title: New in-capillary electrophoretic kinase assays to evaluate inhibitors of the PI3k/Akt/mTOR signaling pathway.
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.
authors.affiliation: Institut de Chimie Organique et Analytique (ICOA), CNRS FR 2708, UMR 7311, Université d'Orléans, Orléans, France, reine.nehme@univ-orleans.fr.
authors.forename: Reine
authors.initials: R
authors.lastname: Nehmé
authors.forename: Hala
authors.initials: H
authors.lastname: Nehmé
authors.forename: Thibault
authors.initials: T
authors.lastname: Saurat
authors.forename: Marie-Ludivine
authors.initials: ML
authors.lastname: de-Tauzia
authors.forename: Frédéric
authors.initials: F
authors.lastname: Buron
authors.forename: Pierre
authors.initials: P
authors.lastname: Lafite
authors.forename: Pierre
authors.initials: P
authors.lastname: Verrelle
authors.forename: Emmanuel
authors.initials: E
authors.lastname: Chautard
authors.forename: Philippe
authors.initials: P
authors.lastname: Morin
authors.forename: Sylvain
authors.initials: S
authors.lastname: Routier
authors.forename: Hélène
authors.initials: H
authors.lastname: Bénédetti
mesh_terms: D000244:Adenosine Diphosphate / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry; D000730:Androstadienes / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D019075:Electrophoresis, Capillary / Q000379:methods*; D004791:Enzyme Inhibitors / Q000737:chemistry; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D020128:Inhibitory Concentration 50; D049449:Luminescence; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D047428:Protein Kinase Inhibitors / Q000737:chemistry; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D015398:Signal Transduction; D058570:TOR Serine-Threonine Kinases / Q000378:metabolism*; D000077191:Wortmannin
chemical_list: D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000730:Androstadienes; D004791:Enzyme Inhibitors; D019869:Phosphatidylinositol 3-Kinases; D047428:Protein Kinase Inhibitors; D051057:Proto-Oncogene Proteins c-akt; D058570:TOR Serine-Threonine Kinases; D000077191:Wortmannin; C546842:MTOR protein, human