A Comparison of Protein Kinases Inhibitor Screening Methods Using Both Enzymatic Activity and Binding Affinity Determination

Rudolf, Amalie Frederikke; Skovgaard, Tine; Knapp, Stefan; Jensen, Lars Juhl; Berthelsen, Jens

doi:10.1371/journal.pone.0098800

Cited by 75 publications

(88 citation statements)

References 26 publications

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“…New chemical approaches and molecular insights into the development of highly selective kinase inhibitors that target the conserved ATP-binding site are urgently needed. Binding assays are the most accurate and robust method to measure potency and selectivity of ATP-competitive kinase inhibitors [27] . Binding assays available at commercial vendors are routinely used to profile kinase inhibitors for their selectivity across the human kinome [28] .…”

Section: Discussionmentioning

confidence: 99%

Identification and Optimization of 4‐Anilinoquinolines as Inhibitors of Cyclin G Associated Kinase

et al. 2017

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4-Anilinoquinolines were identified as potent and narrow-spectrum inhibitors of the cyclin G associated kinase (GAK), an important regulator of viral and bacterial entry into host cells. Optimization of the 4-anilino group and the 6,7-quinoline substituents produced GAK inhibitors with nanomolar activity, over 50 000-fold selectivity relative to other members of the numb-associated kinase (NAK) subfamily, and a compound (6,7-dimethoxy-N-(3,4,5-trimethoxyphenyl)quinolin-4-amine; 49) with a narrow-spectrum kinome profile. These compounds may be useful tools to explore the therapeutic potential of GAK in prevention of a broad range of infectious and systemic diseases.

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Section: Discussionmentioning

confidence: 99%

Identification and Optimization of 4‐Anilinoquinolines as Inhibitors of Cyclin G Associated Kinase

et al. 2017

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“…Determination of the secretion level of such signaling molecules can thus be used to characterize the actual functional cell state in a comprehensive way and, most importantly, to screen for the effects of drugs applied to restore the normal state of the cells [5]. Such an experimental approach is thus quite different when compared to the evaluation of more drastic endpoints like cell death [6] or more selective effects such as inhibitor screening [7] or to computational methods [8]. Secretome analyses may greatly improve our understanding for drug actions and thus support the development of targeted drug applications.…”

Section: Introductionmentioning

confidence: 99%

Quantification of cytokines secreted by primary human cells using multiple reaction monitoring: evaluation of analytical parameters

et al. 2015

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Determination of secreted proteins provides highly valuable information about cell functions. While the typical methods for the determination of biologically relevant but low abundant molecular species still rely on the use of specific antibodies, mass spectrometry-based methods are now gaining sufficient sensitivity to cope with such challenges as well. In the current study, we have identified several cytokines and chemokines which were induced in primary human umbilical vein endothelial cells upon inflammatory activation. Based on the high-resolution mass spectrometry data obtained with a Q Exactive orbitrap, we built an MRM method to quantify the most relevant molecules selected from the screening experiment. All experimental data are available via ProteomeXchange, PXD002211/12, and Panorama ( www.panoramaweb.org ). Using nano-flow Chip-HPLC coupled to a 6490 triple-quadrupole MS for MRM analyses, we achieved calibration curves covering a linear range of four orders of magnitude and detection limits in the low attomol per microliter concentration range. Carryover was consistently less than 0.005 %, the accuracy was between 80 and 120 %, and the median coefficient of variation for LC/MS was only 2.2 %. When including the variance of quantification introduced by cell culture and digestion, the coefficient of variation was less than 20 % for most peptides. With appropriate marker molecules, we monitored typical variations introduced by cell culture caused by differences in cell numbers, proliferative states, and cell death. As a result, here, we present a robust and efficient MRM-based assay for the accurate and sensitive determination of cytokines and chemokines representative for functional cell states and including comprehensive quality controls. Graphical Abstract Work flow diagram: Data processing steps beginning with orbitrap-based shotgun data acquisition and MaxQuant data analysis, followed by peptide and transition selection for MRM analysis using Skyline and experimental validation using triple quadrupole MS.

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“…Another drawback of the previously described screens is that they were based on biochemical or computationally predicted FTO binding and not FTO enzymatic activity. As a result, only a fraction of FTO-binding compounds also show enzymatic inhibition (Rudolf et al, 2014). Thus, it is desirable to develop a screen that directly assays FTO enzymatic activity.…”

Section: Resultsmentioning

confidence: 99%

Fluorescent RNA Aptamers as a Tool to Study RNA-Modifying Enzymes

Svensen

Jaffrey

2016

Cell Chemical Biology

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SUMMARY RNA-modifying enzymes are difficult to assay due to the absence of fluorometric substrates. Here we show that the Broccoli, a previously reported fluorescent RNA-dye complex, can be modified to contain N6-methyladenosine, a prevalent mRNA base modification. Methylated Broccoli is nonfluorescent, but upon demethylation by the RNA demethylases Fat mass and obesity-associated protein (FTO) or ALKBH5 binds and activates the fluorescence of its cognate fluorophore. We describe a high-throughput screen for FTO inhibitors using the fluorogenic methylated Broccoli substrate HTS assay performs robustly with a Z’-factor > 0.8 in the LOPAC1280 library. This allowed the identification of novel high-affinity FTO inhibitors. Several of these compounds were selective for FTO over the related demethylase, ALKBH5, and increase methylation of endogenous FTO target mRNAs in cells. Lastly, we show that Broccoli can be modified to contain other base modifications, suggesting that this approach could be generally applicable for assaying diverse RNA-modifying enzymes.

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A Comparison of Protein Kinases Inhibitor Screening Methods Using Both Enzymatic Activity and Binding Affinity Determination

Cited by 75 publications

References 26 publications

Identification and Optimization of 4‐Anilinoquinolines as Inhibitors of Cyclin G Associated Kinase

Identification and Optimization of 4‐Anilinoquinolines as Inhibitors of Cyclin G Associated Kinase

Quantification of cytokines secreted by primary human cells using multiple reaction monitoring: evaluation of analytical parameters

Fluorescent RNA Aptamers as a Tool to Study RNA-Modifying Enzymes

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