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New in-capillary electrophoretic kinase assays to evaluate inhibitors of the PI3k/Akt/mTOR signaling pathway

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Abstract

Human kinases are one of the most promising targets for cancer therapy. Methods able to measure the effects of drugs on these cell agents remain crucial for biologists and medicinal chemists. The current work therefore sought to develop an in-capillary enzymatic assay based on capillary electrophoresis (CE) to evaluate the inhibition of phosphatidylinositol-3-kinase (PI3K), protein kinase B (Akt), and the mammalian target of rapamycin (mTOR). These kinases belong to the same signaling pathway PI3K/Akt/mTOR. For this proposal, the capillary was used as a nanoreactor in which a few nanoliters of the kinase, its substrate, adenosine triphosphate (ATP), and the potent inhibitor were separately injected. A transverse diffusion of laminar flow profiles (TDLFP) approach was employed to mix the reactants. Adenosine diphosphate (ADP ) was detected online at 254 nm. The CE assay was first developed on the α isoform of PI3K. It was compared to five commercial kits frequently used to assess kinase inhibition, based on time-resolved fluorescence resonance energy transfer (TR-FRET) and bioluminescence. Each assay was evaluated in terms of sensitivity (S/B), reproducibility (Z′), and variability (r 2). This CE method was easily extended to assay the inhibition of the β, γ, and δ isoforms of PI3K, and of the other kinases of the pathway, Akt1 and mTOR, since it is based on in-capillary mixing by TDLFP and on ADP quantification by simple UV absorption. This work shows for the first time the evaluation of inhibitors of the kinases of the PI3K/Akt/mTOR pathway using a common in-capillary CE assay. Several inhibitors with a wide range of affinity toward these enzymes were tested.

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Acknowledgments

This work was supported by “La Ligue Nationale contre le Cancer, Comité du Loiret”, the “Conseil Régional du Centre”, and the “Cancéropôle Grand-Ouest”. Thibault Saurat was the recipient of an INCa/Région Centre PhD fellowship. We are very grateful to Pr. Philippe Bougnoux for his constant support and encouragement.

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Authors and Affiliations

  1. Institut de Chimie Organique et Analytique (ICOA), CNRS FR 2708, UMR 7311, Université d’Orléans, Orléans, France

    Reine Nehmé, Hala Nehmé, Thibault Saurat, Frédéric Buron, Pierre Lafite, Philippe Morin & Sylvain Routier

  2. Centre de Biophysique Moléculaire, CNRS UPR 4301, Université d’Orléans et INSERM, 45071, Orléans, France

    Thibault Saurat, Marie-Ludivine de-Tauzia & Hélène Bénédetti

  3. Centre Jean Perrin, Laboratoire de Radio-Oncologie Expérimentale, 58 Rue Montalembert, 63011, Clermont-Ferrand, France

    Pierre Verrelle & Emmanuel Chautard

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  1. Reine Nehmé
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  2. Hala Nehmé
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  3. Thibault Saurat
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Correspondence to Reine Nehmé.

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Nehmé, R., Nehmé, H., Saurat, T. et al. New in-capillary electrophoretic kinase assays to evaluate inhibitors of the PI3k/Akt/mTOR signaling pathway. Anal Bioanal Chem 406, 3743–3754 (2014). https://doi.org/10.1007/s00216-014-7790-z

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  • DOI: https://doi.org/10.1007/s00216-014-7790-z

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