scholarly journals A Nonradioactive Fluorimetric SPE-Based Ceramide Kinase Assay Using NBD-C6-Ceramide

2012 ◽  
Vol 2012 ◽  
pp. 1-9 ◽  
Author(s):  
Helena Van Overloop ◽  
Gerd Van der Hoeven ◽  
Paul P. Van Veldhoven
Keyword(s):  
High Throughput Screening ◽  
Kinase Inhibitors ◽  
Solid Phase ◽  
Protein Kinase Inhibitors ◽  
Kinase Assay ◽  
Cellular Processes ◽  
Ceramide Kinase ◽  
C6 Ceramide ◽  
Ceramide 1

Ceramide kinase (CERK) has been implicated in important cellular processes such as inflammation and apoptosis. Its activity is usually measured using radiolabeled ceramide or [γ-32P]-ATP, followed by extraction, thin-layer chromatography, and detection of the formed labeled ceramide-1-phosphate. To eliminate the use of radioactivity, we developed similarly but independently from the approach by Don and Rosen (2008), a fluorescence-based ceramide kinase assay, using N-[7-(4-nitrobenz-2-oxa-1,3-diazole)]-6-aminohexanoyl-sphingenine (NBD-C6-ceramide) as substrate. ItsKmvalue (4 μM) was comparable to that of N-hexanoyl-sphingenine (C6-ceramide). The produced fluorescent NBD-C6-ceramide-1-phosphate was captured by means of solid-phase extraction on an aminopropyl phase, resulting in a fast and sensitive CERK measurement. By performing this assay in a 96-well format, it is also suitable for high-throughput screening (HTS) to search for CERK modulators. A limited screen revealed that some protein kinase inhibitors (e.g., U-0126;IC504 μM) and ceramide analogues (e.g., fenretinide, AMG-9810;IC501.1 μM) affect CERKin vitro.

scholarly journals Discovery of Novel Gq-Biased LPA1 Negative Allosteric Modulators

2017 ◽  
Vol 22 (7) ◽  
pp. 859-866
Author(s):  
Yuji Shimizu ◽  
Masaharu Nakayama
Keyword(s):  
G Protein ◽  
High Throughput Screening ◽  
Kinase Inhibitors ◽  
Protein Kinase Inhibitors ◽  
Allosteric Modulators ◽  
Label Free ◽  
Cellular Processes ◽  
Recombinant Cells ◽  
And Migration ◽  
Cellular Phenotypes

Lysophosphatidic acid (LPA) activates the G-protein-coupled receptor LPA1, which regulates various cellular processes, including cell proliferation and migration. Although LPA1 transduces cellular responses via Gq, Gi, and G12/13, associations between these signaling molecules and cellular phenotypes remain poorly characterized due to the lack of signal-specific pharmacological tools. Here, we characterized novel signal-biased modulators using multiple assays, including label-free impedance assays. LPA caused dramatic changes in cellular impedance in LPA1-expressing recombinant cells, which were susceptible to G-protein and protein kinase inhibitors. Subsequently, Gq-biased LPA1 negative allosteric modulators (NAMs) were identified using high-throughput screening, and a nonbiased antagonist differently affected the LPA-induced cellular impedance. These NAMs provide pharmacological tools for further investigations of the biology of LPA1.

scholarly journals Cell Treatment and Lysis in 96-Well Filter-Bottom Plates for Screening Bcr-Abl Activity and Inhibition in Whole-Cell Extracts

2010 ◽  
Vol 15 (4) ◽  
pp. 434-440 ◽  
Author(s):  
Michael R. Mand ◽  
Ding Wu ◽  
Darren R. Veach ◽  
Stephen J. Kron
Keyword(s):  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Solid Phase ◽  
Kinase Assay ◽  
Cell Extracts ◽  
Complementary Approach ◽  
Plate Cell ◽  
High Throughput Screens

Although conventional high-throughput screens performed in vitro with purified protein kinases are powerful tools to discover new kinase inhibitors, they are far from ideal for determining efficacy in vivo. As a complementary approach, cell-based, target-driven secondary screens may help predict in vivo compound potency and specificity as well as evaluate bioavailability and toxicity. Here the authors report a simple protocol for treating K562 Bcr-Abl-expressing cells with small-molecule kinase inhibitors in 96-well filter-bottom plates followed by in-plate cell lysis. The lysates were assayed via a solid-phase kinase assay, allowing determination of apparent IC50 for known Bcr-Abl inhibitors as well as facilitating the screening of a small kinase inhibitor library. This approach may have further applications in generating lysates for analyzing kinase activity and inhibition in other nonadherent suspension cell lines.

Alkaloids as Anticancer Agents: A Review of Chinese Patents in Recent 5 Years

2020 ◽  
Vol 15 (1) ◽  
pp. 2-13 ◽  
Author(s):  
Hongyu Tao ◽  
Ling Zuo ◽  
Huanli Xu ◽  
Cong Li ◽  
Gan Qiao ◽  
...  
Keyword(s):  
Anticancer Activity ◽  
Anticancer Agents ◽  
Kinase Inhibitors ◽  
Protein Kinase Inhibitors ◽  
Cdk Inhibitors ◽  
Comprehensive Overview ◽  
P53 Expression ◽  
Study Results

Background: In recent years, many novel alkaloids with anticancer activity have been found in China, and some of them are promising for developing as anticancer agents. Objective: This review aims to provide a comprehensive overview of the information about alkaloid anticancer agents disclosed in Chinese patents, and discusses their potential to be developed as anticancer drugs used clinically. Methods: Anticancer alkaloids disclosed in Chinese patents in recent 5 years were presented according to their mode of actions. Their study results published on PubMed, and SciDirect databases were presented. Results: More than one hundred anticancer alkaloids were disclosed in Chinese patents and their mode of action referred to arresting cell cycle, inhibiting protein kinases, affecting DNA synthesis and p53 expression, etc. Conclusion: Many newly found alkaloids displayed potent anticancer activity both in vitro and in vivo, and some of the anticancer alkaloids acted as protein kinase inhibitors or CDK inhibitors possess the potential for developing as novel anticancer agents.

scholarly journals Targeting IDH1/2 mutant cancers with combinations of ATR and PARP inhibitors

NAR Cancer ◽  
2021 ◽  
Vol 3 (2) ◽  
Author(s):  
Amrita Sule ◽  
Jinny Van Doorn ◽  
Ranjini K Sundaram ◽  
Sachita Ganesa ◽  
Juan C Vasquez ◽  
...  
Keyword(s):  
Kinase Inhibitors ◽  
Parp Inhibitor ◽  
Parp Inhibitors ◽  
Protein Kinase Inhibitors ◽  
Isocitrate Dehydrogenase 1 ◽  
Normal Product ◽  
Synthetic Lethal Interactions ◽  
Mutant Cells

Abstract Mutations in the isocitrate dehydrogenase-1 and -2 (IDH1/2) genes were first identified in glioma and acute myeloid leukemia (AML), and subsequently found in multiple other tumor types. These neomorphic mutations convert the normal product of enzyme, α-ketoglutarate (αKG), to the oncometabolite 2-hydroxyglutarate (2HG). Our group recently demonstrated that 2HG suppresses the high-fidelity homologous recombination (HR) DNA repair pathway, resulting in a state referred to as ‘BRCAness’, which confers exquisite sensitivity to poly(ADP-ribose) polymerase (PARP) inhibitors. In this study, we sought to elucidate sensitivity of IDH1/2-mutant cells to DNA damage response (DDR) inhibitors and, whether combination therapies could enhance described synthetic lethal interactions. Here, we report that ATR (ataxia telangiectasia and Rad3-related protein kinase) inhibitors are active against IDH1/2-mutant cells, and that this activity is further potentiated in combination with PARP inhibitors. We demonstrate this interaction across multiple cell line models with engineered and endogenous IDH1/2 mutations, with robust anti-tumor activity in vitro and in vivo. Mechanistically, we found ATR and PARP inhibitor treatment induces premature mitotic entry, which is significantly elevated in the setting of IDH1/2-mutations. These data highlight the potential efficacy of targeting HR defects in IDH1/2-mutant cancers and support the development of this combination in future clinical trials.

scholarly journals Application of a PBPK Model of Rivaroxaban to Prospective Simulations of Drug-Drug-Disease Interactions with Protein Kinase Inhibitors in CA-VTE

2021 ◽  
Author(s):  
Eleanor Jing Yi Cheong ◽  
Daniel Zhi Wei Ng ◽  
Sheng Yuan Chin ◽  
Ziteng Wang ◽  
Eric Chun Yong Chan
Keyword(s):  
Protein Kinase ◽  
Kinase Inhibitors ◽  
Pbpk Model ◽  
Organic Anion ◽  
Protein Kinase Inhibitors ◽  
Organic Anion Transporter ◽  
Pbpk Modelling ◽  
Anion Transporter

Background and Purpose Rivaroxaban is emerging as a viable anticoagulant for the pharmacological management of cancer associated venous thromboembolism (CA-VTE). Being eliminated via CYP3A4/2J2-mediated metabolism and organic anion transporter 3 (OAT3)/P-glycoprotein-mediated renal secretion, rivaroxaban is susceptible to drug-drug interactions (DDIs) with protein kinase inhibitors (PKIs), erlotinib and nilotinib. Physiologically based pharmacokinetic (PBPK) modelling was applied to interrogate the DDIs for dose adjustment of rivaroxaban in CA-VTE. Experimental Approach The inhibitory potencies of erlotinib and nilotinib on CYP3A4/2J2-mediated metabolism of rivaroxaban were characterized. Using prototypical OAT3 inhibitor ketoconazole, in vitro OAT3 inhibition assays were optimized to ascertain the in vivo relevance of derived inhibitory constants (K). DDIs between rivaroxaban and erlotinib or nilotinib were investigated using iteratively verified PBPK model. Key Results Mechanism-based inactivation (MBI) of CYP3A4-mediated rivaroxaban metabolism by both PKIs and MBI of CYP2J2 by erlotinib were established. The importance of substrate specificity and nonspecific binding to derive OAT3-inhibitory K values of ketoconazole and nilotinib for the accurate prediction of DDIs was illustrated. When simulated rivaroxaban exposure variations with concomitant erlotinib and nilotinib therapy were evaluated using published dose-exposure equivalence metrics and bleeding risk analyses, dose reductions from 20 mg to 15 mg and 10 mg in normal and mild renal dysfunction, respectively, were warranted. Conclusion and Implications We established the PBPK-DDI platform to prospectively interrogate and manage clinically relevant interactions between rivaroxaban and PKIs in patients with underlying renal impairment. Rational dose adjustments were proposed, attesting to the capacity of PBPK modelling in facilitating precision medicine.

scholarly journals Covalent inhibitors of EGFR family protein kinases induce degradation of human Tribbles 2 (TRIB2) pseudokinase in cancer cells

2018 ◽  
Vol 11 (549) ◽  
pp. eaat7951 ◽  
Author(s):  
Daniel M. Foulkes ◽  
Dominic P. Byrne ◽  
Wayland Yeung ◽  
Safal Shrestha ◽  
Fiona P. Bailey ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Small Molecule ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Growth Factor Receptor ◽  
Drug Repurposing ◽  
Protein Kinase Inhibitors ◽  
Cellular Mechanisms ◽  
Cellular Analysis

A major challenge associated with biochemical and cellular analysis of pseudokinases is a lack of target-validated small-molecule compounds with which to probe function. Tribbles 2 (TRIB2) is a cancer-associated pseudokinase with a diverse interactome, including the canonical AKT signaling module. There is substantial evidence that human TRIB2 promotes survival and drug resistance in solid tumors and blood cancers and therefore is of interest as a therapeutic target. The unusual TRIB2 pseudokinase domain contains a unique cysteine-rich C-helix and interacts with a conserved peptide motif in its own carboxyl-terminal tail, which also supports its interaction with E3 ubiquitin ligases. We found that TRIB2 is a target of previously described small-molecule protein kinase inhibitors, which were originally designed to inhibit the canonical kinase domains of epidermal growth factor receptor tyrosine kinase family members. Using a thermal shift assay, we discovered TRIB2-binding compounds within the Published Kinase Inhibitor Set (PKIS) and used a drug repurposing approach to classify compounds that either stabilized or destabilized TRIB2 in vitro. TRIB2 destabilizing agents, including the covalent drug afatinib, led to rapid TRIB2 degradation in human AML cancer cells, eliciting tractable effects on signaling and survival. Our data reveal new drug leads for the development of TRIB2-degrading compounds, which will also be invaluable for unraveling the cellular mechanisms of TRIB2-based signaling. Our study highlights that small molecule–induced protein down-regulation through drug “off-targets” might be relevant for other inhibitors that serendipitously target pseudokinases.

scholarly journals New tools for carbohydrate sulfation analysis: heparan sulfate 2-O-sulfotransferase (HS2ST) is a target for small-molecule protein kinase inhibitors

2018 ◽  
Vol 475 (15) ◽  
pp. 2417-2433 ◽  
Author(s):  
Dominic P. Byrne ◽  
Yong Li ◽  
Krithika Ramakrishnan ◽  
Igor L. Barsukov ◽  
Edwin A. Yates ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Heparan Sulfate ◽  
Small Molecule ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Protein Kinase Inhibitors ◽  
Fluorescent Substrate ◽  
Raf Kinase ◽  
Shift Analysis

Sulfation of carbohydrate residues occurs on a variety of glycans destined for secretion, and this modification is essential for efficient matrix-based signal transduction. Heparan sulfate (HS) glycosaminoglycans control physiological functions ranging from blood coagulation to cell proliferation. HS biosynthesis involves membrane-bound Golgi sulfotransferases, including HS 2-O-sulfotransferase (HS2ST), which transfers sulfate from the cofactor PAPS (3′-phosphoadenosine 5′-phosphosulfate) to the 2-O position of α-l-iduronate in the maturing polysaccharide chain. The current lack of simple non-radioactive enzyme assays that can be used to quantify the levels of carbohydrate sulfation hampers kinetic analysis of this process and the discovery of HS2ST inhibitors. In the present paper, we describe a new procedure for thermal shift analysis of purified HS2ST. Using this approach, we quantify HS2ST-catalysed oligosaccharide sulfation using a novel synthetic fluorescent substrate and screen the Published Kinase Inhibitor Set, to evaluate compounds that inhibit catalysis. We report the susceptibility of HS2ST to a variety of cell-permeable compounds in vitro, including polyanionic polar molecules, the protein kinase inhibitor rottlerin and oxindole-based RAF kinase inhibitors. In a related study, published back-to-back with the present study, we demonstrated that tyrosyl protein sulfotranferases are also inhibited by a variety of protein kinase inhibitors. We propose that appropriately validated small-molecule compounds could become new tools for rapid inhibition of glycan (and protein) sulfation in cells, and that protein kinase inhibitors might be repurposed or redesigned for the specific inhibition of HS2ST.

scholarly journals Identification of Two Kinase Inhibitors with Synergistic Toxicity with Low-Dose Hydrogen Peroxide in Colorectal Cancer Cells In vitro

Cancers ◽  
2020 ◽  
Vol 12 (1) ◽  
pp. 122 ◽  
Author(s):  
Eric Freund ◽  
Kim-Rouven Liedtke ◽  
Lea Miebach ◽  
Kristian Wende ◽  
Amanda Heidecke ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Colorectal Cancer ◽  
Hydrogen Peroxide ◽  
Protein Kinase ◽  
Tumor Cells ◽  
Low Dose ◽  
Kinase Inhibitors ◽  
Protein Kinase Inhibitors ◽  
Colorectal Cancer Cells

Colorectal carcinoma is among the most common types of cancers. With this disease, diffuse scattering in the abdominal area (peritoneal carcinosis) often occurs before diagnosis, making surgical removal of the entire malignant tissue impossible due to a large number of tumor nodules. Previous treatment options include radiation and its combination with intraperitoneal heat-induced chemotherapy (HIPEC). Both options have strong side effects and are often poor in therapeutic efficacy. Tumor cells often grow and proliferate dysregulated, with enzymes of the protein kinase family often playing a crucial role. The present study investigated whether a combination of protein kinase inhibitors and low-dose induction of oxidative stress (using hydrogen peroxide, H2O2) has an additive cytotoxic effect on murine, colorectal tumor cells (CT26). Protein kinase inhibitors from a library of 80 substances were used to investigate colorectal cancer cells for their activity, morphology, and immunogenicity (immunogenic cancer cell death, ICD) upon mono or combination. Toxic compounds identified in 2D cultures were confirmed in 3D cultures, and additive cytotoxicity was identified for the substances lavendustin A, GF109203X, and rapamycin. Toxicity was concomitant with cell cycle arrest, but except HMGB1, no increased expression of immunogenic markers was identified with the combination treatment. The results were validated for GF109203X and rapamycin but not lavendustin A in the 3D model of different colorectal (HT29, SW480) and pancreatic cancer cell lines (MiaPaca, Panc01). In conclusion, our in vitro data suggest that combining oxidative stress with chemotherapy would be conceivable to enhance antitumor efficacy in HIPEC.

scholarly journals Kinase Inhibitors of Novel Pyridopyrimidinone Candidates: Synthesis and In Vitro Anticancer Properties

2019 ◽  
Vol 2019 ◽  
pp. 1-10 ◽  
Author(s):  
Nagy M. Khalifa ◽  
Mohamed A. Al-Omar ◽  
Hamad M. Alkahtani ◽  
Ahmed H. Bakheit
Keyword(s):  
Kinase Inhibitors ◽  
Human Cancer ◽  
Ethyl Cyanoacetate ◽  
Diethyl Malonate ◽  
Aromatic Aldehydes ◽  
Docking Studies ◽  
Braf V600e ◽  
Kinase Assay ◽  
Human Cancer Cell Lines

A new class of pyridopyrimidinone compounds containing different nitrogenous heterocycles were synthesized starting from the key precursor 2-hydrazinyl-5-phenyl-7-(pyridin-3-yl)pyrido[2,3-d]pyrimidin-4(3H)-one via condensation with series of aromatic aldehydes and cyclization using different reagents as ethyl acetoacetate, ethyl cyanoacetate, diethyl malonate, and ammonium isothiocyanate. The bioassay results showed compound 6 to be highly effective towards three human cancer cell lines (HepG2, PC-3, and HCT-116) in vitro with promising activity values (IC50: 0.5 μM) relative to the standard doxorubicin (IC50: 0.6 μM). Kinase inhibitory evaluation of compound 6 displays hopeful inhibitory action against BRAF V600E, EGFR, and PDGFRβ at100 μM. The molecular docking studies supported the initial kinase assay.

scholarly journals Genistein reduces tumor necrosis factor alpha-induced plasminogen activator inhibitor-1 transcription but not urokinase expression in human endothelial cells

Blood ◽  
1994 ◽  
Vol 84 (9) ◽  
pp. 2984-2991 ◽  
Author(s):  
VW van Hinsbergh ◽  
M Vermeer ◽  
P Koolwijk ◽  
J Grimbergen ◽  
T Kooistra
Keyword(s):  
Endothelial Cells ◽  
Protein Kinase ◽  
Plasminogen Activator ◽  
Kinase Inhibitors ◽  
Protein Kinase Inhibitors ◽  
Tnf Alpha ◽  
Necrosis Factor Alpha ◽  
Tyrosine Protein Kinase ◽  
Pai 1

Abstract The plasminogen activator inhibitor PAI-1 is markedly elevated in vivo and in vitro upon exposure to the inflammatory mediators tumor necrosis factor alpha (TNF alpha), interleukin-1 (IL-1), and bacterial lipopolysaccharide. Here we report that the isoflavone compound genistein prevents the increase in synthesis of PAI-1 induced by these inflammatory mediators in human endothelial cells in vitro, and partially reduces the basal PAI-1 production by these cells. These effects of genistein were accompanied by a decrease in PAI-1 mRNA and in a suppression of the PAI-1 transcription rate as shown by run-on assay. A specific action of genistein, probably by inhibiting a tyrosine protein kinase, is likely, because the structural genistein analogue daidzein, which has a low tyrosine protein kinase inhibitor activity, did not inhibit PAI-1 synthesis. Vanadate, a tyrosine protein phosphatase inhibitor, increased PAI-1 production. The effect of genistein on PAI-1 synthesis was rather selective. Herbimycin A also reduced PAI-1 synthesis, but several other tyrosine protein kinase inhibitors, namely tyrphostin A47, methyl-2,5-dihydroxy-cinnamate, and compound 5, were unable to do so. All these tyrosine protein kinase inhibitors reduced basic fibroblast growth factor (b-FGF)-induced [3H]thymidine incorporation in endothelial cells. This indicates that the effect of genistein on PAI-1 transcription proceeds independently of its effect on mitogenesis. In contrast to TNF-alpha-induced PAI-1 production, the transcription and synthesis of urokinase-type plasminogen activator (u-PA) was not inhibited by genistein. A TNF- alpha-mutant (Trp32Thr86TNF alpha) that specifically recognizes the 55- kD TNF-receptor, mimicked the effects of TNF alpha on both PAI-1 and u- PA. Because genistein affected PAI-1, but not u-PA induced by this mutant, involvement of different TNF-receptors cannot underlie the difference in the effects of genistein on PAI-1 and u-PA synthesis. Because genistein also inhibited PAI-1 induction by thrombin and IL-4, it is likely that genistein does not act on a TNF alpha-receptor- coupled protein kinase but on the signal transduction pathway enhancing PAI-1 transcription. Our results suggest that the TNF alpha-induced signal transduction pathway of PAI-1 transcription involves a genistein- sensitive step that is not involved in the induction of u-PA by TNF alpha. Given the limited sensitivity to several other tyrosine protein kinase inhibitors, this genistein-sensitive step may be a potential target for pharmacologic intervention to reduce elevated plasma PAI-1 levels.

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