Development of a HTRF® Kinase Assay for Determination of Syk Activity

Harbert, Christopher; Marshall, Jeannette; Soh, Sharon; Steger, Krista

doi:10.2174/1875397300801010020

Cited by 14 publications

(10 citation statements)

References 17 publications

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“…After 10 min incubation at room temperature, the assay plates were measured in the TR-FRET detection mode with a ViewLux plate reader. The results were calculated as a ratio of the acceptor fluorescence intensity divided by the donor fluorescence intensity [ 16 ].…”

Section: Methodsmentioning

confidence: 99%

Comparison on Functional Assays for Gq-Coupled GPCRs by Measuring Inositol Monophospate-1 and Intracellular Calcium in 1536-Well Plate Format

Liu¹,

Titus²,

Southall³

et al. 2008

Curr Chem Genomics

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Cell-based functional assays used for compound screening and lead optimization play an important role in drug discovery for G-protein coupled receptors (GPCRs). Cell-based assays can define the role of a compound as an agonist, antagonist or inverse agonist and can provide detailed information about the potency and efficacy of a compound. In addition, cell-based screens can be used to identify allosteric modulators that interact with sites other than the binding site of the endogenous ligand. Intracellular calcium assays which use a fluorescent calcium binding dye (such as Fluo-3, Fluo-4 or Fura-2) have been used in compound screening campaigns to measure the activity of Gq-coupled GPCRs. However, such screening methodologies require a special instrumentation to record the rapid change in intracellular free calcium concentration over time. The radioactive inositol 1,4,5- triphosphate (IP3) assay measures 3H-inositol incorporation and is another traditional assay for the assessment of Gq-coupled GPCR activity, but it is not suitable for screening of large size compound collections because it requires a cell wash step and generates radioactive waste. To avoid these limitations, we have optimized and miniaturized a TR-FRET based IP-One assay that measures inositol monophosphate in a 1536-well plate format. This assay is homogenous, non-radioactive and does not require a kinetic readout. It has been tested with the cell lines expressing M1 acetylcholine, FFAR1, vasopressin V1b, or Neuropeptide S receptors. The activities of antagonists determined in the IP-One assay correlated well with these measured in the intracellular calcium assay while the correlation of agonist activities might vary from cell line to cell line. This IP-One assay offers an alternative method for high throughput screening of Gq-coupled GPCRs without using costly kinetic plate readers.

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Section: Methodsmentioning

confidence: 99%

Comparison on Functional Assays for Gq-Coupled GPCRs by Measuring Inositol Monophospate-1 and Intracellular Calcium in 1536-Well Plate Format

Liu¹,

Titus²,

Southall³

et al. 2008

Curr Chem Genomics

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show abstract

“…1A , [ 17 ]). In vitro, the incubation of purified kinases with biotinylated peptide substrates leads to substrate phosphorylation quantifiable by TR-FRET [ 28 ]. We sought to translate this biochemical assay into a cellular format by first coexpressing Ser/Thr-kinases with reengineered cellular STK-peptide substrates –subsequently referred to as cSTK-pep– and then quantifying intracellular peptide phosphorylation using a simple homogeneous addition-only protocol.…”

Section: Resultsmentioning

confidence: 99%

Cellular Ser/Thr-Kinase Assays Using Generic Peptide Substrates

Adams¹,

Wang²,

Mak³

et al. 2008

Curr Chem Genomics

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High-throughput cellular profiling has successfully stimulated early drug discovery pipelines by facilitating targeted as well as opportunistic lead finding, hit annotation and SAR analysis. While automation-friendly universal assay formats exist to address most established drug target classes like GPCRs, NHRs, ion channels or Tyr-kinases, no such cellular assay technology is currently enabling an equally broad and rapid interrogation of the Ser/Thr-kinase space. Here we present the foundation of an emerging cellular Ser/Thr-kinase platform that involves a) coexpression of targeted kinases with promiscuous peptide substrates and b) quantification of intracellular substrate phosphorylation by homogeneous TR-FRET. Proof-of-concept data is provided for cellular AKT, B-RAF and CamK2δ assays. Importantly, comparable activity profiles were found for well characterized B-Raf inhibitors in TR-FRET assays relying on either promiscuous peptide substrates or a MEK1(WT) protein substrate respectively. Moreover, IC50-values correlated strongly between cellular TR-FRET assays and a gold standard Ba/F3 proliferation assay for B-Raf activity. Finally, we expanded our initial assay panel by screening a kinase-focused cDNA library and identified starting points for >20 cellular Ser/Thr-kinase assays.

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“…Homogeneous time-resolved fluorescence (HTRF) assays were conducted to evaluate the inhibition of JAKs by different compounds [12]. The assays were performed with the HTRF KinEASE kit (Cisbio Bioassays, Codolet, France), according to the manufacturer’s instructions.…”

Section: Methodsmentioning

confidence: 99%

Discovery and evaluation of ZT55, a novel highly-selective tyrosine kinase inhibitor of JAK2V617F against myeloproliferative neoplasms

Chen

Yang

et al. 2019

J Exp Clin Cancer Res

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BackgroundThe JAK2-STAT signaling pathway plays a critical role in myeloproliferative neoplasms (MPN). An activating mutation in JAK2 (V617F) is present in ~ 95% of polycythemia vera, essential thrombocythemia, and primary myelofibrosis cases. This study aims to explore the selective JAK2V617F inhibitor, evaluate the efficacy and possible mechanism of ZT55 on MPN.MethodsHTRF assays were conducted to evaluate the selective inhibition of ZT55 for JAKs. Cell apoptosis, proliferation, and cycle arrest assays were performed to examine the effect of ZT55 on HEL cell line with JAK2V617F mutation in vitro. Western analysis was used to monitor the expression and activity of proteins on JAK2/STAT pathway. A mice xenograft model was established to evaluate the antitumor efficacy of ZT55 in vivo. Peripheral blood samples from patients with the JAK2V617F mutation were collected to estimate the effect of ZT55 on erythroid colony formation by colony-forming assay.ResultsWe found that ZT55 showed a selective inhibition of a 0.031 μM IC50 value against JAK2. It exhibited potent effects on the cellular JAK-STAT pathway, inhibiting tyrosine phosphorylation in JAK2V617F and downstream STAT3/5 transcription factors. ZT55 inhibited the proliferation of the JAK2V617F-expressing HEL cell line, leading to cell cycle arrest at the G2/M phase and induction of caspase-dependent apoptosis. Notably, ZT55 also significantly suppressed the growth of HEL xenograft tumors in vivo. Further evaluation indicated that ZT55 blocked erythroid colony formation of peripheral blood hematopoietic progenitors from patients carrying the JAK2V617F mutation.ConclusionThese results suggest that ZT55 is a highly-selective JAK2 inhibitor that can induce apoptosis of human erythroleukemia cells by inhibiting the JAK2-STAT signaling.Electronic supplementary materialThe online version of this article (10.1186/s13046-019-1062-x) contains supplementary material, which is available to authorized users.

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Development of a HTRF® Kinase Assay for Determination of Syk Activity

Cited by 14 publications

References 17 publications

Comparison on Functional Assays for Gq-Coupled GPCRs by Measuring Inositol Monophospate-1 and Intracellular Calcium in 1536-Well Plate Format

Comparison on Functional Assays for Gq-Coupled GPCRs by Measuring Inositol Monophospate-1 and Intracellular Calcium in 1536-Well Plate Format

Cellular Ser/Thr-Kinase Assays Using Generic Peptide Substrates

Discovery and evaluation of ZT55, a novel highly-selective tyrosine kinase inhibitor of JAK2V617F against myeloproliferative neoplasms

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