Measuring Kinase Activity—A Global Challenge

Cann, Marissa L.; McDonald, Ian M.; East, Michael P.; Johnson, Gary L.; Graves, Lee M.

doi:10.1002/jcb.26103

Cited by 12 publications

(13 citation statements)

References 98 publications

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“…MIB-MS is a powerful method to evaluate kinase inhibitor specificity by competition binding assays. 27 Composed of type I kinase inhibitors, kinases bind to MIBs through interactions with the ATP pocket. Incubating cells (or cell lysates) with a small-molecule kinase inhibitor can allow us to identify direct kinase targets by occupation of the ATP binding site and prevention of subsequent MIB binding.…”

Section: Kinase Inhibitor Specificity Profiling By Mib-msmentioning

confidence: 99%

Application of Integrated Drug Screening/Kinome Analysis to Identify Inhibitors of Gemcitabine-Resistant Pancreatic Cancer Cell Growth

et al. 2018

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Continuous exposure of a pancreatic cancer cell line MIA PaCa-2 (Mia) to gemcitabine resulted in the formation of a gemcitabine-resistant subline (Mia). In an effort to discover kinase inhibitors that inhibited Mia growth, Mia cells were exposed to kinase inhibitors (PKIS-1 library) in a 384-well screening format. Three compounds (UNC10112721A, UNC10112652A, and UNC10112793A) were identified that inhibited the growth of Mia cells by more than 50% (at 50 nM). Two compounds (UNC10112721A and UNC10112652A) were classified as cyclin-dependent kinase (CDK) inhibitors, whereas UNC10112793A was reported to be a PLK inhibitor. Dose-response experiments supported the efficacy of these compounds to inhibit growth and increase apoptosis in 2D cultures of these cells. However, only UNC10112721A significantly inhibited the growth of 3D spheroids composed of Mia cells and GFP-tagged cancer-associated fibroblasts. Multiplexed inhibitor bead (MIB)-mass spectrometry (MS) kinome competition experiments identified CDK9, CLK1-4, DYRK1A, and CSNK1 as major kinase targets for UNC10112721A in Mia cells. Another CDK9 inhibitor (CDK-IN-2) replicated the growth inhibitory effects of UNC10112721A, whereas inhibitors against the CLK, DYRK, or CSNK1 kinases had no effect. In summary, these studies describe a coordinated approach to discover novel kinase inhibitors, evaluate their efficacy in 3D models, and define their specificity against the kinome.

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Section: Kinase Inhibitor Specificity Profiling By Mib-msmentioning

confidence: 99%

Application of Integrated Drug Screening/Kinome Analysis to Identify Inhibitors of Gemcitabine-Resistant Pancreatic Cancer Cell Growth

et al. 2018

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“…While next-generation sequencing technology already allows rapid genomic characterization of patient tumor samples and enables identification of cancer driver mutations, methods for fast prediction of drug efficacies for each unique genetic background are still lacking. The majority of currently applied technologies for determination of kinase activities rely either on purified kinases, commonly limited to the kinase domain, or cell lysates [25][26][27][28][29]. Disruption of the cellular environment, however, might have unforeseeable effects on kinase functions, as the concerted interplay between activating and deactivating inputs, provided by the cellular environment, represents the fundamental mode of kinase activity regulation.…”

Section: Personalized Medicinementioning

confidence: 99%

Tracking mutation and drug-driven alterations of oncokinase conformations

Feichtner

Kugler

Schwaighofer

et al. 2022

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SummaryNumerous kinases act as central nodes of cellular signaling networks. As such, many of these enzymes function as molecular switches for coordinating spatiotemporal signal transmission. Typically, it is the compartmentalized phosphorylation of protein substrates which relays the transient input signal to determine decisive physiological cell responses. Genomic alterations affect kinase abundance and/or their activities which contribute to the malignant transformation, progression, and metastasis of human cancers. Thus, major drug discovery efforts have been made to identify lead molecules targeting clinically relevant oncokinases. The concept of personalized medicine aims to apply the therapeutic agent with the highest efficacy towards a patient-specific mutation. Here, we discuss the implementation of a cell-based reporter system which may foster the decision-making process to identify the most promising lead-molecules. We present a modular kinase conformation (KinCon) biosensor platform for live-cell analyses of kinase activity states. This biosensor facilitates the recording of kinase activity conformations of the wild-type and the respective mutated kinase upon lead molecule exposure. We reflect proof-of-principle studies demonstrating how this technology has been extended to profile drug properties of the full-length kinases BRAF and MEK1 in intact cells. Further, we pinpoint how this technology may open new avenues for systematic and patient-tailored drug discovery efforts. Overall, this precision-medicine-oriented biosensor concept aims to determine kinase inhibitor specificity and anticipate their drug efficacies.

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“…Currently, a collection of cell‐free and cell‐based assays are available to identify the most promising kinase inhibitors . The underlying read‐outs primarily focus on the individual kinase domain or on the kinase‐mediated phospho‐transfer to the protein substrate . Noninvasive cell‐based reporter assays for systematically studying the regulation, mode of action, and inhibition of full‐length kinases and their carcinogenic mutations are needed .…”

Section: Targeting the Kinomementioning

confidence: 99%

KinCon: Cell‐based recording of full‐length kinase conformations

et al. 2020

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The spectrum of kinase alterations displays distinct functional characteristics and requires kinase mutation-oriented strategies for therapeutic interference.Besides phosphotransferase activity, protein abundance, and intermolecular interactions, particular patient-mutations promote pathological kinase conformations. Despite major advances in identifying lead molecules targeting clinically relevant oncokinase functions, still many kinases are neglected and not part of drug discovery efforts. One explanation is attributed to challenges in

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Measuring Kinase Activity—A Global Challenge

Cited by 12 publications

References 98 publications

Application of Integrated Drug Screening/Kinome Analysis to Identify Inhibitors of Gemcitabine-Resistant Pancreatic Cancer Cell Growth

Application of Integrated Drug Screening/Kinome Analysis to Identify Inhibitors of Gemcitabine-Resistant Pancreatic Cancer Cell Growth

Tracking mutation and drug-driven alterations of oncokinase conformations

KinCon: Cell‐based recording of full‐length kinase conformations

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