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16 pages, 4547 KiB

Open AccessArticle

Isoquinolinamine FX-9 Exhibits Anti-Mitotic Activity in Human and Canine Prostate Carcinoma Cell Lines

by Jan Torben Schille, Ingo Nolte, Eva-Maria Packeiser, Laura Wiesner, Jens Ingo Hein, Franziska Weiner, Xiao-Feng Wu, Matthias Beller, Christian Junghanss and Hugo Murua Escobar

Int. J. Mol. Sci. 2019, 20(22), 5567; https://doi.org/10.3390/ijms20225567 - 7 Nov 2019

Cited by 4 | Viewed by 3133

Abstract

Current therapies are insufficient for metastatic prostate cancer (PCa) in men and dogs. As human castrate-resistant PCa shares several characteristics with the canine disease, comparative evaluation of novel therapeutic agents is of considerable value for both species. Novel isoquinolinamine FX-9 exhibits antiproliferative activity [...] Read more.

Current therapies are insufficient for metastatic prostate cancer (PCa) in men and dogs. As human castrate-resistant PCa shares several characteristics with the canine disease, comparative evaluation of novel therapeutic agents is of considerable value for both species. Novel isoquinolinamine FX-9 exhibits antiproliferative activity in acute lymphoblastic leukemia cell lines but has not been tested yet on any solid neoplasia type. In this study, FX-9′s mediated effects were characterized on two human (PC-3, LNCaP) and two canine (CT1258, 0846) PCa cell lines, as well as benign solid tissue cells. FX-9 significantly inhibited cell viability and induced apoptosis with concentrations in the low micromolar range. Mediated effects were highly comparable between the PCa cell lines of both species, but less pronounced on non-malignant chondrocytes and fibroblasts. Interestingly, FX-9 exposure also leads to the formation and survival of enlarged multinucleated cells through mitotic slippage. Based on the results, FX-9 acts as an anti-mitotic agent with reduced cytotoxic activity in benign cells. The characterization of FX-9-induced effects on PCa cells provides a basis for in vivo studies with the potential of valuable transferable findings to the benefit of men and dogs. Full article

(This article belongs to the Special Issue Drug Discovery, Development and Regulatory Affairs)

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18 pages, 3294 KiB

Open AccessArticle

N-Methylparoxetine Blocked Autophagic Flux and Induced Apoptosis by Activating ROS-MAPK Pathway in Non-Small Cell Lung Cancer Cells

by Kun Wang, Bonan Chen, Ting Yin, Yujuan Zhan, Yuhua Lu, Yilin Zhang, Jiawei Chen, Weijie Wu, Shikun Zhou, Wenli Mao, Yuhui Tan, Biaoyan Du, Xiaodong Liu, Hiuting Idy HO and Jianyong Xiao

Int. J. Mol. Sci. 2019, 20(14), 3415; https://doi.org/10.3390/ijms20143415 - 11 Jul 2019

Cited by 22 | Viewed by 5239

Abstract

The main mechanistic function of most chemotherapeutic drugs is mediated by inducing mitochondria-dependent apoptosis. Tumor cells usually respond to upregulate autophagy to eliminate impaired mitochondria for survival. Hypothetically, inhibiting autophagy might promote mitochondria-dependent apoptosis, thus enhancing the efficacy of chemotherapeutic therapies. We previously [...] Read more.

The main mechanistic function of most chemotherapeutic drugs is mediated by inducing mitochondria-dependent apoptosis. Tumor cells usually respond to upregulate autophagy to eliminate impaired mitochondria for survival. Hypothetically, inhibiting autophagy might promote mitochondria-dependent apoptosis, thus enhancing the efficacy of chemotherapeutic therapies. We previously identified N-methylparoxetine (NMP) as an inducer of mitochondrial fragmentation with subsequent apoptosis in non-small cell lung cancer (NSCLC) cells. We discovered that ROS was accumulated in NMP-treated NSCLC cells, followed by c-Jun N-terminal kinase (JNK) and p38 MAP kinase (p38) activation. This was reversed by the application of a reactive oxygen species (ROS) scavenger, N-acetylcysteine (NAC), leading to a reduction in apoptosis. Our data suggested that NMP induced apoptosis in NSCLC cells by activating mitogen-activated protein kinase (MAPK) pathway. We further speculated that the remarkable increase of ROS in NMP-treated NSCLC cells might result from an inhibition of autophagy. Our current data confirmed that NMP blocked autophagy flux at late stage wherein lysosomal acidification was inhibited. Taken together, this study demonstrated that NMP could exert dual apoptotic functions—mitochondria impairment and, concomitantly, autophagy inhibition. NMP-related excessive ROS accumulation induced apoptosis by activating the MAPK pathway in NSCLC cells. Full article

(This article belongs to the Special Issue Drug Discovery, Development and Regulatory Affairs)

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16 pages, 1162 KiB

Open AccessArticle

A Pilot Study towards the Impact of Type 2 Diabetes on the Expression and Activities of Drug Metabolizing Enzymes and Transporters in Human Duodenum

by Sophie Gravel, Benoit Panzini, Francois Belanger, Jacques Turgeon and Veronique Michaud

Int. J. Mol. Sci. 2019, 20(13), 3257; https://doi.org/10.3390/ijms20133257 - 2 Jul 2019

Cited by 8 | Viewed by 2837

Abstract

To characterize effects of type 2 diabetes (T2D) on mRNA expression levels for 10 Cytochromes P450 (CYP450s), two carboxylesterases, and three drug transporters (ABCB1, ABCG2, SLCO2B1) in human duodenal biopsies. To compare drug metabolizing enzyme activities of four CYP450 isoenzymes in duodenal biopsies [...] Read more.

To characterize effects of type 2 diabetes (T2D) on mRNA expression levels for 10 Cytochromes P450 (CYP450s), two carboxylesterases, and three drug transporters (ABCB1, ABCG2, SLCO2B1) in human duodenal biopsies. To compare drug metabolizing enzyme activities of four CYP450 isoenzymes in duodenal biopsies from patients with or without T2D. mRNA levels were quantified (RT-qPCR) in human duodenal biopsies obtained from patients with (n = 20) or without (n = 16) T2D undergoing a scheduled gastro-intestinal endoscopy. CYP450 activities were determined following incubation of biopsy homogenates with probe substrates for CYP2B6 (bupropion), CYP2C9 (tolbutamide), CYP2J2 (ebastine), and CYP3A4/5 (midazolam). Covariables related to inflammation, T2D, demographic, and genetics were investigated. T2D had no major effects on mRNA levels of all enzymes and transporters assessed. Formation rates of metabolites (pmoles mg protein⁻¹ min⁻¹) determined by LC-MS/MS for CYP2C9 (0.48 ± 0.26 vs. 0.41 ± 0.12), CYP2J2 (2.16 ± 1.70 vs. 1.69 ± 0.93), and CYP3A (5.25 ± 3.72 vs. 5.02 ± 4.76) were not different between biopsies obtained from individuals with or without T2D (p > 0.05). No CYP2B6 specific activity was measured. TNF-α levels were higher in T2D patients but did not correlate with any changes in mRNA expression levels for drug metabolizing enzymes or transporters in the duodenum. T2D did not modulate expression or activity of tested drug metabolizing enzymes and transporters in the human duodenum. Previously reported changes in drug oral clearances in patients with T2D could be due to a tissue-specific disease modulation occurring in the liver and/or in other parts of the intestines. Full article

(This article belongs to the Special Issue Drug Discovery, Development and Regulatory Affairs)

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17 pages, 2302 KiB

Open AccessArticle

Isolated Compounds from Turpinia formosana Nakai Induce Ossification

by Zuha Imtiyaz, Yi-Fang Wang, Yi-Tzu Lin, Hui-Kang Liu and Mei-Hsien Lee

Int. J. Mol. Sci. 2019, 20(13), 3119; https://doi.org/10.3390/ijms20133119 - 26 Jun 2019

Cited by 13 | Viewed by 3268

Abstract

Bone metabolism is a homeostatic process, imbalance in which leads to the onset of diseases such as osteoporosis and osteopenia. Although several drugs are currently available to treat such conditions, they are associated with severe side effects and do not enhance bone formation. [...] Read more.

Bone metabolism is a homeostatic process, imbalance in which leads to the onset of diseases such as osteoporosis and osteopenia. Although several drugs are currently available to treat such conditions, they are associated with severe side effects and do not enhance bone formation. Thus, identifying alternative treatment strategies that focus on enhancing bone formation is essential. Herein, we explored the osteogenic potential of Turpinia formosana Nakai using human osteoblast (HOb) cells. The plant extract was subjected to various chromatographic techniques to obtain six compounds, including one new compound: 3,3′-di-O-methylellagic acid-4-O-α-l-arabinofuranoside (1). Compounds 3,3′-di-O-methylellagic acid-4-O-α-l-arabinofuranoside (1), gentisic acid 5-O-β-d-(6′-O-galloyl) glucopyranoside (2), strictinin (3), and (-)-epicatechin-3-O-β-d-allopyranoside (6) displayed no significant cytotoxicity toward HOb cells, and thus their effects on various osteogenic markers were analyzed. Results showed that 1–3 and 6 significantly increased alkaline phosphatase (ALP) activity up to 120.0, 121.3, 116.4, and 125.1%, respectively. Furthermore, 1, 2, and 6 also markedly enhanced the mineralization process with respective values of up to 136.4, 118.9, and 134.6%. In addition, the new compound, 1, significantly increased expression levels of estrogen receptor-α (133.4%) and osteogenesis-related genes of Runt-related transcription factor 2 (Runx2), osteopontin (OPN), bone morphogenetic protein (BMP)-2, bone sialoprotein (BSP), type I collagen (Col-1), and brain-derived neurotropic factor (BDNF) by at least 1.5-fold. Our results demonstrated that compounds isolated from T. formosana possess robust osteogenic potential, with the new compound, 1, also exhibiting the potential to enhance the bone formation process. We suggest that T. formosana and its isolated active compounds deserve further evaluation for development as anti-osteoporotic agents. Full article

(This article belongs to the Special Issue Drug Discovery, Development and Regulatory Affairs)

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Structure of compounds isolated from T. formosana. Full article ">Figure 2
Cytotoxicity and induction ALP activity by isolated compounds in HOb cells. Cells were seeded in a 96-well plate, and after 24 h, 100 µM samples were added. (A) After five days, an MTT assay was performed to analyze cell viability. (B) After three days, ALP activity was detected by performing an ALP assay using bicinchoninic acid (BCA) protein to normalize protein expression of cells. Data are expressed as the mean ± SD, * p ≤ 0.05, ** p ≤ 0.01 compared to the control, and all experiments were performed in triplicate. Full article ">Figure 3
Isolated compounds increase mineralization in HOb cells. Cells were seeded in a 48-well plate for three days, after which fresh osteoblast differentiation media (ODM) miner containing inducers and samples were added. Subsequently, every two days, fresh ODM miner, inducers, and samples were added until the 11th day. Mineralization was then detected by performing an alizarin red assay. (A) Pictures show mineral deposition after addition of the alizarin red dye, and (B) the graph shows quantitative data obtained after adding 10% cetylpyridinium chloride as a de-stain to dissolve the crystals. Data are expressed as the mean ± SD (** p ≤ 0.01 compared to the control), and all experiments were performed in triplicate. Full article ">Figure 4
Effect of isolated compounds on ER expression in HOb cells. Cells were seeded in a 96-well plate and after 24 h, 100 µM of samples and 1 µM estradiol (positive control) were added. After five days, (A) ER-α/β expression levels were detected. (B) The dose-dependent effect of 1 on the expression of ER-α was analyzed. Data are expressed as the mean ± SD (** p ≤ 0.01 compared to the control), and all experiments were performed in triplicate. Full article ">Figure 4 Cont.
Effect of isolated compounds on ER expression in HOb cells. Cells were seeded in a 96-well plate and after 24 h, 100 µM of samples and 1 µM estradiol (positive control) were added. After five days, (A) ER-α/β expression levels were detected. (B) The dose-dependent effect of 1 on the expression of ER-α was analyzed. Data are expressed as the mean ± SD (** p ≤ 0.01 compared to the control), and all experiments were performed in triplicate. Full article ">Figure 5
Effect of active compounds on bone formation-related genes in HOb cells. Cells were seeded in 6-cm dishes and after 24 h, fresh ODM containing (A) 100 µM of 1 and 1 µM puerarin (the positive control), or (B) 100 µM of 2, 3, and 6 was added to cells. This was followed by mRNA isolation and reverse transcription. Expression levels were detected by performing a real-time PCR. Data are expressed as the mean ± SD (* p ≤ 0.05, ** p ≤ 0.01 compared to the control), and all experiments were performed in triplicate. Full article ">

21 pages, 6126 KiB

Open AccessArticle

Virtual Screening Guided Design, Synthesis and Bioactivity Study of Benzisoselenazolones (BISAs) on Inhibition of c-Met and Its Downstream Signalling Pathways

by Siqi Zhang, Qiaoling Song, Xueting Wang, Zhiqiang Wei, Rilei Yu, Xin Wang and Tao Jiang

Int. J. Mol. Sci. 2019, 20(10), 2489; https://doi.org/10.3390/ijms20102489 - 20 May 2019

Cited by 6 | Viewed by 3500

Abstract

c-Met is a transmembrane receptor tyrosine kinase and an important therapeutic target for anticancer drugs. In this study, we designed a small library containing 300 BISAs molecules that consisted of carbohydrates, amino acids, isothiourea, tetramethylthiourea, guanidine and heterocyclic groups and screened c-Met targeting [...] Read more.

c-Met is a transmembrane receptor tyrosine kinase and an important therapeutic target for anticancer drugs. In this study, we designed a small library containing 300 BISAs molecules that consisted of carbohydrates, amino acids, isothiourea, tetramethylthiourea, guanidine and heterocyclic groups and screened c-Met targeting compounds using docking and MM/GBSA. Guided by virtual screening, we synthesised a series of novel compounds and their activity on inhibition of the autophosphorylation of c-Met and its downstream signalling pathway proteins were evaluated. We found a panel of benzisoselenazolones (BISAs) obtained by introducing isothiourea, tetramethylthiourea and heterocyclic groups into the C-ring of Ebselen, including 7a, 7b, 8a, 8b and 12c (with IC₅₀ values of less than 20 μM in MET gene amplified lung cancer cell line EBC-1), exhibited more potent antitumour activity than Ebselen by cell growth assay combined with in vitro biochemical assays. In addition, we also tested the antitumour activity of three cancer cell lines without MET gene amplification/activation, including DLD1, MDA-MB-231 and A549. The neuroblastoma SK-N-SH cells with HGF overexpression which activates MET signalling are sensitive to MET inhibitors. The results reveal that our compounds may be nonspecific multitarget kinase inhibitors, just like type-II small molecule inhibitors. Western blot analysis showed that these inhibitors inhibited autophosphorylation of c-MET, and its downstream signalling pathways, such as PI3K/AKT and MARK/ERK. Results suggest that bensoisoselenones can be used as a scaffold for the design of c-Met inhibiting drug leads, and this study opens up new possibilities for future antitumour drug design. Full article

(This article belongs to the Special Issue Drug Discovery, Development and Regulatory Affairs)

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11 pages, 2524 KiB

Open AccessArticle

Lipid Modulating Anti-oxidant Stress Activity of Gastrodin on Nonalcoholic Fatty Liver Disease Larval Zebrafish Model

by Owais Ahmad, Bing Wang, Kejian Ma, Yang Deng, Maoru Li, Liping Yang, Yuqi Yang, Jingyun Zhao, Lijun Cheng, Qinyang Zhou and Jing Shang

Int. J. Mol. Sci. 2019, 20(8), 1984; https://doi.org/10.3390/ijms20081984 - 23 Apr 2019

Cited by 41 | Viewed by 4993

Abstract

Non-alcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) is the most common chronic liver disease in the world. However, there are still no drugs for NAFLD/NASH in the market. Gastrodin (GAS) is a bioactive compound that is extracted from Gastrodia elata, which [...] Read more.

Non-alcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) is the most common chronic liver disease in the world. However, there are still no drugs for NAFLD/NASH in the market. Gastrodin (GAS) is a bioactive compound that is extracted from Gastrodia elata, which is used as an active compound on nervous system diseases. Recent reports showed that GAS and Gastrodia elata possess anti-oxidant activity and lipid regulating effects, which makes us curious to reveal the anti-NAFLD effect of GAS. A high cholesterol diet (HCD) was used to induce a NAFLD larval zebrafish model, and the lipid regulation and anti-oxidant effects were tested on the model. Furthermore, qRT-PCR studied the underlying mechanism of GAS. To conclude, this study revealed a lipid regulation and anti-oxidant insights of GAS on NAFLD larval zebrafish model and provided a potential therapeutic compound for NAFLD treatment. Full article

(This article belongs to the Special Issue Drug Discovery, Development and Regulatory Affairs)

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Full article ">Figure 1
Effect of Gastrodin (GAS) on high cholesterol diet (HCD) induced larval zebrafish. (A) Chemical structure of GAS; (B) Experimental outline of the feeding protocol. Full article ">Figure 2
Effect of GAS on HCD induced larval zebrafish. (A) Mortality of larval zebrafish (n = 3); (B) Weight of larval zebrafish (n = 30). Bar indicate means ± SD. n.s. indicate no significant; *** p < 0.001 represent as compared with the control. ## p < 0.01, ### p < 0.001 represent compared with Model. p < 0.05 was considered to statistically significant, as calculated by One-way ANOVA, followed by Tukey’s test. Full article ">Figure 3
Lipid is regulating the effect of GAS on HCD induced larval zebrafish. (A) Nile red stain of larval zebrafish; (B) Triglyceride (TG) levels; and (C) total cholesterol (TC) levels of larval zebrafish in each group. (D) hematoxylin and eosin (HE) staining of larval zebrafish liver, macrovesicular steatosis and the differences mentioned with red arrows. Bar indicate means ± SD. *** p < 0.001 represent compared with the control. # p < 0.05, ### p < 0.001 represent compared with Model. p < 0.05 was considered as statistically significant, calculated by One-way ANOVA, followed by Tukey’s test. (n = 30). Full article ">Figure 4
The anti-oxidant stress effect of GAS on HCD induced larval zebrafish. (A) The ROS production showed in fluorescence image and merged with a light field image. (B,C) Quantitation of reactive oxygen species C malondialdehyde (ROS. C. MDA) of each treated larval zebrafish group. Bar indicate means ± SD. *** p < 0.001 represent compared with the control. # p < 0.05, ### p < 0.001 represent compared with Model. p < 0.05 was considered as statistically significant, as calculated by One-way ANOVA followed by Tukey’s test. (n = 30). Full article ">Figure 5
mRNA expression profile of GAS on HCD induced larval zebrafish and Molecular Mechanism of GAS. (A) mRNA expression of lipogenesis and lipid-lowering of larval zebrafish. (B) mRNA expression of inflammation, Fibrosis and oxidant stress of larval zebrafish. (C)molecular mechanisms of lipid metabolism modulation by GAS. Bar indicate means ± SD. n.s. indicate no significant; *** p < 0.001 represent compared with the control. # p < 0.05, ### p < 0.001 represent compared with Model. p < 0.05 was considered as statistically significant, as calculated by One-way ANOVA followed by Tukey’s test. (n = 6). Full article ">

21 pages, 2651 KiB

Open AccessArticle

Targeted Capture of Chinese Hamster Ovary Host Cell Proteins: Peptide Ligand Discovery

by R. Ashton Lavoie, Alice di Fazio, R. Kevin Blackburn, Michael B. Goshe, Ruben G. Carbonell and Stefano Menegatti

Int. J. Mol. Sci. 2019, 20(7), 1729; https://doi.org/10.3390/ijms20071729 - 8 Apr 2019

Cited by 27 | Viewed by 5323

Abstract

The growing integration of quality-by-design (QbD) concepts in biomanufacturing calls for a detailed and quantitative knowledge of the profile of impurities and their impact on the product safety and efficacy. Particularly valuable is the determination of the residual level of host cell proteins [...] Read more.

The growing integration of quality-by-design (QbD) concepts in biomanufacturing calls for a detailed and quantitative knowledge of the profile of impurities and their impact on the product safety and efficacy. Particularly valuable is the determination of the residual level of host cell proteins (HCPs) secreted, together with the product of interest, by the recombinant cells utilized for production. Though often referred to as a single impurity, HCPs comprise a variety of species with diverse abundance, size, function, and composition. The clearance of these impurities is a complex issue due to their cell line to cell line, product-to-product, and batch-to-batch variations. Improvements in HCP monitoring through proteomic-based methods have led to identification of a subset of “problematic” HCPs that are particularly challenging to remove, both at the product capture and product polishing steps, and compromise product stability and safety even at trace concentrations. This paper describes the development of synthetic peptide ligands capable of capturing a broad spectrum of Chinese hamster ovary (CHO) HCPs with a combination of peptide species that allow for advanced mixed-mode binding. Solid phase peptide libraries were screened for identification and characterization of peptides that capture CHO HCPs while showing minimal binding of human IgG, utilized here as a model product. Tetrameric and hexameric ligands featuring either multipolar or hydrophobic/positive amino acid compositions were found to be the most effective. Tetrameric multipolar ligands exhibited the highest targeted binding ratio (ratio of HCP clearance over IgG loss), more than double that of commercial mixed-mode and anion exchange resins utilized by industry for IgG polishing. All peptide resins tested showed preferential binding to HCPs compared to IgG, indicating potential uses in flow-through mode or weak-partitioning-mode chromatography. Full article

(This article belongs to the Special Issue Drug Discovery, Development and Regulatory Affairs)

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Full article ">Figure 1
Conceptual diagram of “polyclonal” synthetic HCP-binding resins. Targeted HCP capture is not only possible, but standard practice for HCP quantification using HCP ELISA via polyclonal α-HCP antibodies, as depicted on the left. We propose the generation of a set of diverse ligands to mimic the broad capture of varied HCP species with low binding of IgG, as shown on the right, to allow for targeted capture without the expense and variability introduced by antibody-based ligands. Full article ">Figure 2
Distribution of amino acid residues for lead tetrameric HCP-binding peptide candidates identified via manually sorted solid-phase fluorescent screening by combinatorial position. Full article ">Figure 3
Distribution of amino acid residues for lead hexameric HCP-binding peptide candidates identified by ClonePix 2 sorted solid-phase fluorescent screening by combinatorial position. Full article ">Figure 4
Protein removal (N = 3 for each condition) by hexameric hydrophobic positive and multipolar (6HP and 6MP, respectively) and tetrameric hydrophobic positive and multipolar (4HP and 4MP, respectively) lead HCP-binding peptide ligands coupled to Toyopearl Amino-650M resin in static binding mode, as compared to commercial resins Capto Adhere and Capto Q. Panels A.1–F.1 indicate total protein removal as measured using a Bradford assay. Panels A.2–F.2 indicate CHO-K1 host cell protein removed as measured using a Cygnus CHO HCP ELISA, 3G assay kit. Panels A.3–F.3 indicate monoclonal antibody removed as measured using a Thermo Fisher EasyTiter kit. Each resin was screened in multiple buffer conditions (A panels = pH 6, 20 mM NaCl; B = pH 7, 20 mM NaCl; C = pH 8, 20 mM NaCl; D = pH 6, 150 mM NaCl; E = pH 7, 150 mM NaCl; F = pH 8, 150 mM NaCl); and for two load conditions: ≈5 mg HCP loaded per mL resin, and ≈10 mg HCP loaded per mL resin. Full article ">Figure 5
Resin HCP targeted binding ratio (TBR) by resin and buffer condition (N = 3). Resin HCP TBR is defined as the percent of HCP removed compared to the feed stream divided by the percent of mAb removed compared to the feed stream in static binding mode. In this analysis, HCP TBR > 1 indicates preferential binding to HCP as compared to IgG, and HCP TBR < 1 indicates preferential binding to IgG. Full article ">Figure A1
Host cell protein bubble plot distribution by approximate molar percent abundance, theoretical molecular weight, theoretical isoelectric point, and grand average of hydropathy clarified for null CHO-S cell culture harvest material used in this work as the HCP population fluorescently tagged for solid phase peptide library screening for null CHO-S abundance. Full article ">Figure A2
Head-to-head comparison of peptide-coupled resins to Toyopearl AF-Amino-650M and Toyopearl HW-65F polymethacrylate control resins. A representative clarified CHO-K1 mAb production harvest was directly loaded onto the resins at a load of ≈5 mg HCP/ mL resin, where HCP, IgG, and total protein removal in the flow-through were further compared to determine the impact of the peptide coupling. Using an analysis of variance for each assay as a function of resin type, a strong correlation between protein binding and the functional group for each of the resins was found (p < 0.0001, =0.0002, <0.0001 for total protein removal, HCP removal, and IgG removal, respectively). Full article ">

28 pages, 10797 KiB

Open AccessArticle

IMM-H004 Protects against Cerebral Ischemia Injury and Cardiopulmonary Complications via CKLF1 Mediated Inflammation Pathway in Adult and Aged Rats

by Qidi Ai, Chen Chen, Shifeng Chu, Yun Luo, Zhao Zhang, Shuai Zhang, Pengfei Yang, Yan Gao, Xiaoling Zhang and Naihong Chen

Int. J. Mol. Sci. 2019, 20(7), 1661; https://doi.org/10.3390/ijms20071661 - 3 Apr 2019

Cited by 16 | Viewed by 3580

Abstract

(1) Background: Chemokine-like factor 1 (CKLF1) is a chemokine with potential to be a target for stroke therapy. Compound IMM-H004 is a novel coumarin derivative screened from a CKLF1/C-C chemokine receptor type 4 (CCR4) system and has been reported to improve cerebral ischemia/reperfusion [...] Read more.

(1) Background: Chemokine-like factor 1 (CKLF1) is a chemokine with potential to be a target for stroke therapy. Compound IMM-H004 is a novel coumarin derivative screened from a CKLF1/C-C chemokine receptor type 4 (CCR4) system and has been reported to improve cerebral ischemia/reperfusion injury. This study aims to investigate the protective effects of IMM-H004 on cerebral ischemia injury and its infectious cardiopulmonary complications in adult and aged rats from the CKLF1 perspective. (2) Methods: The effects of IMM-H004 on the protection was determined by 2,3,5-triphenyltetrazolium chloride (TTC) staining, behavior tests, magnetic resonance imaging (MRI) scans, enzyme-linked immunosorbent assay (ELISA), Nissl staining, histo-pathological examination, and cardiopulmonary function detection. Immunohistological staining, immunofluorescence staining, quantitative real-time PCR (qPCR), and western blotting were used to elucidate the underlying mechanisms. (3) Results: IMM-H004 protects against cerebral ischemia induced brain injury and its cardiopulmonary complications, inhibiting injury, and inflammation through CKLF1-dependent anti-inflammation pathway in adult and aged rats. IMM-H004 downregulates the amount of CKLF1, suppressing the followed inflammatory response, and further protects the damaged organs from ischemic injury. (4) Conclusions: The present study suggested that the protective mechanism of IMM-H004 is dependent on CKLF1, which will lead to excessive inflammatory response in cerebral ischemia. IMM-H004 could also be a therapeutic agent in therapy for ischemic stroke and cardiopulmonary complications in the aged population. Full article

(This article belongs to the Special Issue Drug Discovery, Development and Regulatory Affairs)

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15 pages, 2799 KiB

Open AccessArticle

Carnosol as a Nrf2 Activator Improves Endothelial Barrier Function Through Antioxidative Mechanisms

by Xi Li, Qiao Zhang, Ning Hou, Jing Li, Min Liu, Sha Peng, Yuxin Zhang, Yinzhen Luo, Bowen Zhao, Shifeng Wang, Yanling Zhang and Yanjiang Qiao

Int. J. Mol. Sci. 2019, 20(4), 880; https://doi.org/10.3390/ijms20040880 - 18 Feb 2019

Cited by 32 | Viewed by 4128

Abstract

Oxidative stress is the main pathogenesis of diabetic microangiopathy, which can cause microvascular endothelial cell damage and destroy vascular barrier. In this study, it is found that carnosol protects human microvascular endothelial cells (HMVEC) through antioxidative mechanisms. First, we measured the antioxidant activity [...] Read more.

Oxidative stress is the main pathogenesis of diabetic microangiopathy, which can cause microvascular endothelial cell damage and destroy vascular barrier. In this study, it is found that carnosol protects human microvascular endothelial cells (HMVEC) through antioxidative mechanisms. First, we measured the antioxidant activity of carnosol. We showed that carnosol pretreatment suppressed tert-butyl hydroperoxide (t-BHP)-induced cell viability, affected the production of lactate dehydrogenase (LDH) as well as reactive oxygen species (ROS), and increased the produce of nitric oxide (NO). Additionally, carnosol promotes the protein expression of vascular endothelial cadherin (VE-cadherin) to keep the integrity of intercellular junctions, which indicated that it protected microvascular barrier in oxidative stress. Meanwhile, we investigated that carnosol can interrupt Nrf2-Keap1 protein−protein interaction and stimulated antioxidant-responsive element (ARE)-driven luciferase activity in vitro. Mechanistically, we showed that carnosol promotes the expression of heme oxygenase 1(HO-1) and nuclear factor-erythroid 2 related factor 2(Nrf2). It can also promote the expression of endothelial nitric oxide synthase (eNOS). Collectively, our data support the notion that carnosol is a protective agent in HMVECs and has the potential for therapeutic use in the treatments of microvascular endothelial cell injury. Full article

(This article belongs to the Special Issue Drug Discovery, Development and Regulatory Affairs)

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Full article ">Figure 1
Chemical structure of carnosol. Full article ">Figure 2
Detection of antioxidant activity of carnosol by ABTS radical scavenging activity. * p < 0.05, **** p < 0.0001, 0.25% DMSO-treated as negative control group, TBHQ (10 μM) was used as a positive control group, compared with negative group. Results are expressed as mean ± SD (n = 3). Full article ">Figure 3
To evaluate the protective effect of carnosol in t-BHP-induced endothelial injury model. (a) Evaluating the cell viability of carnosol by CCK-8 assay. (b) The cell viability of carnosol pretreated cells after t-BHP-treated for 3 h. (c) The levels of the release of LDH were measured using LDH kits. (d) the green fluorescence is Annexin V-FITC staining positive cell, and the red fluorescence is propidium iodide (PI) staining positive cell at lower magnification (10×). Apoptotic cells were stained only by green fluorescence, necrotic cells were stained with green and red fluorescence, and normal cells were not stained with fluorescence. (e,f) Detection of apoptosis by flow cytometry. Apoptotic cells were distributed in Q2 and Q4 regions. ** p < 0.01, **** p < 0.0001, ns: no significant difference. 200 μM t-BHP-treated as model group, TBHQ (10 μM) was used as a positive control group, 0.25% DMSO-treated as negative control group, compared with model group. Results are expressed as mean ± SD (n = 3). Full article ">Figure 4
Immunofluorescence staining of VE-cadherin at 20× magnification. VE-cadherin shows a green line on the cell membranes. We continued to enlarge twice in the lower right corner of the merge images. The last row graphs show the relative intensity profiles of fluorescent signal intensities of VE-cadherin (green) and nucleu (blue) along the white line scans depicted in the immunofluorescent images. The X-axis indicates the distance of the line and the Y-axis represents the fluorescence intensity. The red arrows indicate the location of the peak. Full article ">Figure 5
Effects of carnosol in ROS and NO assays. (a) The levels of intracellular ROS were measured using DCFH-DA fluorescent probe. (b–c) The levels of intracellular NO were measured using DAF-FM DA fluorescent probe. It was used a fluorescence microscope to take photos at 20× magnification. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns: no significant difference. 200 μM t-BHP-treated as model group, TBHQ (10 μM) was used as a positive control group, 0.25% DMSO-treated as a negative control group, all groups were compared with the model group (no carnosol, no TBHQ, with 200 μM t-BHP). Full article ">Figure 6
Docking mode of carnosol in the binding site of Keap1 and ARE-mediated luciferase activity. (a) Top view of the docking mode of carnosol in the binding site of Keap1. (b) The potential interaction between carnosol and Keap1 is via hydrogen bonds and hydrophobic. Potential hydrogen bonds were depicted in green dot lines and hydrophobic were depicted in purple dot lines. (c) Dose-response curves of ARE agonist (t-BHP) served as a positive control in this study. (d) Carnosol induced ARE-luciferase activity in a dose-dependent manner. Results are expressed as mean ± SD (n = 3). Full article ">Figure 7
Effects of carnosol on cytoprotective gene and protein expression in HMVEC cells. (a–c) The mRNA levels of cytoprotective genes, such as HO-1, Nrf2 and eNOS were evaluated using real-time RT-PCR, with GAPDH as an internal control. (d) HMVEC cells were treated with 10 μM carnosol for 0, 2, 4, 8, 12 and 24 h (top) or with various concentrations of t-CA for 24 h (bottom). The protein levels of HO-1 and Nrf2 were detected with immunoblotting. (e) Levels of HO-1 expression with 10 μM carnosol for 0, 2, 4, 8, 12 and 24 h (top). Levels of HO-1 expression with various concentrations of t-CA for 24 h(bottom). (f) Levels of Nrf2 expression with 10 μM carnosol for 0, 2, 4, 8, 12 and 24 h (top). Levels of Nrf2 expression with various concentrations of t-CA for 24 h (bottom). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, 200 μM t-BHP-treated as model group, TBHQ (10 μM) was used as a positive control group, 0.25% DMSO-treated as a negative control group, compared with the model group. Results are expressed as mean ± SD (n = 3). Full article ">

13 pages, 2514 KiB

Open AccessArticle

Daphnetin: A Novel Anti-Helicobacter pylori Agent

by Genzhu Wang, Jing Pang, Xinxin Hu, Tongying Nie, Xi Lu, Xue Li, Xiukun Wang, Yun Lu, Xinyi Yang, Jiandong Jiang, Congran Li, Yan Q Xiong and Xuefu You

Int. J. Mol. Sci. 2019, 20(4), 850; https://doi.org/10.3390/ijms20040850 - 15 Feb 2019

Cited by 31 | Viewed by 5714

Abstract

Background: Antibiotic-resistant H. pylori was increasingly found in infected individuals, which resulted in treatment failure and required alternative therapeutic strategies. Daphnetin, a coumarin-derivative compound, has multiple pharmacological activities. Methods: The mechanism of daphnetin on H. pylori was investigated focusing on its effect on [...] Read more.

Background: Antibiotic-resistant H. pylori was increasingly found in infected individuals, which resulted in treatment failure and required alternative therapeutic strategies. Daphnetin, a coumarin-derivative compound, has multiple pharmacological activities. Methods: The mechanism of daphnetin on H. pylori was investigated focusing on its effect on cell morphologies, transcription of genes related to virulence, adhesion, and cytotoxicity to human gastric epithelial (GES-1) cell line. Results: Daphnetin showed good activities against multidrug resistant (MDR) H. pylori clinical isolates, with minimal inhibitory concentration (MIC) values ranging from 25 to 100 μg/mL. In addition, daphnetin exposure resulted in H. pylori morphological changes. Moreover, daphnetin caused increased translocation of phosphatidylserine (PS), DNA damage, and recA expression, and RecA protein production vs. control group. Of great importance, daphnetin significantly decreased H. pylori adhesion to GES-1 cell line vs. control group, which may be related to the reduced expression of colonization related genes (e.g., babA and ureI). Conclusions: These results suggested that daphnetin has good activity against MDR H. pylori. The mechanism(s) of daphnetin against H. pylori were related to change of membrane structure, increase of DNA damage and PS translocation, and decrease of H. pylori attachment to GES-1 cells. Full article

(This article belongs to the Special Issue Drug Discovery, Development and Regulatory Affairs)

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Graphical abstract

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Full article ">Figure 1
Structure of daphnetin (Molecular Weight 178.143 g/mol). Full article ">Figure 2
The morphology of H. pylori cells with/without daphnetin exposure observed by SEM. Control (A,D); H. pylori were treated with 6.25 μg/mL (B,E) or 12.5 μg/mL of daphnetin (C,F). Magnification: A–C = 4000; D–F = 40000. Full article ">Figure 3
The morphology of H. pylori cells with/without daphnetin exposure observed by TEM. Control (A–C); H. pylori treated with 12.5 μg/mL daphnetin (D–F). Magnification: A and D = 3000; B and E = 5000; and C and F = 8000. Full article ">Figure 4
Detection of DNA damage and recA expression in H. pylori. DNA damage detected using TUNEL by flow cytometry (A) and confocal (B). (C) The expression of recA in H. pylori with/without daphnetin exposure. (** p < 0.001 vs. control). The expression of the study genes without daphnetin exposure was normalized as 1. Full article ">Figure 5
The transcription of babA (A) and ureI (B) in H. pylori with/without daphnetin exposure. The expression of the study genes without daphnetin exposure was normalized as 1. Inhibitory effect of daphnetin on adhesion of H. pylori to GES-1 cells (C). The level of adherence of H. pylori was detected by confocal (magnification: 600). All the data were presented as mean and standard deviations (SD). * p < 0.05, ** p < 0.01 vs. control. Full article ">Figure 6
Hypothesized model of the mechanism(s) of daphnetin against H. pylori. Daphnetin exposure caused DNA damage and subsequently induced recA expression. In addition, recA negatively regulated babA transcription. To our best knowledge, no study indicated a direct interaction between recA and urel. Lower babA and urel transcription, and their respective protein production could reduce H. pylori adherence to GES-1 cells. Moreover, daphnetin exhibited effect on membrane changes (e.g., outer membrane structural change and increased PS exposure). In the current study, no significant impact of daphnetin on membrane permeability and depolarization was observed (dotted line indicates no statistical significance between control and daphnetin exposure groups). Full article ">

17 pages, 3745 KiB

Open AccessArticle

Network Pharmacology-Based Investigation of Protective Mechanism of Aster tataricus on Lipopolysaccharide-Induced Acute Lung Injury

by Yijun Chen, Jiaojiao Dong, Jie Liu, Wenjuan Xu, Ziyi Wei, Yueting Li, Hao Wu and Hongbin Xiao

Int. J. Mol. Sci. 2019, 20(3), 543; https://doi.org/10.3390/ijms20030543 - 28 Jan 2019

Cited by 26 | Viewed by 4169

Abstract

Acute lung injury (ALI) is a common clinical condition that badly influences people’s health. Recent studies indicated that Aster tataricus (RA) had potential effects on ALI, but the effective components and their mechanism is not clear. In this study, we found that the [...] Read more.

Acute lung injury (ALI) is a common clinical condition that badly influences people’s health. Recent studies indicated that Aster tataricus (RA) had potential effects on ALI, but the effective components and their mechanism is not clear. In this study, we found that the Fraction-75 eluted from RA extract could significantly protect the lipopolysaccharide (LPS)-induced ALI in mice, including alleviating the severity of lung pathology, attenuating the pulmonary edema, and reducing the release of inflammatory cells. Further ingredient analyses demonstrated that there were mainly 16 components in it, among which 10 components were collected according to their relative peak area and oral bioavailability. Next, the components-disease targets network suggested that the candidate components had extensive associations with 49 known therapeutic targets of ALI, among which 31 targets could be regulated by more than one component. Herein, GO functional and pathway analysis revealed that the common targets were associated with four biological processes, including the inflammatory response to stimulus, cellular process, chemokine biosynthetic process and immune system process. Furthermore, the ELISA validation indicated that the candidate components in RA extract may protect the LPS-induced ALI mainly through inhibiting the release of inflammatory cytokines and promoting the repair of vascular endothelial. Full article

(This article belongs to the Special Issue Drug Discovery, Development and Regulatory Affairs)

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15 pages, 3441 KiB

Open AccessArticle

YC-1 Prevents Tumor-Associated Tissue Factor Expression and Procoagulant Activity in Hypoxic Conditions by Inhibiting p38/NF-κB Signaling Pathway

by Kan-Yen Hsieh, Chien-Kei Wei and Chin-Chung Wu

Int. J. Mol. Sci. 2019, 20(2), 244; https://doi.org/10.3390/ijms20020244 - 9 Jan 2019

Cited by 11 | Viewed by 5210

Abstract

Tissue factor (TF) expressed in cancer cells has been linked to tumor-associated thrombosis, a major cause of mortality in malignancy. Hypoxia is a common feature of solid tumors and can upregulate TF. In this study, the effect of YC-1, a putative inhibitor of [...] Read more.

Tissue factor (TF) expressed in cancer cells has been linked to tumor-associated thrombosis, a major cause of mortality in malignancy. Hypoxia is a common feature of solid tumors and can upregulate TF. In this study, the effect of YC-1, a putative inhibitor of hypoxia-inducible factor-1α (HIF-1α), on hypoxia-induced TF expression was investigated in human lung cancer A549 cells. YC-1 selectively prevented hypoxia-induced TF expression and procoagulant activity without affecting the basal TF levels. Surprisingly, knockdown or pharmacological inhibition of HIF-1α failed to mimic YC-1′s effect on TF expression, suggesting other mechanisms are involved. NF-κB, a transcription factor for TF, and its upstream regulator p38, were activated by hypoxia exposure. Treatment of hypoxic A549 cells with YC-1 prevented the activation of both NF-κB and p38. Inhibition of p38 suppressed hypoxia-activated NF-κB, and inhibited TF expression and activity to similar levels as treatment with an NF-κB inhibitor. Furthermore, stimulation of p38 by anisomycin reversed the effects of YC-1. Taken together, our results suggest that YC-1 prevents hypoxia-induced TF in cancer cells by inhibiting the p38/NF-κB pathway, this is distinct from the conventional anticoagulants that systemically inhibit blood coagulation and may shed new light on approaches to treat tumor-associated thrombosis. Full article

(This article belongs to the Special Issue Drug Discovery, Development and Regulatory Affairs)

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Graphical abstract
Full article ">Figure 1
YC-1 inhibits hypoxia-induced TF expression in human cancer cell lines. Human lung cancer A549, breast cancer MDA-MB-231, and oral cancer Ca9-22 cells were pretreated with DMSO (vehicle control) or YC-1 (10–100 μM) for 1 h, and then incubated under hypoxic (A) or normoxic (B) conditions for 24 h. The protein expression of TF was evaluated by Western blotting. (C) A549 cells were pretreated with DMSO or YC-1 and exposed to hypoxia or normoxia for 4 h. The mRNA levels of TF were determined by real-time PCR. All results are presented as mean ± SEM (n = 3). * = p < 0.05, ** = p < 0.01, *** = p < 0.001. Full article ">Figure 2
YC-1 reduces hypoxia-induced TF procoagulant activity in A549 cells. A549 cells were pretreated with DMSO or YC-1 for 1 h, and then incubated under hypoxic or normoxic conditions for 24 h. (A) The cell surface TF activity was measured by a coupled amidolytic assay of TF-dependent factor Xa generation; (B) Cancer cell-induced plasma clotting was determined by tilt tube assay. Anti-TF antibody (20 μg/mL) was used as positive control. (C) A549 cells treated with DMSO or YC-1 (100 μM) in normoxia or hypoxia were harvested (1 × 105 cells/mL) and mixed with platelet suspension (3 × 108 platelets/mL). Platelet aggregation was induced by adding human plasma (0.25%). All results are presented as mean ± SEM (n = 3). * = p < 0.05, ** = p <0.01, *** = p < 0.001. Full article ">Figure 3
YC-1 inhibits hypoxia-induced TF via a HIF-1α-independent manner in A549 cells. (A,B) YC-1 inhibits HIF-1α accumulations and decreases HIF-1α nuclear translocation in response to hypoxia. A549 cancer cells were treated with DMSO or YC-1 for 1 h and incubated under normoxia or hypoxia for 24 h. Whole-cell lysates (A) and the nuclear fractions (B) were subjected to Western blotting for HIF-1α; In (B), ORC2 was used as a loading control in the nuclear fractions; (C) YC-1 inhibits hypoxia-induced upregulation of VEGF. A549 cancer cells pretreated with DMSO or YC-1 were exposed to hypoxia for 4 h. The mRNA levels were determined by real-time PCR. All results were presented as mean ± SEM (n = 3). ** = p < 0.01. Knockdown (D) or pharmacological inhibition (E) of HIF-1α fails to prevent hypoxia-induced TF expression. A549 cancer cells were transiently transfected with si-HIF-1α (50 nM) or negative control siRNA (D), or treated with the HIF-1α inhibitor CAY10585 (10 μM) for 1 h (E), followed by exposure to hypoxia for 24 h. The protein levels of TF in the cell lysates were determined by Western blotting. Full article ">Figure 4
YC-1 activates cyclic nucleotide-dependent protein kinases in A549 cells. (A) A549 cells were pretreated with YC-1 for 1 h and incubated under normoxia or hypoxia for another 1 h. Cell lysates were subjected to Western blotting for phospho-VASP. (B,C) A549 cells were pretreated with the PDE inhibitor IBMX (100 μM), the sGC inhibitor ODQ (10 μM), or the PKA inhibitor H89 (5 μM) for 30 min, then treated with YC-1 or BAY 41-2272 (BAY) for 1 h and incubated under hypoxia for another 24 h. TF expression was determined by Western blotting. Full article ">Figure 5
YC-1 inhibits hypoxia-induced NF-κB activation in A549 cells. A549 cancer cells were pretreated with YC-1 for 1 h and exposed to normoxia or hypoxia. Protein levels of NF-κB p65 in the cytosolic and nuclear fractions (A) and the phosphorylation of NF-κB p65 and IκBα in the whole-cell lysates (B) were determined by Western blotting. (C) A549 cells were pretreated with the NF-κB inhibitor RO 106-9920 for 1 h and exposed to hypoxia for 24 h. TF expression was determined by Western blotting. Full article ">Figure 6
YC-1 prevents hypoxia-induced TF through inhibition of the p38/NF-κB pathway. (A) Effects of specific inhibitors of MAPKs and Akt on hypoxia-induced TF expression. A549 cells were treated with U0126 (10 μM), SP600125 (10 μM), SB202190 (10 μM), and wortmannin (0.1 μM) for 1 h, and exposed to hypoxia for 24 h. Cell lysates were subjected to Western blotting for TF. (B,C) YC-1 inhibits hypoxia-stimulated p38 activation. Cells pretreated with YC-1 were exposed to hypoxia for 15 min (B) or 2 h (C). Cell lysates were subjected to Western blotting for p38 and MAPKAPK2. (D) The effect of YC-1 on ERK activation in hypoxic conditions. A549 cells were treated as in (B), and the cell lysates were subjected to Western blotting for ERK. (E) The p38 inhibitor prevents hypoxia-induced NF-κB activation. A549 cells treated with SB202190 (10 μM) or YC-1 (50 μM) were exposed to hypoxia for 2 h. Cell lysates were subjected to Western blotting for NF-κB. (F) The p38 and NF-κB inhibitors prevent hypoxia-induced TF activity. A549 cells treated with SB202190 (10 μM) or Ro 106-9920 (10 μM) were exposed to hypoxia for 24 h, then the TF-dependent factor Xa generation was determined. Data are presented as mean ± SEM (n = 3). * = p < 0.05, ** = p < 0.01. (G,H) The p38 activator rescues YC-1′s effect on hypoxia-induced TF expression and procoagulant activity. A549 cells were pretreated with YC-1 (50 μM) in the absence or presence of anisomycin (0.1 μM) and exposed to hypoxia for 24 h. The protein levels (G) and activity (H) of TF were determined by Western blotting and TF-dependent factor Xa generation, respectively. Data are presented as mean ± SEM (n = 3). ** = p < 0.01, *** = p < 0.001. Full article ">

12 pages, 2940 KiB

Open AccessArticle

R-Fluoxetine Increases Melanin Synthesis Through a 5-HT1A/2A Receptor and p38 MAPK Signaling Pathways

by Li Liu, Mengsi Fu, Siran Pei, Liangliang Zhou and Jing Shang

Int. J. Mol. Sci. 2019, 20(1), 80; https://doi.org/10.3390/ijms20010080 - 25 Dec 2018

Cited by 21 | Viewed by 5838

Abstract

Fluoxetine, a member of the class of selective serotonin reuptake inhibitors, is a racemic mixture and has an anxiolytic effect in rodents. Previously, we have shown that fluoxetine can up-regulate melanin synthesis in B16F10 melanoma cells and normal human melanocytes (NMHM). However, the [...] Read more.

Fluoxetine, a member of the class of selective serotonin reuptake inhibitors, is a racemic mixture and has an anxiolytic effect in rodents. Previously, we have shown that fluoxetine can up-regulate melanin synthesis in B16F10 melanoma cells and normal human melanocytes (NMHM). However, the role of r-fluoxetine and s-fluoxetine, in the regulation of melanin synthesis, is still unknown. Here, we show how r-fluoxetine plays a critical role in fluoxetine enhancing melanogenesis, both in vivo and vitro, by up-regulating tyrosinase (TYR) and the microphthalmia-associated transcription factor (MITF) expression, whereas, s-fluoxetine does not show any effect in the vivo and vitro systems. In addition, we found that r-fluoxetine induced melanin synthesis through the serotonin1A receptor (5-HT1A) and serotonin 2A receptor (5-HT2A). Furthermore, r-fluoxetine increased the phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK), without affecting the phosphorylation of extracellularly responsive kinase (ERK1/2) and c-Jun N-terminal kinase (JNK). These data suggest that r-fluoxetine may be used as a drug for skin hypopigmentation disorders. Full article

(This article belongs to the Special Issue Drug Discovery, Development and Regulatory Affairs)

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15 pages, 6294 KiB

Open AccessArticle

Search of Allosteric Inhibitors and Associated Proteins of an AKT-like Kinase from Trypanosoma cruzi

by Rodrigo Ochoa, Cristian Rocha-Roa, Marcel Marín-Villa, Sara M. Robledo and Rubén E. Varela-M

Int. J. Mol. Sci. 2018, 19(12), 3951; https://doi.org/10.3390/ijms19123951 - 8 Dec 2018

Cited by 7 | Viewed by 3747

Abstract

Proteins associated to the PI3K/AKT/mTOR signaling pathway are widely used targets for cancer treatment, and in recent years they have also been evaluated as putative targets in trypanosomatids parasites, such as Trypanosoma cruzi. Here, we performed a virtual screening approach to find [...] Read more.

Proteins associated to the PI3K/AKT/mTOR signaling pathway are widely used targets for cancer treatment, and in recent years they have also been evaluated as putative targets in trypanosomatids parasites, such as Trypanosoma cruzi. Here, we performed a virtual screening approach to find candidates that can bind regions on or near the Pleckstrin homology domain of an AKT-like protein in T. cruzi. The compounds were also evaluated in vitro. The in silico and experimental results allowed us to identify a set of compounds that can potentially alter the intracellular signaling pathway through the AKT-like kinase of the parasite; among them, a derivative of the pyrazolopyridine nucleus with an IC₅₀ of 14.25 ± 1.00 μM against amastigotes of T. cruzi. In addition, we built a protein–protein interaction network of T. cruzi to understand the role of the AKT-like protein in the parasite, and look for additional proteins that can be postulated as possible novel molecular targets for the rational design of compounds against T. cruzi. Full article

(This article belongs to the Special Issue Drug Discovery, Development and Regulatory Affairs)

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Figure 1
(A) Structural model chosen of the TcAKT-like protein; the Pleckstrin homology (PH) domain is shown in blue, while the rest of the protein is shown in gray in a schematic way. (B) Selected drug pockets located near the PH domain of TcAKT-like predicted by the PockDrug (yellow surface) and metaPocket (red spheres) tools. (C) Analysis of the stereochemistry (angles Ψ and Φ) of the model selected for the TcAKT-like protein; 95% of the residues are in favored and allowed regions. (D) Evaluation of the overall quality of the TcAKT-like model; the model presented a Z-score (−8.42, highlighted with a red dot) similar to the structures of the same size resolved experimentally. Full article ">Figure 2
Selected compounds of the virtual screening coupled in the predicted binding site for the TcAKT-like protein (left). Similar structural fragments between the coupled compounds. The naphthalene fragment could function as an anchor for each compound (right). Full article ">Figure 3
(A) Interaction network of T. cruzi strain CL Breiner. The nodes (proteins) are presented in black and the score associated with the interaction is presented in colors, blue being more reliable than orange. (B) Interaction network of T. cruzi strain CL Breiner. The axes (interactions) are represented in black and on a scale of orange (less connected) to blue (more connected) the nodes (proteins) of the network. The size of the circle indicates its degree of connectivity (degree). Full article ">Figure 4
(A) Protein–protein interaction network with the AKT-like protein signaled in yellow, where the axes that connect with the closest neighboring nodes (located in the center of the network) are highlighted in red. (B) Protein–protein interaction network with proteins represented as white nodes, and in blue are those that were mapped with the PI3K/AKT/mTOR pathway of human proteins. Full article ">Figure 5
Interactions between the UBMC-6 compound and the TcAKT-like protein. In the upper part, the docking of the compound in the predicted binding site is observed in 3D. The compound UBMC-6 is shown in cyan color and its fragments called R1 and R2 are shown in black; the protein is shown on a hydrophobic surface, where red is hydrophobic and blue is hydrophilic. In the lower part, the 2D interactions between the UBMC-6 compound and the TcAKT-like protein are shown. The blue shadows on the amino acids represent that they are or not on the surface of the protein; the larger the size, the more exposed it is. Full article ">

Review

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18 pages, 2042 KiB

Open AccessReview

Discontinued Drugs for the Treatment of Cardiovascular Disease from 2016 to 2018

by Tingting Li, Sida Jiang, Bingwei Ni, Qiuji Cui, Qinan Liu and Hongping Zhao

Int. J. Mol. Sci. 2019, 20(18), 4513; https://doi.org/10.3390/ijms20184513 - 12 Sep 2019

Cited by 17 | Viewed by 3715

Abstract

Cardiovascular drug research and development (R&D) has been in active state and continuously attracts attention from the pharmaceutical industry. However, only one individual drug can eventually reach the market from about the 10,000 compounds tested. It would be useful to learn from these [...] Read more.

Cardiovascular drug research and development (R&D) has been in active state and continuously attracts attention from the pharmaceutical industry. However, only one individual drug can eventually reach the market from about the 10,000 compounds tested. It would be useful to learn from these failures when developing better strategies for the future. Discontinued drugs were identified from a search performed by Thomson Reuters Integrity. Additional information was sought through PubMed, ClinicalTrials.gov, and pharmaceutical companies search. Twelve compounds discontinued for cardiovascular disease treatment after reaching Phase I–III clinical trials from 2016 to 2018 are detailed in this manuscript, and the reasons for these failures are reported. Of these, six candidates (MDCO-216, TRV027, ubenimex, sodium nitrite, losmapimod, and bococizumab) were dropped for lack of clinical efficacy, the other six for strategic or unspecified reasons. In total, three candidates were discontinued in Phase I trials, six in Phase II, and three in Phase III. It was reported that the success rate of drug R&D utilizing selection biomarkers is higher. Four candidate developments (OPC-108459, ONO-4232, GSK-2798745, and TAK-536TCH) were run without biomarkers, which could be used as surrogate endpoints in the 12 cardiovascular drugs discontinued from 2016 to 2018. This review will be useful for those involved in the field of drug discovery and development, and for those interested in the treatment of cardiovascular disease. Full article

(This article belongs to the Special Issue Drug Discovery, Development and Regulatory Affairs)

17 pages, 823 KiB

Open AccessReview

Flexible and Expedited Regulatory Review Processes for Innovative Medicines and Regenerative Medical Products in the US, the EU, and Japan

by Sumimasa Nagai

Int. J. Mol. Sci. 2019, 20(15), 3801; https://doi.org/10.3390/ijms20153801 - 3 Aug 2019

Cited by 54 | Viewed by 8233

Abstract

Several expedited regulatory review projects for innovative drugs and regenerative medical products have been developed in the US, the EU, and Japan. Each regulatory agency has elaborated an original regulatory framework and adopted regulatory projects developed by the other regulatory agencies. For example, [...] Read more.

Several expedited regulatory review projects for innovative drugs and regenerative medical products have been developed in the US, the EU, and Japan. Each regulatory agency has elaborated an original regulatory framework and adopted regulatory projects developed by the other regulatory agencies. For example, the Food and Drug Administration (FDA) first developed the breakthrough therapy designation, and then the Pharmaceuticals and Medical Devices Agency (PMDA) and European Medicines Agency (EMA) introduced the Sakigake designation and the priority medicines (PRIME) designation, respectively. In addition, the necessity of the product being first development in Japan is the original feature of the Sakigake designation, while actively supporting the development of advanced-therapy medicinal products (ATMPs) by academia or small/medium-sized sponsors is the original feature of the PRIME; these particular features are different from the breakthrough therapy designation in the US. In this review article, flexible and expedited review processes for new drugs, and cell and gene therapies in the US, the EU, and Japan are described. Moreover, all the drugs and regenerative medical products that were granted conditional approval or Sakigake designation in Japan are listed and analyzed herein. Full article

(This article belongs to the Special Issue Drug Discovery, Development and Regulatory Affairs)

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16 pages, 2055 KiB

Open AccessReview

Effects of Anti-Calcitonin Gene-Related Peptide for Migraines: A Systematic Review with Meta-Analysis of Randomized Clinical Trials

by I-Hsin Huang, Po-Chien Wu, En-Yuan Lin, Chien-Yu Chen and Yi-No Kang

Int. J. Mol. Sci. 2019, 20(14), 3527; https://doi.org/10.3390/ijms20143527 - 18 Jul 2019

Cited by 25 | Viewed by 4490

Abstract

We aimed to evaluate the response rate of migraines by using anti-calcitonin gene-related peptide (anti-CGRP) for patients with migraines. We searched three main medical databases up to 29 March 2019. No restriction on language and publication time were applied. Eligible trials included randomized [...] Read more.

We aimed to evaluate the response rate of migraines by using anti-calcitonin gene-related peptide (anti-CGRP) for patients with migraines. We searched three main medical databases up to 29 March 2019. No restriction on language and publication time were applied. Eligible trials included randomized clinical trials investigating a 50%, 75%, and 100% response rate of migraine patients after anti-CGRP intervention. The collected data were dichotomous, and risk ratios (RRs) with a 95% confidence interval (CI) were used to present the quantitative synthesis results. The systematic review identified 16 eligible randomized clinical trials (RCTs) with 9439 patients. Eight of the 16 trials with 2516 patients reported a 50% response rate, and the pooled results showed a significant benefit from anti-CGRP. However, the effects seem to gradually reduce from the first month (RR 1.99, 95% CI 1.59 to 2.49) to the third month (RR 1.48, 95% CI 1.26 to 1.75) of treatment. The magnitude of effect was influenced by the type of anti-CGRP, according to the test for differences between subgroups (I-square = 53%). The funnel plots and Egger’s tests did not show serious small study effects in the results. In conclusion, the current evidences confirmed that anti-CGRP treatment can reduce migraine pain in the short term (within three months), but the long-term effect should be investigated in the future. Moreover, its effects may be influenced by the type and dose of anti-CGRP. Therefore, future studies should make direct comparisons among anti-CGRP medications. Full article

(This article belongs to the Special Issue Drug Discovery, Development and Regulatory Affairs)

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15 pages, 266 KiB

Open AccessReview

Effect of Different Classes of Antihypertensive Drugs on Endothelial Function and Inflammation

by Isabella Viana Gomes Silva, Roberta Carvalho de Figueiredo and Danyelle Romana Alves Rios

Int. J. Mol. Sci. 2019, 20(14), 3458; https://doi.org/10.3390/ijms20143458 - 14 Jul 2019

Cited by 105 | Viewed by 7023

Abstract

Hypertension is characterized by structural and functional changes in blood vessels that travel with increased arterial stiffness, vascular inflammation, and endothelial dysfunction. Some antihypertensive drugs have been shown to improve endothelial function and reduce levels of inflammatory markers regardless of the effect of [...] Read more.

Hypertension is characterized by structural and functional changes in blood vessels that travel with increased arterial stiffness, vascular inflammation, and endothelial dysfunction. Some antihypertensive drugs have been shown to improve endothelial function and reduce levels of inflammatory markers regardless of the effect of blood pressure lowering. Third-generation β-blockers, such as nebivolol and carvedilol, because they have additional properties, have been shown to improve endothelial function in patients with hypertension. Calcium channel antagonists, because they have antioxidant effects, may improve endothelial function and vascular inflammation.The Angiotensin Receptor Blocker (ARBs) are able to improve endothelial dysfunction and vascular inflammation in patients with hypertension and other cardiovascular diseases. Angiotensin converting enzyme (ACE) inhibitors have shown beneficial effects on endothelial function in patients with hypertension and other cardiovascular diseases, however there are few studies evaluating the effect of treatment with this class on the reduction of C-reactive protein (CRP) levels. Further studies are needed to assess whether treatment of endothelial dysfunction and vascular inflammation may improve the prognosis of patients with essential hypertension. Full article

(This article belongs to the Special Issue Drug Discovery, Development and Regulatory Affairs)

33 pages, 2598 KiB

Open AccessReview

QT Assessment in Early Drug Development: The Long and the Short of It

by Robert M. Lester, Sabina Paglialunga and Ian A. Johnson

Int. J. Mol. Sci. 2019, 20(6), 1324; https://doi.org/10.3390/ijms20061324 - 15 Mar 2019

Cited by 45 | Viewed by 11941

Abstract

The QT interval occupies a pivotal role in drug development as a surface biomarker of ventricular repolarization. The electrophysiologic substrate for QT prolongation coupled with reports of non-cardiac drugs producing lethal arrhythmias captured worldwide attention from government regulators eventuating in a series of [...] Read more.

The QT interval occupies a pivotal role in drug development as a surface biomarker of ventricular repolarization. The electrophysiologic substrate for QT prolongation coupled with reports of non-cardiac drugs producing lethal arrhythmias captured worldwide attention from government regulators eventuating in a series of guidance documents that require virtually all new chemical compounds to undergo rigorous preclinical and clinical testing to profile their QT liability. While prolongation or shortening of the QT interval may herald the appearance of serious cardiac arrhythmias, the positive predictive value of an abnormal QT measurement for these arrhythmias is modest, especially in the absence of confounding clinical features or a congenital predisposition that increases the risk of syncope and sudden death. Consequently, there has been a paradigm shift to assess a compound’s cardiac risk of arrhythmias centered on a mechanistic approach to arrhythmogenesis rather than focusing solely on the QT interval. This entails both robust preclinical and clinical assays along with the emergence of concentration QT modeling as a primary analysis tool to determine whether delayed ventricular repolarization is present. The purpose of this review is to provide a comprehensive understanding of the QT interval and highlight its central role in early drug development. Full article

(This article belongs to the Special Issue Drug Discovery, Development and Regulatory Affairs)

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Full article ">Figure 1
Cardiac Action Potential Phases. Major inward and outward cardiac ion channels affecting the five phases of the cardiac action potential. Note that these phases represent time dependent intervals based upon the ingress and egress of the various ions and their impact on transmembrane voltage. Reproduced from [<a href="#B14-ijms-20-01324" class="html-bibr">14</a>]. Full article ">Figure 2
Electrogradiogram (ECG) Principal Waveforms and Intervals. Reproduced from [<a href="#B44-ijms-20-01324" class="html-bibr">44</a>]. Full article ">Figure 3
Shortest and Longest QT interval by Lead Selection.ECG lead data obtained from 4429 participants. Distribution of shortest and longest QT interval by lead in counts (x-axis) and as a percentage (y-axis). Full article ">Figure 4
ECG quality metrics. Full article ">Figure 5
ECG tools. Top panel- Representation of 12 lead median beat using the “vertical separation” tool to enable better visualization and accurate placement of fiducial markers at the onset and offset of PR, QRS and QT intervals. Bottom panel- Representative 12 lead median beat with “vector magnitude” overlay in green which can facilitate accurate placement of fiducial markers at the onset and offset of PR, QRS and QT intervals. Full article ">Figure 6
QTc values for congenital syndromes vs. normal healthy adults. Full article ">Figure 7
Principal waveforms and intervals of ventricular depolarization and repolarization on the surface ECG. Repolarization phase shaded in blue. Reproduced with minor edits from [<a href="#B77-ijms-20-01324" class="html-bibr">77</a>]. Full article ">

22 pages, 2474 KiB

Open AccessReview

Iron Metabolism in Cancer

by Yafang Wang, Lei Yu, Jian Ding and Yi Chen

Int. J. Mol. Sci. 2019, 20(1), 95; https://doi.org/10.3390/ijms20010095 - 27 Dec 2018

Cited by 178 | Viewed by 13385

Abstract

Demanded as an essential trace element that supports cell growth and basic functions, iron can be harmful and cancerogenic though. By exchanging between its different oxidized forms, iron overload induces free radical formation, lipid peroxidation, DNA, and protein damages, leading to carcinogenesis or [...] Read more.

Demanded as an essential trace element that supports cell growth and basic functions, iron can be harmful and cancerogenic though. By exchanging between its different oxidized forms, iron overload induces free radical formation, lipid peroxidation, DNA, and protein damages, leading to carcinogenesis or ferroptosis. Iron also plays profound roles in modulating tumor microenvironment and metastasis, maintaining genomic stability and controlling epigenetics. in order to meet the high requirement of iron, neoplastic cells have remodeled iron metabolism pathways, including acquisition, storage, and efflux, which makes manipulating iron homeostasis a considerable approach for cancer therapy. Several iron chelators and iron oxide nanoparticles (IONPs) has recently been developed for cancer intervention and presented considerable effects. This review summarizes some latest findings about iron metabolism function and regulation mechanism in cancer and the application of iron chelators and IONPs in cancer diagnosis and therapy. Full article

(This article belongs to the Special Issue Drug Discovery, Development and Regulatory Affairs)

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Iron and epigenetic regulation. Iron can modulate heterochromatin assembly mediated by Fenton reactions and induce global histone methylation changes through iron-dependent JmjC-domain-containing epigenetic modifying enzymes in cancer cells. Multiple miRNAs have been demonstrated to regulate iron metabolism-related proteins. DNA methylation, histone acetylation/methylation modification, and some transcription factors such as NRF2 and MZF-1 function corporately to maintain cellular iron metabolism in cancer. TfR, transferrin receptor 1; IRP2, iron regulatory protein; FPN, ferroportin; DMT1, divalentmetal transporter 1; ER, endoplasmic reticulum; HDAC, histone deacetylase; HEPH, hephaestin; Elp3, elongator complex protein 3; 5mC, 5-methylcytosine; TET, ten-eleven translocation protein. Full article ">Figure 2
Iron handling in the tumor microenvironment. Tumor microenvironment compartments play a critical role in controlling iron metabolism. Inflammatory cytokines upregulate Lcn2 via NF-κB pathway. After releasing out of the cell, Lcn2 sequesters iron and stabilize MMP-9, promoting cell survival and matrix degradation leading to EMT. M2 macrophages are major sites of taking up, metabolizing, storing, and exporting iron. They supply iron to accelerate tumor growth by multiple transport pathways. Tumor-associated fibroblasts contribute to hepcidin induction via paracrine IL-6/BMP/SMAD signaling. Circulating T cells has accumulated H-ferritin to maintain proper immune functions. Th1 cells and NKT cells can secret cytokines like IFN-γ and TNF to the environment, which increase DMT1 whereas decrease FPN level, thus resulting in iron sequestration in the MPS. Tumor-associated fibroblasts induce hepcidin expression via paracrine IL-6-BMP-SMAD signaling. Lcn2, Lipocalin 2; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; MMP-9, matrix metalloproteinases-9; EMT, epithelial-mesenchymal transition; IL-6, interleukin-6; NKT, natural killer T cells; IFN—γ, interferon-γ; TNF, tumor necrosis factor; FPN, ferroportin; MPS, mononuclear phagocyte system. Full article ">

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