title: Intracellular Targets of Paullones. Identification following affinity purification on immobilized inhibitor.
abstract: Numerous inhibitors of cyclin-dependent kinases and glycogen synthase kinase-3 (GSK-3) are being developed in view of their potential applications against cancers and neurodegenerative disorders. Among these, paullones constitute a family of potent and apparently selective cyclin-dependent kinase and GSK-3 inhibitors. However, their actual intracellular targets remain to be identified. To address this issue we have immobilized a paullone, gwennpaullone, on an agarose matrix. Extracts from various cell types and tissues were screened for proteins interacting with this matrix. This approach validated GSK-3alpha and GSK-3beta as major intracellular paullone targets and also mitochondrial, but not cytoplasmic, malate dehydrogenase (MDH). Mitochondrial MDH was indeed inhibited by micromolar concentrations of paullones. Mitochondrial MDH was the major paullone-binding protein in the parasitic protozoon Leishmania mexicana, and paullones inhibited growth of the parasite. This simple batchwise affinity chromatography approach constitutes a straightforward method for the identification of intracellular targets of this particular class of novel anti-mitotic compounds. It has revealed an unexpected target, mitochondrial MDH, the inhibition of which may participate in the pharmacological effects of paullones.
authors.affiliation: Station Biologique de Roscoff, CNRS, BP 74, Bretagne, France. knockaer@sb-roscoff.fr
authors.forename: Marie
authors.initials: M
authors.lastname: Knockaert
authors.forename: Karen
authors.initials: K
authors.lastname: Wieking
authors.forename: Sophie
authors.initials: S
authors.lastname: Schmitt
authors.forename: Maryse
authors.initials: M
authors.lastname: Leost
authors.forename: Karen M
authors.initials: KM
authors.lastname: Grant
authors.forename: Jeremy C
authors.initials: JC
authors.lastname: Mottram
authors.forename: Conrad
authors.initials: C
authors.lastname: Kunick
authors.forename: Laurent
authors.initials: L
authors.lastname: Meijer
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001381:Azepines / Q000302:isolation & purification / Q000494:pharmacology*; D001552:Benzazepines; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000037:antagonists & inhibitors*; D002846:Chromatography, Affinity; D004791:Enzyme Inhibitors / Q000302:isolation & purification / Q000494:pharmacology*; D038362:Glycogen Synthase Kinase 3; D038341:Glycogen Synthase Kinases; D007211:Indoles / Q000302:isolation & purification / Q000494:pharmacology*; D007894:Leishmania mexicana / Q000201:enzymology; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid
chemical_list: D001381:Azepines; D001552:Benzazepines; D004791:Enzyme Inhibitors; D007211:Indoles; C462503:gwennpaullone; D038341:Glycogen Synthase Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D038362:Glycogen Synthase Kinase 3