Generation and Characterization of ATP Analog-specific Protein Kinase Cδ

Kumar, Varun; Weng, Yi Chinn; Geldenhuys, Werner J.; Wang, Dan; Han, Xiqian; Messing, Robert O.; Chou, Wen Hai

doi:10.1074/jbc.m114.598698

Cited by 10 publications

(10 citation statements)

References 53 publications

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“…By extending the box around the ATP binding pocket of DAPK1 kinase which had the ATP bound to the model, we were able to find a docking solution that fits the hypothesis that compound 1 is able to allow for the ATP/kinase transition activity to occur in a more favorable state as compared to the kinase alone. 18 As can be seen from this study, compound 1 is able to allow for an optimized positioning of the ATP for the gamma phosphate transfer to occur. This docking pose in part may explain the kinase activation by compound 1.…”

mentioning

confidence: 80%

“…Since compound 1 activates/stimulates DAPK1, we posited that ATP would be in the ATP binding pocket and allosterically modulate it. 17 Using MOE 2016 we docked ATP into the binding site of DAPK1 kinase similar to our previously published methods 18 . Using this model as our starting point, compound 1 was docked into the ATP binding pocket area with the OEDOCKING 3.2.0.3/FRED program as part of the OpenEye drug discovery suit (www.eyesopen.com).…”

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confidence: 99%

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High-content screen using zebrafish (Danio rerio) embryos identifies a novel kinase activator and inhibitor

Geldenhuys

Bergeron

Mullins

et al. 2017

Bioorganic & Medicinal Chemistry Letters

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In this report we utilized zebrafish (Danio rerio) embryos in a phenotypical high-content screen (HCS) to identify novel leads in a cancer drug discovery program. We initially validated our HCS model using the flavin adenosine dinucleotide (FAD) containing endoplasmic reticulum (ER) enzyme, endoplasmic reticulum oxidoreductase (ERO1) inhibitor EN460. EN460 showed a dose response effect on the embryos with a dose of 10 μM being significantly lethal during early embryonic development. The HCS campaign which employed a small library identified a promising lead compound, a naphthyl-benzoic acid derivative coined compound 1 which had significant dosage and temporally dependent effects on notochord and muscle development in zebrafish embryos. Screening a 369 kinase member panel we show that compound 1 is a PIM3 kinase inhibitor (IC50 = 4.078 μM) and surprisingly a DAPK1 kinase agonist/activator (EC50 = 39.525 μM). To our knowledge this is the first example of a small molecule activating DAPK1 kinase. We provide a putative model for increased phosphate transfer in the ATP binding domain when compound 1 is virtually docked with DAPK1. Our data indicate that observable phenotypical changes can be used in future zebrafish screens to identify compounds acting via similar molecular signaling pathways.

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confidence: 80%

mentioning

confidence: 99%

High-content screen using zebrafish (Danio rerio) embryos identifies a novel kinase activator and inhibitor

Geldenhuys

Bergeron

Mullins

et al. 2017

Bioorganic & Medicinal Chemistry Letters

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“…This latter method was employed in developing the analog‐sensitive protein kinase Cδ that can utilize N‐6‐benzyl ATP and is vulnerable to inhibition by PP1 analogs (Fig. D) .…”

Section: Direct Tagging Of Kinase Substratesmentioning

confidence: 99%

The significant others: Global search for direct kinase substrates using chemical approaches

Shah

Kim

2019

IUBMB Life

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Protein kinases function as key signaling hubs in the intricate network of biochemical signaling processes in the living cell. More than two‐thirds of the human proteome is estimated to be phosphorylated at ~960,000 phosphosites, which makes it challenging to identify the direct contribution of any desired kinase in generating this phosphoproteome. In this review, we discuss some of the methods that have been developed over the years for global identification of kinase substrates. The methods are essentially categorized into two classes, namely, (i) direct tagging of kinase substrates and (ii) indirect phosphoproteomics‐based approaches. We discuss the advantages and limitations entailed to each of the method introduced, with a special emphasis on the analog‐sensitive (as) kinase approach method. © 2019 IUBMB Life, 71(6):721–737, 2019

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“…The Shokat method uses site-directed mutagenesis to enlarge a kinase's ATP binding pocket. While not affecting kinase activity, the mutation specifically allows bulky synthetic ATP analogs, such as N 6 -Benzyl-ATP (A*TP), to fit into the enlarged ATP binding pocket (11)(12)(13)(14)(15)(16). Moreover, A*TP was further modified to create A*TP-γ-S by replacing an O with a S in the γ-phosphate group.…”

Section: Introductionmentioning

confidence: 99%

A Chemical-genetics and Nanoparticle Enabled Approach forin vivoProtein Kinase Analysis

Chen

Liu

Hilliard

et al. 2020

Preprint

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The human kinome contains >500 protein kinases, and regulates up to 30% of all human proteins. Kinase study is currently hindered by a lack of in vivo analysis approaches, mainly due to two factors: our inability to distinguish the kinase reaction of interest from those of other members of the kinome in live cells and the cell impermeability of the ATP analogs. Herein, we aimed to overcome this issue by combining the widely used chemical genetic method developed by Dr. Kevan Shokat and colleagues with nanoparticle-mediated intracellular delivery of the ATP analog. The critical AKT1 protein kinase, which has been successfully studied with the Shokat method, was used as our initial prototype. Briefly, following the Shokat method, enlargement of the ATP binding pocket was performed by mutating the gate-keeper Methionine residue to a Glycine, prompting the mutant AKT1 to preferentially use the bulky ATP analog N 6 -Benzyl-ATP-γ-S (A*TPγS) and, thus, differentiating AKT1-catalyzed and other phosphorylation events. The LPC nanoparticle was used for efficient intracellular delivery of A*TPγS, overcoming the cell impermeability issue. We demonstrated that the mutant, but not the wild type, AKT1 was able to use the delivered A*TPγS for autophosphorylation as well as phosphorylating its substrates in live cells. Thus, an in vivo protein kinase analysis method has been developed. The strategy should be widely applicable to other protein kinases.

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Generation and Characterization of ATP Analog-specific Protein Kinase Cδ

Cited by 10 publications

References 53 publications

High-content screen using zebrafish (Danio rerio) embryos identifies a novel kinase activator and inhibitor

High-content screen using zebrafish (Danio rerio) embryos identifies a novel kinase activator and inhibitor

The significant others: Global search for direct kinase substrates using chemical approaches

A Chemical-genetics and Nanoparticle Enabled Approach forin vivoProtein Kinase Analysis

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