Mécanismes d'action des inhibiteurs de kinases cyclines-dépendantes : identification de leurs cibles intracellulaires par chromatographie d'affinité

Abstract : Among the estimated 850 human protein kinases, the family of cyclin-dependent kinases (CDK) has been extensively studied because of its essential cellular functions. CDKs play a key role in ceil cycle control, transcription, differentiation, apoptosis and in neuronal cell functions. The observation that CDKs are frequently deregulated in cancers has stimulated an active search for chemical inhibitors of this class of kinases. 60 far, more than 50 compounds have been identified, on the basis of their ability to inhibit in vitro purified CDK activity. These compounds display antiproliferative, anti-neurodegenerative, anti-viral and anti-parasitic properties. The anticancer potential of the most promising compounds is currently evaluated in pre-clinical and clinical trials. The aim of this study is to understand the intracellular mechanism of action of CDK inhibitors. We chose to approach this question by investigating their in vivo selectivity, i.e. the scope of their real intracellular targets. lndeed, CDK inhibitors have been identified on their ability to inhibit purified CDKs in vitro but their actual intracellular targets remain unverified. To address this question, we developed an affinity chromatography approach allowing to purify, from cellular extracts, the intracellular targets of the inhibitor. This technique was used to study the selectivity of three CDK inhibitors belonging to different chemical classes: the purine purvalanol, paullones and indirubin-3’-monoxime. For each compound, the expected targets were recovered from the matrix. However this approach has also allowed the identification of several unexpected interactions between CDK inhibitors and other enzymes, kinases (p42/p44 MAPKs with purvalanol) or other (malate dehydrogenase with paullones and glyceraldehyde-3-phosphate dehydrogenase with indirubin). In each case, the molecular basis of the interaction between the inhibitor and its new target was investigated by testing the in vitro sensitivity 0f the target enzyme to the compound. Based on these results, we next investigated more in detail how the interaction between MAPKs and purvalanol participated to the cellular effects of the drug. Lastly, this affinity chromatography approach was used to understand two particular cellular effects of purvalanol: its antiviral activity against herpes simplex virus (HSV-1) and the induction of supernumerary hair cells in Organs of Corti of rat embryos. In conclusion, this affinity approach has confirmed the strong interactions between CDK inhibitors and the enzymatic targets they have been optimised on. In addition, this technique has allowed the identification of several unexpected targets for CDK inhibitors, the identity of which could not be deduced from in vitro classical selectivity studies, and the inhibition of which may participate to the observed cellular effects of the compounds.
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Marie Knockaert. Mécanismes d'action des inhibiteurs de kinases cyclines-dépendantes : identification de leurs cibles intracellulaires par chromatographie d'affinité. Biochimie, Biologie Moléculaire. Université de Bretagne Occidentale, 2002. Français. ⟨tel-01116136⟩



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