overrepresented in EV from PC9R cells. Treatment of recipient cells with EV harvested from PC9R cells increased phosphorylation of signaling molecules, and enhanced proliferation, invasion, and drug resistance to gefitinib-induced apoptosis. Dose- and time-dependent pharmaceutical inhibition of AKT/mTOR pathway overcame drug resistance of PC9R cells and those of H1975 exhibiting EGFR T790M mutation. Our findings provide new insight into an oncogenic EV protein signature regulating tumor microenvironment, and will aid in the development of novel diagnostic strategies for prediction and assessment of gefitinib resistance.

Formononetin attenuates osteoclastogenesis via suppressing the RANKL-induced activation of NF-κB, c-Fos, and nuclear factor of activated T-cells cytoplasmic 1 signaling pathway.

Formononetin (1), a plant-derived phytoestrogen, possesses bone protective properties. To address the potential therapeutic efficacy and mechanism of action of 1, we investigated its antiosteoclastogenic activity and its effect on nuclear factor-kappaB ligand (RANKL)-induced bone-marrow-derived macrophages (BMMs). Compound 1 markedly inhibited RANKL-induced osteoclast differentiation in the absence of cytotoxicity, by regulating the expression of osteoprotegerin (OPG) and RANKL in BMMs and in cocultured osteoblasts. Compound 1 significantly inhibited RANKL-induced tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, IL-6, monocyte chemoattractant protein-1 (MCP-1), regulated on activation normal T cell expressed and secreted (RANTES), and macrophage inflammatory protein-1α (MIP-1α) in a concentration-dependent manner. These effects were accompanied by a decrease in RANKL-induced activation of the NF-κB p65 subunit, degradation of inhibitor κBα (IκBα), induction of NF-κB, and phosphorylation of AKT, extracellular-signal regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (p38 MAPK). NF-κB siRNA suppressed AKT, ERK, JNK, and p38 MAPK phosphorylation. Furthermore, 1 significantly suppressed c-Fos and nuclear factor of activated T-cells cytoplasmic 1 (NFATc1), key transcription factors during osteoclastogenesis. SP600125, a specific inhibitor of JNK, reduced RANKL-induced expression of phospho-c-Jun, c-Fos, and NFATc1 and inhibited osteoclast formation. These results suggested that 1 acted as an antiresorption agent by blocking osteoclast activation.

Arginine enhances osteoblastogenesis and inhibits adipogenesis through the regulation of Wnt and NFATc signaling in human mesenchymal stem cells.

Arginine, an α-amino acid, has been reported to exert beneficial effects that ameliorate health problems and prevent excessive fat deposition. In this study, we investigated whether the activation of cell signaling by arginine can induce osteogenic differentiation and modulate excessive adipogenic differentiation in human mesenchymal stem cells (MSCs). Arginine potently induced the expression of type Iα1 collagen, osteocalcin, and ALP in a dose-dependent manner without causing cytotoxicity. Arginine significantly increased the mRNA expression of the osteogenic transcription factors runt-related transcription factor 2 (Runx2), DIx5, and osterix. Furthermore, arginine demonstrated its antiadipogenicity by decreasing adipocyte formation and triglyceride (TG) content in MSCs and inhibiting the mRNA expression of the adipogenic transcription factors peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer-binding protein α (C/EBPα), and fatty acid binding protein 4 (Fabp4). This effect was associated with increased expression of Wnt5a, and nuclear factor of activated T-cells (NFATc), and was abrogated by antagonists of Wnt and NFATc, which indicated a role of Wnt and NFATc signaling in the switch from adipogenesis to osteoblastogenesis induced by arginine. In conclusion, this is the first report of the dual action of arginine in promoting osteogenesis and inhibiting adipocyte formation through involving Wnt5a and NFATc signaling pathway.

Identification and characterization of proteins isolated from microvesicles derived from human lung cancer pleural effusions.

Microvesicles (MVs, also known as exosomes, ectosomes, microparticles) are released by various cancer cells, including lung, colorectal, and prostate carcinoma cells. MVs released from tumor cells and other sources accumulate in the circulation and in pleural effusion. Although recent studies have shown that MVs play multiple roles in tumor progression, the potential pathological roles of MV in pleural effusion, and their protein composition, are still unknown. In this study, we report the first global proteomic analysis of highly purified MVs derived from human nonsmall cell lung cancer (NSCLC) pleural effusion. Using nano-LC-MS/MS following 1D SDS-PAGE separation, we identified a total of 912 MV proteins with high confidence. Three independent experiments on three patients showed that MV proteins from PE were distinct from MV obtained from other malignancies. Bioinformatics analyses of the MS data identified pathologically relevant proteins and potential diagnostic makers for NSCLC, including lung-enriched surface antigens and proteins related to epidermal growth factor receptor signaling. These findings provide new insight into the diverse functions of MVs in cancer progression and will aid in the development of novel diagnostic tools for NSCLC.

Combined phospho- and glycoproteome enrichment in nephrocalcinosis tissues of phytate-fed rats.

RATIONALE: Protein post-translational modifications (PTMs) are directly involved in protein function and cellular activities. Among them, glycosylation and phosphorylation are particularly important modifications on proteins located at extracellular and intracellular domains, respectively. However, the combined detection using phospho- and glycoproteomics is limited mainly due to protocol differences. METHODS: In this study, we developed a novel method for both phospho- and glycoproteome detection from a single sample batch, in which a titanium dioxide cartridge was used to capture the phosphoproteome, and the flow-through solution was processed for capturing N-linked glycopeptides using hydrazide resin. RESULTS: By using 1 mg of protein from kidney tissue lysates from normal and diseased rats, we concurrently identified 437 glycosites/358 phosphosites and 468 glycosites/369 phosphosites in normal and disease kidneys, respectively, by liquid chromatography/tandem mass spectrometric analysis. CONCLUSIONS: Compared with individual PTM analyses, the combined PTM analysis clearly provides more broad implications for PTMs related to the pathological status and discovery of biomarker candidates. Furthermore, the combined protocol thoroughly showed its advantages in enrichment efficiency and biological interpretation compared with current methods.

Proteomic analysis of microvesicles derived from human mesenchymal stem cells.

Mesenchymal stem cells (MSCs) have emerged as a promising means for treating degenerative or incurable diseases. Recent studies have shown that microvesicles (MVs) from MSCs (MSC-MVs) contribute to recovery of damaged tissues in animal disease models. Here, we profiled the MSC-MV proteome to investigate their therapeutic effects. LC-MS/MS analysis of MSC-MVs identified 730 MV proteins. The MSC-MV proteome included five positive and two variable known markers of MSCs, but no negative marker, as well as 43 surface receptors and signaling molecules controlling self-renewal and differentiation of MSCs. Functional enrichment analysis showed that cellular processes represented by the MSC-MV proteins include cell proliferation, adhesion, migration, and morphogenesis. Integration of MSC's self-renewal and differentiation-related genes and the proteome of MSC-conditioned media (MSC-CM) with the MSC-MV proteome revealed potential MV protein candidates that can be associated with the therapeutic effects of MSC-MVs: (1) surface receptors (PDGFRB, EGFR, and PLAUR); (2) signaling molecules (RRAS/NRAS, MAPK1, GNA13/GNG12, CDC42, and VAV2); (3) cell adhesion (FN1, EZR, IQGAP1, CD47, integrins, and LGALS1/LGALS3); and (4) MSC-associated antigens (CD9, CD63, CD81, CD109, CD151, CD248, and CD276). Therefore, the MSC-MV proteome provides a comprehensive basis for understanding the potential of MSC-MVs to affect tissue repair and regeneration.

Melittin suppresses VEGF-A-induced tumor growth by blocking VEGFR-2 and the COX-2-mediated MAPK signaling pathway.

Melittin (1) is a major polypeptide in honey bee venom that has been used traditionally against chronic inflammation and cancer. However, its molecular mechanism has not been determined. In this study, the antitumor effect of 1 was compared with that of NS398, a cyclooxygenase-2 (COX-2) inhibitor, in vivo and in vitro. Subcutaneous injection of 1 at 0.5 and 5 mg/kg suppressed significantly vascular endothelial growth factor (VEGF)-A-transfected highly metastatic Lewis lung cancer (VEGF-A-hm LLC) tumor growth by 25% and 57%, respectively. Also, 1 inhibited significantly the number of vessels around VEGF-A-hm LLC cells. The results were superior to those obtained in the mice treated with NS398. Compound 1 dose-dependently inhibited proliferation and tube formation in human umbilical vein endothelial cells (VEGF-A-HUVECs), without affecting cell viability in native HUVECs. In addition, 1 decreased the expression of VEGF receptor-2 (VEGFR-2), COX-2, and prostaglandin E2 (PGE2) in VEGF-A-transfected HUVECs. These effects were accompanied by a reduction of the phosphorylation of extracellular signal-regulated kinase 1/2 and c-jun N-terminal kinase, whereas it increased the phosphorylation of p38 mitogen-activated protein kinase (MAPK). SB203580 abolished the downregulation of COX-2 and VEGFR-2 and the inhibition of cell proliferation by 1. The antitumor activity of 1 may be associated with antiangiogenic actions via inhibiting VEGFR-2 and inflammatory mediators involved in the MAPK signaling pathway.

Standardized butanol fraction of WIN-34B suppresses cartilage destruction via inhibited production of matrix metalloproteinase and inflammatory mediator in osteoarthritis human cartilage explants culture and chondrocytes.

BACKGROUND: WIN-34B is a novel Oriental medicine, which represents the n-butanol fraction prepared from dried flowers of Lonicera japonica Thunb and dried roots of Anemarrhena asphodeloides BUNGE. The component herb of WIN-34B is used for arthritis treatment in East Asian countries. The aim of this study was to determine the cartilage-protective effects and mechanisms of WIN-34B and its major phenolic compounds, chlorogenic acid and mangiferin, in osteoarthritis (OA) human cartilage explants culture and chondrocytes. METHODS: The investigation focused on whether WIN-34B and its standard compounds protected cartilage in interleukin (IL)-1ß-stimulated cartilage explants culture and chondrocytes derived from OA patients. Also, the mechanisms of WIN-34B on matrix metalloproteinases (MMPs), tissue inhibitor of matrix metalloproteinases (TIMPs), inflammatory mediators, and mitogen-activated protein kinases (MAPKs) pathways were assessed. RESULTS: WIN-34B was not cytotoxic to cultured cartilage explants or chondrocytes. WIN-34B dose-dependently inhibited the release of glycosaminoglycan and type II collagen, increased the mRNA expression of aggrecan and type II collagen, and recovered the intensity of proteoglycan and collagen by histological analysis in IL-1ß-stimulated human cartilage explants culture. The cartilage protective effect of WIN-34B was similar to or better than that of chlorogenic acid and mangiferin. Compared to chlorogenic acid and mangiferin, WIN-34B displayed equal or greater decreases in the levels of MMP-1, MMP-3, MMP-13, ADAMTS-4, and ADAMTS-5, and markedly up-regulated TIMP-1 and TIMP-3. WIN-34B inhibited inflammatory mediators involved in cartilage destruction, such as prostaglandin E2, nitric oxide, tumor necrosis factor-alpha, and IL-1ß. The phosphorylation of extracellular signal-regulated kinase, c-Jun N-terminal kinase (JNK), and p38 was significantly reduced by WIN-34B treatment, while phosphorylation of JNK was only inhibited by chlorogenic acid or mangiferin in IL-1ß-stimulated chondrocytes. CONCLUSIONS: WIN-34B is potentially valuable as a treatment for OA by virtue of its suppression of MMPs, ADAMTSs, and inflammatory mediators, and it's up-regulation of TIMP-1 and TIMP-3 involved in the MAPK pathway.

Targeting regulation of ATP synthase 5 alpha/beta dimerization alleviates senescence.

Senescence is a distinct set of changes in the senescence-associated secretory phenotype (SASP) and leads to aging and age-related diseases. Here, we screened compounds that could ameliorate senescence and identified an oxazoloquinoline analog (KB1541) designed to inhibit IL-33 signaling pathway. To elucidate the mechanism of action of KB1541, the proteins binding to KB1541 were investigated, and an interaction between KB1541 and 14-3-3ζ protein was found. Specifically, KB1541 interacted with 14-3-3ζ protein and phosphorylated of 14-3-3ζ protein at serine 58 residue. This phosphorylation increased ATP synthase 5 alpha/beta dimerization, which in turn promoted ATP production through increased oxidative phosphorylation (OXPHOS) efficiency. Then, the increased OXPHOS efficiency induced the recovery of mitochondrial function, coupled with senescence alleviation. Taken together, our results demonstrate a mechanism by which senescence is regulated by ATP synthase 5 alpha/beta dimerization upon fine-tuning of KB1541-mediated 14-3-3ζ protein activity.