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Lanthascreen eu kinase binding assay

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The LanthaScreen Eu Kinase Binding Assay is a fluorescence-based assay used to measure the binding affinity of compounds to kinases. It utilizes a europium-labeled kinase and a fluorescently-labeled ligand to detect the interaction between the compound and the kinase.

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Lab products found in correlation

Infinite m1000 pro, by Tecan (1 mentions) Z lytescreen, by Thermo Fisher Scientific (1 mentions) Safire2 plate reader, by Tecan (1 mentions) Low volume 384 well plates, by Corning (1 mentions) Prestoblue cell viability reagent, by Thermo Fisher Scientific (1 mentions) Envision multilabel reader, by PerkinElmer (1 mentions) Z lyte kinase assay, by Thermo Fisher Scientific (1 mentions)

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LanthaScreen™ (34 citations)

9 protocols using lanthascreen eu kinase binding assay

1

In Vitro Evaluation of DYRK1A Inhibitor Compound 1

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Example 1

Compound 1, a 1,3,4-thiadiazine compound identified in a screening assay, was evaluated for testing of in vitro DYRK1A activity at 30 μM concentration at Life Technologies using a FRET-based LanthaScreen® Eu Kinase Binding Assay. Compound 1 had an IC50 of 9.41 μM against DYRK1A (Kd of 7.5 μM against DYRK1A). This data was confirmed by a second assay, KINOMEscan® (Fabian et al., “A Small Molecule-kinase Interaction Map for Clinical Kinase Inhibitors,” Nat. Biotechnol. 23(3):329-336 (2005), which is incorporated by reference in its entirety), which measures DYRK1A binding. The results obtained were consistent with those of the Life Technologies inhibition assay with the Kd of 7.3 μM for Compound 1 in the DiscoverX assay.

US11547712B2. Kinase inhibitor compounds and compositions and methods of use (2023-01-10). ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI [US]. Inventors: Robert Devita [US], Andrew Stewart [US], Avner Schlessinger [US], Kunal Kumar [US], Peter Man-Un Ung [US], Hui Wang [US], Hailing Li [US].
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2

DYRK1A Kinase Binding Assay

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Compounds were tested for DYRK1A binding activity by a commercial kinase profiling services, Life Technologies which uses the FRET-based LanthaScreen® Eu Kinase Binding Assay. [38 (link)] Compounds were screened for DYRK1A activity at concentrations of 1000 nM and 300 nM in duplicates. The IC50 was determined by 10 point LanthaScreen® Eu Kinase Binding Assay [38 (link)] in duplicates.
Kumar K., Wang P., A. Swartz E., Khamrui S., Secor C., B. Lazarus M., Sanchez R., F. Stewart A, & DeVita R.J. (2020). Structure–Activity Relationships and Biological Evaluation of 7-Substituted Harmine Analogs for Human β-Cell Proliferation. Molecules, 25(8), 1983.
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3

Kinase Inhibition Profiling Assays

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The IC50 values against ALK5, TGFBR2, casein kinase 1 delta (CK1δ), and epsilon (CK1ε) of selected compounds were determined using LanthaScreen Eu Kinase binding assay (Life Technologies, Rockville, MD). The assays were performed following the manufacturers’ protocols in white 384‐well plates (Cat. No. 3572; Corning Life Sciences, Acton, MA). SB203580 and TA‐01 were tested as controls in all assays. The compounds were dissolved in DMSO (Hybri‐Max Cat. No. D2650; Sigma‐Aldrich, St. Louis, MO, 5 mM stocks) and diluted to a final concentration of 1% (vol/vol) DMSO for all assays. Each data point was carried out in triplicate. The assay was run according to the protocol developed by Invitrogen (Carlsbad, CA). Tecan Infinite M1000 Pro microplate reader was used for fluorescence measurements (LanthaScreen Eu Kinase binding assay; excitation λ = 317 nm, emission λ = 665, 620 nm). Inhibition curves and IC50 values were generated by nonlinear regression analysis and data represent mean ± SEM. Results are included in Supporting information Table S1.
Zhong Q., Laco F., Liao M., Woo T.L., Oh S.K, & Chai C.L. (2018). Influencing the Fate of Cardiac and Neural Stem Cell Differentiation Using Small Molecule Inhibitors of ALK5. Stem Cells Translational Medicine, 7(10), 709-720.
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4

Kinase Profiling of INX-315

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INX-315 was first evaluated in the SelectScreen Biochemical Kinase Profiling at Thermo Fisher Scientific (Madison, WI). The primary screen consisted of INX-315 dosed at a single concentration of 100 nmol/L in 1% DMSO across each platform within the SelectScreen array of assays, LanthaScreen Eu Kinase Binding Assay; AdaptaScreen; and Z'LyteScreen using preestablished conditions at Thermo Fisher Scientific as shown in Supplementary Table S2. All target kinases that responded to greater than 90% inhibition in primary screen were retested as individuals for IC50 determination shown in Supplementary Fig. S1. The follow-up screen consisted of 10-point dose–response curves for INX-315 from 1 μmol/L to 0.0495 nmol/L.
Dietrich C., Trub A., Ahn A., Taylor M., Ambani K., Chan K.T., Lu K.H., Mahendra C.A., Blyth C., Coulson R., Ramm S., Watt A.C., Matsa S.K., Bisi J., Strum J., Roberts P, & Goel S. (2023). INX-315, a Selective CDK2 Inhibitor, Induces Cell Cycle Arrest and Senescence in Solid Tumors. Cancer Discovery, 14(3), 446-467.
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5

Evaluating Vemurafenib Derivatives for Cell Proliferation

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For cell proliferation/viability assays, cells were plated at 5000 cells per well in 96-well plates (Corning assay well plates, Corning, NY, USA) and grown for 24 hrs. Cells were then treated with increasing concentrations of vemurafenib derivatives (0 nM, 0.001 nM, 0.01 nM, 0.1 nM, 1 nM, 10 nM, 100 nM, 1 µM, 10 µM, 50 µM) for 72 hrs. PrestoBlue® Cell Viability Reagent (ThermoFisher Scientific) was added as a 10X solution and plates were read out on a Tecan Safire 2 Platereader (Tecan Trading AG, Switzerland) after 10-30 min. The binding affinity of vemurafenib derivatives to BRAFV600E was assayed using the LanthaScreen® Eu Kinase Binding Assay (ThermoFisher Scientific) following the manufacturer's protocol. All reagents and BRAF recombinant human protein (sold as BRAF[V599E], which is equivalent to BRAFV600E) were purchased from ThermoFisher Scientific/Life technologies. Low volume 384-well plates (Corning, NY, USA) were used and read out using a Perkin Elmer EnVision Multilabel Reader (Waltham, MA, USA).
Mikula H., Stapleton S., Kohler R.H., Vinegoni C, & Weissleder R. (2017). Design and Development of Fluorescent Vemurafenib Analogs for In Vivo Imaging. Theranostics, 7(5), 1257-1265.
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6

Kinase Inhibitor Screening of PLX8725

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Briefly, a LanthaScreen™ Eu Kinase Binding Assay (Thermo Fisher Scientific) was used to detect and characterize the kinase inhibitor activity (i.e., % inhibition and IC50) of the Plexxikon compound PLX8725 versus multiple MAP kinases including MAP2K1, MAP2K2, MAP2K4, MAP2K5 and MAP2K6 as previously described [13 (link)].
Stead D, & Park S.F. (2000). Roles of Fe Superoxide Dismutase and Catalase in Resistance of Campylobacter coli to Freeze-Thaw Stress. Applied and Environmental Microbiology, 66(7), 3110-3112.
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7

Measuring DYRK1A Kinase Activity

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The assay to measure DYRK1A kinase activity utilizes components from a LanthaScreen™ Eu Kinase Binding Assay (Thermo Fisher Scientific). Assay components DYRK1A-GST protein (catalog #PR7189B), Kinase Tracer 236 and Europium-anti GST antibody were from Life Technologies (Thermo Fisher Scientific). 384-well low volume black round bottom polystyrene non-binding surface microplates were from Corning, NY (catalog # 4514). Unless otherwise stated, all reagents and compounds were purchased from Thermo Fisher Scientific (Waltham, MA) or Sigma Aldrich (St. Louis, MO) at the highest level of purity possible.
Tarpley M., Caligan T.B., Onyenwoke R.U, & Williams K.P. (2021). Optimization and validation of a DYRK1A TR-FRET assay for high-throughput screening. MethodsX, 8, 101383.
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8

Kinase Inhibitor IC50 Determination

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Each inhibitor’s potency was determined by generating 10-point IC50 curves from a 4-fold dilution series in DMSO (1 mM). Curves were generated from the compound concentration and the corresponding percent (%) inhibition calculated for each concentration tested. For PLK4, we used the LanthaScreen Eu Kinase Binding Assay (Thermo Fisher Scientific, Carlsbad, CA, USA) that utilizes an Alexa Fluor conjugated “tracer” and an Eu-labeled anti-tag antibody to measure binding of a compound to the kinase target. For AURKs (AURKA, AURKB and AURKC) we used the Z’ LYTE Kinase Assay (Thermo Fisher Scientific, Carlsbad, CA, USA) that determines the differential sensitivity of phosphorylated and non-phosphorylated peptide substrates to proteolytic cleavage using a FRET-based readout.
Suri A., Bailey A.W., Tavares M.T., Gunosewoyo H., Dyer C.P., Grupenmacher A.T., Piper D.R., Horton R.A., Tomita T., Kozikowski A.P., Roy S.M, & Sredni S.T. (2019). Evaluation of Protein Kinase Inhibitors with PLK4 Cross-Over Potential in a Pre-Clinical Model of Cancer. International Journal of Molecular Sciences, 20(9), 2112.
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9

Inhibitory Potencies of JAK and TGF-β Kinases

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JAK1, JAK2, JAK3, and TYK2 50% inhibitory concentrations (IC50s) for MMB and RUX were assessed in a commercial kinase potency screen performed by Carna Biosciences (Kobe, Japan). To obtain IC50 values, MMB and RUX were incubated as 10-point titrations to cover cpd concentrations from 0.3 nM to 10 μM.
ACVR1, BMPR1a, and transforming growth factor β receptor 1 (TGFBR1) IC50 values were obtained using the SelectScreen biochemical kinase profiling service (LanthaScreen Eu Kinase Binding Assay; Thermo Fisher). To obtain IC50 values, MMB and RUX were incubated as 10-point titration, threefold serial dilutions to cover cpd concentrations from 0.5 nM to 10 μM.
Asshoff M., Petzer V., Warr M.R., Haschka D., Tymoszuk P., Demetz E., Seifert M., Posch W., Nairz M., Maciejewski P., Fowles P., Burns C.J., Smith G., Wagner K.U., Weiss G., Whitney J.A, & Theurl I. (2017). Momelotinib inhibits ACVR1/ALK2, decreases hepcidin production, and ameliorates anemia of chronic disease in rodents. Blood, 129(13), 1823-1830.
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