Glycogen Synthase Kinase 3-β (GSK-3β) and Casein Kinase 1-δ (CK-1δ) are both highly conserved serine/threonine protein kinases that seem to be involved in neuroinflammation and in the development and progression of several disorders of the central nervous system (CNS), such as Alzheimer’s disease (AD) and Parkinson’s disease (PD). In particular, both are related to the hyperphosphorylation of tau-protein, one of the major hallmark of AD and other taupathies. So, the inhibition of GSK-3β and or CK-1δ could represent a strategy for the treatment of these unmet diseases. The main purpose of this thesis is the synthesis of new chemical entities as GSK-3β and/or CK-1δ inhibitors as potential therapeutic agents for neurodegenerative disorders. Since all kinases present an ATP-binding site, we can deduce that adenine-like nuclei could be suitable scaffolds to design novel ATP-competitive inhibitors. In particular, we focused our attention on fused 5 and 6 membered rings: [1,2,4]triazolo[1,5-a]triazines (TT) and [1,2,4]triazolo[1,5-c]pyrimidines (TP), two classes of heterocyclic derivatives well known in literature. Both TT and TP scaffolds were obtained following the synthetic pathways reported in literature. Computational studies helped us to decide the pattern of substitutions to introduce on the above-mentioned nuclei: in particular, we modified positions 2, 5 and 7 of the TT nucleus and 2, 5, 7 and 8 of the TP one. After chemical characterization through 1H-NMR, 13C-NMR and ESI-MS, all the synthetized derivatives were assayed against GSK-3β and/or CK-1δ. On the most promising compounds, kinetic experiments, to investigate the inhibitory mechanism towards the kinases, and PAMPA assay for predicting the brain permeability were performed. Different substituents were inserted in both TT and TP nuclei and good inhibitors of GSK-3β and/or CK-1δ were obtained with IC50 values in the submicromolar range. Moreover, the insertion of a Michael acceptor moiety (eg. cyanacrylamide) in position 2 of the TT scaffold led to a potent GSK-3β irreversible inhibitor (IC50=0.17 μM) that inhibited also CK-1δ in an ATP competitive manner with an IC50 value of 0.68 μM, and for these reasons it could be considered a good dual inhibitor of GSK-3β and CK-1δ. Its reactivity in front of thiols was evaluated through UV-spectrometry and HPLC-MS methodology and its binding mode to GSK-3β has been investigated, both theoretically and experimentally. In fact, kinetic assays showed a mixed ATP-competitive/non-ATP-competitive behavior of the TT derivative; on the other hand, time-dependent experiments confirmed that a covalent interaction with GSK-3β took place. Interestingly, despite the dual inhibitor has not shown an optimal BBB-permeability, it resulted able to increase the survival of cells treated with 6-OHDA or MPTP, two models of Parkinson’s disease (PD), and also to enhance β-catenin expression.

DESIGN, SYNTHESIS AND CHARACTERIZATION OF GSK-3β AND CK-1δ INHIBITORS

REDENTI, SARA
2017-05-04

Abstract

Glycogen Synthase Kinase 3-β (GSK-3β) and Casein Kinase 1-δ (CK-1δ) are both highly conserved serine/threonine protein kinases that seem to be involved in neuroinflammation and in the development and progression of several disorders of the central nervous system (CNS), such as Alzheimer’s disease (AD) and Parkinson’s disease (PD). In particular, both are related to the hyperphosphorylation of tau-protein, one of the major hallmark of AD and other taupathies. So, the inhibition of GSK-3β and or CK-1δ could represent a strategy for the treatment of these unmet diseases. The main purpose of this thesis is the synthesis of new chemical entities as GSK-3β and/or CK-1δ inhibitors as potential therapeutic agents for neurodegenerative disorders. Since all kinases present an ATP-binding site, we can deduce that adenine-like nuclei could be suitable scaffolds to design novel ATP-competitive inhibitors. In particular, we focused our attention on fused 5 and 6 membered rings: [1,2,4]triazolo[1,5-a]triazines (TT) and [1,2,4]triazolo[1,5-c]pyrimidines (TP), two classes of heterocyclic derivatives well known in literature. Both TT and TP scaffolds were obtained following the synthetic pathways reported in literature. Computational studies helped us to decide the pattern of substitutions to introduce on the above-mentioned nuclei: in particular, we modified positions 2, 5 and 7 of the TT nucleus and 2, 5, 7 and 8 of the TP one. After chemical characterization through 1H-NMR, 13C-NMR and ESI-MS, all the synthetized derivatives were assayed against GSK-3β and/or CK-1δ. On the most promising compounds, kinetic experiments, to investigate the inhibitory mechanism towards the kinases, and PAMPA assay for predicting the brain permeability were performed. Different substituents were inserted in both TT and TP nuclei and good inhibitors of GSK-3β and/or CK-1δ were obtained with IC50 values in the submicromolar range. Moreover, the insertion of a Michael acceptor moiety (eg. cyanacrylamide) in position 2 of the TT scaffold led to a potent GSK-3β irreversible inhibitor (IC50=0.17 μM) that inhibited also CK-1δ in an ATP competitive manner with an IC50 value of 0.68 μM, and for these reasons it could be considered a good dual inhibitor of GSK-3β and CK-1δ. Its reactivity in front of thiols was evaluated through UV-spectrometry and HPLC-MS methodology and its binding mode to GSK-3β has been investigated, both theoretically and experimentally. In fact, kinetic assays showed a mixed ATP-competitive/non-ATP-competitive behavior of the TT derivative; on the other hand, time-dependent experiments confirmed that a covalent interaction with GSK-3β took place. Interestingly, despite the dual inhibitor has not shown an optimal BBB-permeability, it resulted able to increase the survival of cells treated with 6-OHDA or MPTP, two models of Parkinson’s disease (PD), and also to enhance β-catenin expression.
SPALLUTO, GIAMPIERO
29
2015/2016
Settore CHIM/06 - Chimica Organica
Università degli Studi di Trieste
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11368/2908172
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