Intracellular targets of paullones : identification by affinity chromatography using immobilized inhibitor.

Knockaert, Marie and Wieking, Karen and Schmitt, Sophie and Leost, Maryse and Grant, Karen M. and Mottram, Jeremy C. and Kunick, Conrad and Meijer, Laurent (2002) Intracellular targets of paullones : identification by affinity chromatography using immobilized inhibitor. Journal of Biological Chemistry, 277 (28). pp. 25493-25501. ISSN 1083-351X

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-3 and GSK-3 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.