Substrate specificity and inhibitor profile of human recombinant p56lck from a baculovirus expression vector

Flotow, Horst; Purton, Tracey; Whitaker, D P; Williams, D. H.; Wilkinson, S. E.

doi:10.1007/bf02252937

Cited by 5 publications

(3 citation statements)

References 13 publications

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“…T cell PKC was partially purified from peripheral blood T cells by ion-exchange chromatography by the method outlined in ref 23. Bovine heart PKA (23), rabbit skeletal muscle phosphorylase kinase (24), and human recombinant p56 lck (25) and ZAP-70 (5) assays were performed as previously described.…”

Section: Methodsmentioning

confidence: 99%

Ro 09-2210 Exhibits Potent Anti-proliferative Effects on Activated T Cells by Selectively Blocking MKK Activity

Purton

et al. 1998

Biochemistry

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By using high throughput screening of microbial broths, we have identified a compound, designated Ro 09-2210, which is able to block anti-CD3 induced peripheral blood T cell activation with an IC50 = 40 nM. Ro 09-2210 was also able to block antigen-induced IL-2 secretion with an IC50 = 30 nM, but was considerably less potent at blocking Ca2+ flux stimulated by anti-CD3 treatment. To determine the mechanism of action of Ro 09-2210, we set up a transient expression system in Jurkat T cells using a variety of reporter gene constructs and showed effective inhibition of phorbol ester/ionomycin-induced NF-AT activation and anti-CD3 induced NF-AT with IC50 = 7.7 and 10 nM, respectively. Ro 09-2210 was also able to inhibit phorbol ester/ionomycin-induced activation of AP1 with IC50 = <10 nM. We further showed that Ro 09-2210 was unable to inhibit c-jun induced expression of AP1-dependent reporter constructs (IC50 > 500 nM), but was able to potently inhibit ras-induced AP1 activation (IC50 = 20 nM). This suggested that Ro 09-2210 was inhibiting an activator of AP-1 which was upstream of c-jun and downstream of ras signaling. To investigate further, we then purified a number of different kinases, including PKC, PhK, ZAP-70, ERK, and MEK 1 (a MKK), and showed that Ro 09-2210 was a selective inhibitor of MEK1 in vitro (IC50 = 59 nM).

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Section: Methodsmentioning

confidence: 99%

Ro 09-2210 Exhibits Potent Anti-proliferative Effects on Activated T Cells by Selectively Blocking MKK Activity

Purton

et al. 1998

Biochemistry

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“…The first in vitro high-throughput screening study to identify enzyme inhibitors was reported in 1994, using a scintillation proximity assay . Although there are earlier reports of high-throughput screening approaches to identify enzyme inhibitors, these all used cell systems expressing the target enzyme. − This first report of in vitro high-throughput screening was followed by reports of a kinase inhibitor screening campaign (using a radioactivity-based assay), and a protease inhibitor screening campaign (using a fluorescence-based assay). Since then the literature has exploded, reaching a peak of 260 publications describing HTS campaigns against specific enzymes (Figure ).…”

Section: Introductionmentioning

confidence: 99%

High-Throughput Screening for the Discovery of Enzyme Inhibitors

Lloyd

2020

J. Med. Chem.

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Enzymes are common targets in high-throughput screening and related campaigns. An analysis of papers published between 1990 and 2018 showed that kinases were the most common enzymes investigated, fluorescence-based assays were the most common readout method, and cancer and bacterial infections were the most common therapeutic areas. High-throughput screening and fragment-screening campaigns published between 2017 and 2019 were analyzed in more depth, giving 75 examples of hit to lead development. Kinases, phosphatases, proteases, and peptidases were the most common targets, fluorescent assays were the most commonly used, and a wide variety of structural features were observed within the derived drugs. Hit frequency was largely independent of library size and positively correlated with Z′ value for the assay. Binding of metal ions to library compounds and substrates is an underappreciated source of false-positive results and unreproducible behavior.

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“…Whether derived from native recognition sequences or synthetically optimized, peptide substrates are used to screen for small‐molecule inhibitors against kinases that are associated with disease (Table I). 17, 28–39 Abltide (EAIYAAPFAKKK)17 is one example of an optimized substrate that was selected by screening a degenerate peptide library against purified kinases in vitro . Abltide demonstrated a higher rate of reactivity with Abl kinase than with closely related kinases such as Src17 and is now routinely used to monitor Abl kinase activity in vitro .…”

Section: Introductionmentioning

confidence: 99%

Peptide reporters of kinase activity in whole cell lysates

Sylvester

Parker

et al. 2010

Biopolymers

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Kinase assays are used to screen for small-molecule inhibitors that may show promise as targeted pharmaceutical therapies. Using cell lysates instead of purified kinases provides a more accurate estimate of inhibitor sensitivity and selectivity in a biological setting. This review summarizes the range of homogeneous (solution-phase) and heterogeneous (solid-supported) formats available for using peptide substrates to monitor kinase activities in cell lysates. With a focus on heterogeneous kinase assays, the peptide substrate Abltide is used as a model to optimize presentation geometries and the modular arrangement of short sequences for kinase recognition. We present results from peptides immobilized on two- and three-dimensional surfaces such as hydrogels on 96-well plates and glass slides, and fluorescent Luminex beads. We discuss methods to increase assay sensitivity using chemifluorescent ELISAs, antibody-based recognition, and label-free mass spectrometry. Monitoring the activity of specific kinases in cell lysates presents challenges that can be overcome by manipulating peptide substrates to optimize assay conditions. In particular, signal-to-background ratios were improved by 1) adding long branched hydrophilic linkers between the substrate and the surface, 2) changing the orientation of peptides relative to the surface, and 3) including peptide ligands in cis or in trans to recruit kinases to the surface. By improving the accessibility of immobilized peptide substrates to kinases in solution, the apparent rate of phosphorylation increased and assays were more sensitive to changes in endogenous kinase activities. These strategies can be generalized to improve the reactivity of most peptide substrates used in heterogeneous kinase assays with cell lysates.

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Substrate specificity and inhibitor profile of human recombinant p56lck from a baculovirus expression vector

Cited by 5 publications

References 13 publications

Ro 09-2210 Exhibits Potent Anti-proliferative Effects on Activated T Cells by Selectively Blocking MKK Activity

Ro 09-2210 Exhibits Potent Anti-proliferative Effects on Activated T Cells by Selectively Blocking MKK Activity

High-Throughput Screening for the Discovery of Enzyme Inhibitors

Peptide reporters of kinase activity in whole cell lysates

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