Bioluminescence Methods for Assaying Kinases in Quantitative High-Throughput Screening (qHTS) Format Applied to Yes1 Tyrosine Kinase, Glucokinase, and PI5P4Kα Lipid Kinase

Davis, Mindy I.; Auld, Douglas S.; Inglese, James

doi:10.1007/978-1-4939-3073-9_4

Cited by 7 publications

(4 citation statements)

References 23 publications

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“…The ADP-Glo TM Kinase Assay (Promega, Madison, WI, USA) was used to test the CK2 inhibition effects of these compounds [30,31]. As indicated by the relevant studies [32], competitive and noncompetitive inhibitors show the increased and similar IC 50 values with increasing concentrations of ATP, respectively. Therefore, we were able to distinguish between ATP competitive and noncompetitive inhibitors of CK2 by exploring whether the IC 50 value of inhibitors will change or not at 10 and 100 µM ATP.…”

Section: Methodsmentioning

confidence: 99%

Identification and Biological Evaluation of CK2 Allosteric Fragments through Structure-Based Virtual Screening

Zhang

et al. 2020

Molecules

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Casein kinase II (CK2) is considered as an attractive cancer therapeutic target, and recent efforts have been made to develop its ATP-competitive inhibitors. However, achieving selectivity with respect to related kinases remains challenging due to the highly conserved ATP-binding pocket of kinases. Allosteric inhibitors, by targeting the much more diversified allosteric site relative to the highly conserved ATP-binding pocket, might be a promising strategy with the enhanced selectivity and reduced toxicity than ATP-competitive inhibitors. The previous studies have highlighted the traditional serendipitousity of discovering allosteric inhibitors owing to the complicate allosteric modulation. In this current study, we identified the novel allosteric inhibitors of CK2α by combing structure-based virtual screening and biological evaluation methods. The structure-based pharmacophore model was built based on the crystal structure of CK2α-compound 15 complex. The ChemBridge fragment library was searched by evaluating the fit values of these molecules with the optimized pharmacophore model, as well as the binding affinity of the CK2α-ligand complexes predicted by Alloscore web server. Six hits forming the holistic interaction mechanism with the αD pocket were retained after pharmacophore- and Alloscore-based screening for biological test. Compound 3 was found to be the most potent non-ATP competitive CK2α inhibitor (IC50 = 13.0 μM) with the anti-proliferative activity on A549 cancer cells (IC50 = 23.1 μM). Our results provide new clues for further development of CK2 allosteric inhibitors as anti-cancer hits.

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Section: Methodsmentioning

confidence: 99%

Identification and Biological Evaluation of CK2 Allosteric Fragments through Structure-Based Virtual Screening

Zhang

et al. 2020

Molecules

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“…Briefly, the transphosphorylation reaction (10 μL) was transferred into a 96well plate (Greiner, solid white, low-binding assay plates) and 10 µL of ADP Glo reagent was added to each well. The reaction was then incubated at room temperature for 40 min to deplete all remaining ATP [63]. The ADP produced by the enzyme/substrate interaction was then converted to ATP by adding 20 µL/well of Kinase Detection Reagent to yield a total assay volume of 40 µL/well.…”

Section: Erk2 Activation and Purificationmentioning

confidence: 99%

BCATc modulates crosstalk between the PI3K/Akt and the Ras/ERK pathway regulating proliferation in triple negative breast cancer

et al. 2020

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The cytosolic branched chain aminotransferase (BCATc) protein has been found to be highly expressed in breast cancer subtypes, including triple negative breast cancer (TNBC), compared with normal breast tissue. The catabolism of branched-chain amino acids (BCAAs) by BCATc leads to the production of glutamate and key metabolites which further drive the TCA cycle, important for cellular metabolism and growth. Upregulation of BCATc has been associated with increased cell proliferation, cell cycle progression and metastasis in several malignancies including breast, gliomas, ovarian and colorectal cancer but the underlying mechanisms are unclear. As nutrient levels of BCAAs, substrates of BCATc, regulate the PI3K/Akt pathway we hypothesized that increased expression of BCATc would contribute to tumour cell growth through upregulation of the insulin/IGF-1 signalling pathway. This pathway is known to potentiate proliferation and metastasis of malignant cells through the activation of PI3K/Akt and the RAS/ERK signalling cascades. Here we show that knockdown of BCATc significantly reduced insulin and IGF-1-mediated proliferation, migration and invasion of TNBC cells. An analysis of this pathway showed that when overexpressed BCATc regulates proliferation through the PI3K/Akt axis, whilst simultaneously attenuating the Ras/Erk pathway indicating that BCATc acts as a conduit between these two pathways. This ultimately led to an increase in FOXO3a, a key regulator of cell proliferation and Nrf2, which mediates redox homeostasis. Together this data indicates that BCATc regulates TNBC cell proliferation, migration and invasion through the IGF-1/insulin PI3K/Akt pathway, culminating in the upregulation of FOXO3a and Nrf2, pointing to a novel therapeutic target for breast cancer treatment.

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“…[19][20][21][22][23][24] Among them, the adenosine diphosphate (ADP) quantitative assay may be the most commonly used platform for HTS nowadays, because ADP is the universal product of all kinases, in contrast to the phosphorylated products, which vary widely and are sometimes difficult to detect. These ADP assays include the ADP-Glo assay (Promega, Madison, WI), in which ADP is converted to ATP and the ATP is measured by means of luciferase/ luciferin reaction; [25][26][27] the Transcreener fluorescence polarization assay (Bellbrook Labs, Madison, WI) using anti-ADP antibody and far-red fluorescence-labeled ADP; [28][29][30] and the ADP-Hunter fluorescence assay (DiscoverX, Fremont, CA), which generates H 2 O 2 from ADP by enzyme coupling reaction followed by production of resorufin. 31,32 These generic ADP detection assay platforms do not employ radiolabeling and are homogeneous, so they are suitable for HTS, but their cost is quite high for large-scale screening, and this may be an issue especially in academia.…”

Section: Introductionmentioning

confidence: 99%

Inexpensive High-Throughput Screening of Kinase Inhibitors Using One-Step Enzyme-Coupled Fluorescence Assay for ADP Detection

et al. 2019

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Protein kinases are attractive targets for both biological research and drug development. Several assay kits, especially for the detection of adenosine diphosphate (ADP), which is universally produced by kinases, are commercially available for high-throughput screening (HTS) of kinase inhibitors, but their cost is quite high for large-scale screening. Here, we report a new enzyme-coupled fluorescence assay for ADP detection, which uses just 10 inexpensive, commercially available components. The assay protocol is very simple, requiring only the mixing of test solutions with ADP detection solution and reading the fluorescence intensity of resorufin produced by coupling reaction. To validate the assay, we focused on CDC2-like kinase 1 (CLK1), a dual-specificity kinase that plays an important role in alternative splicing, and we used the optimized assay to screen an in-house chemical library of about 215,000 compounds for CLK1 inhibitors. We identified and validated 12 potent inhibitors of CLK1, including a novel inhibitory scaffold. The results demonstrate that this assay platform is not only simple and cost-effective, but also sufficiently robust, showing good reproducibility and giving similar results to those obtained with the widely used ADP-Glo bioluminescent assay.

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Bioluminescence Methods for Assaying Kinases in Quantitative High-Throughput Screening (qHTS) Format Applied to Yes1 Tyrosine Kinase, Glucokinase, and PI5P4Kα Lipid Kinase

Cited by 7 publications

References 23 publications

Identification and Biological Evaluation of CK2 Allosteric Fragments through Structure-Based Virtual Screening

Identification and Biological Evaluation of CK2 Allosteric Fragments through Structure-Based Virtual Screening

BCATc modulates crosstalk between the PI3K/Akt and the Ras/ERK pathway regulating proliferation in triple negative breast cancer

Inexpensive High-Throughput Screening of Kinase Inhibitors Using One-Step Enzyme-Coupled Fluorescence Assay for ADP Detection

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