Reversible and covalent protein kinase CK1δ inhibitors: potential neuroprotective agents in neurodegenerative diseases

Besides its well-known role in controlling the circadian rhythm and cancer, protein kinase CK1δ is involved in the onset of neurodegenerative disorders such as Alzheimer’s disease (AD), Parkinson’s disease (PD) and amyotrophic lateral sclerosis (ALS). This pleiotropic role of CK1δ makes it an attractive target for innovative therapeutic approaches. The aim of this PhD project is therefore to develop both CK1δ reversible and covalent inhibitors. The catalytic domain (aa. 1-296) of CK1δ was expressed and purified to obtain a pure, properly folded and enzymatically active recombinant protein for in vitro biochemical studies, to deeply characterize the kinase binding site and the interactions of our inhibitors, as well as to obtain co-crystal structures. As regards the development of ATP-competitive inhibitors, the [1,2,4]triazolo[1,5-a][1,3,5]triazine (TT) nucleus was investigated since it has already successfully demonstrated to mimic the ATP’s adenine ring in interacting with the kinase’s catalytic domain. Starting from a previous series of TT kinase inhibitors having diamine substituents at the 5 position, new triazolo-triazine 5,7-diamines were synthesized. In particular, a new sub-set of molecules in which the terminal amino group was substituted with different aromatic systems or less bulky groups to study the role of the steric hindrance in this position as any additional interactions with the catalytic pocket was designed. After chemical characterization, all the compounds were evaluated towards CK1δ through a luminescence based assay and the blood-brain barrier (BBB) permeability of the most active compounds was predicted using the PAMPA-BBB assay. Also, their cytotoxicity was investigated with the MTT assay. In parallel, covalent inhibitors of CK1δ were designed and synthesized by inserting different electrophilic moieties at the 2-position of the phenyl group of known CK1δ inhibitor scaffold. The catalytic lysine (Lys-38), was identified as the nucleophile target for the covalent bond formation. The synthesized compounds were tested on CK1δ and IC50 data revealed that the aldehyde moiety is the most suitable electrophile (IC50 = 241 nM). Several experiments were conducted to demonstrate the covalent inhibitory mechanism. Results of the assays performed to demonstrate the dependence of the inhibitory activity on time and ATP concentration suggest a mixed ATP-competitive/un-competitive behavior. TSA experiments confirmed the stabilizing effect of the ligand towards CK1δ. Furthermore, the covalent adduct formation was investigated through intact mass spectrometry experiments and first co-crystallization attempts were carried out. 1H-NMR studies allowed to assess the reactivity of the electrophilic warhead towards a lysine’s amino group surrogate. Notably, despite the presence of a polar aldehyde substituent, the covalent compound demonstrated to be able to passively permeate the BBB and the MTT assay resulted in a good cell viability showing also a potential neuroprotective action of the covalent derivative.

Besides its well-known role in controlling the circadian rhythm and cancer, protein kinase CK1δ is involved in the onset of neurodegenerative disorders such as Alzheimer’s disease (AD), Parkinson’s disease (PD) and amyotrophic lateral sclerosis (ALS). This pleiotropic role of CK1δ makes it an attractive target for innovative therapeutic approaches. The aim of this PhD project is therefore to develop both CK1δ reversible and covalent inhibitors. The catalytic domain (aa. 1-296) of CK1δ was expressed and purified to obtain a pure, properly folded and enzymatically active recombinant protein for in vitro biochemical studies, to deeply characterize the kinase binding site and the interactions of our inhibitors, as well as to obtain co-crystal structures. As regards the development of ATP-competitive inhibitors, the [1,2,4]triazolo[1,5-a][1,3,5]triazine (TT) nucleus was investigated since it has already successfully demonstrated to mimic the ATP’s adenine ring in interacting with the kinase’s catalytic domain. Starting from a previous series of TT kinase inhibitors having diamine substituents at the 5 position, new triazolo-triazine 5,7-diamines were synthesized. In particular, a new sub-set of molecules in which the terminal amino group was substituted with different aromatic systems or less bulky groups to study the role of the steric hindrance in this position as any additional interactions with the catalytic pocket was designed. After chemical characterization, all the compounds were evaluated towards CK1δ through a luminescence based assay and the blood-brain barrier (BBB) permeability of the most active compounds was predicted using the PAMPA-BBB assay. Also, their cytotoxicity was investigated with the MTT assay. In parallel, covalent inhibitors of CK1δ were designed and synthesized by inserting different electrophilic moieties at the 2-position of the phenyl group of known CK1δ inhibitor scaffold. The catalytic lysine (Lys-38), was identified as the nucleophile target for the covalent bond formation. The synthesized compounds were tested on CK1δ and IC50 data revealed that the aldehyde moiety is the most suitable electrophile (IC50 = 241 nM). Several experiments were conducted to demonstrate the covalent inhibitory mechanism. Results of the assays performed to demonstrate the dependence of the inhibitory activity on time and ATP concentration suggest a mixed ATP-competitive/un-competitive behavior. TSA experiments confirmed the stabilizing effect of the ligand towards CK1δ. Furthermore, the covalent adduct formation was investigated through intact mass spectrometry experiments and first co-crystallization attempts were carried out. 1H-NMR studies allowed to assess the reactivity of the electrophilic warhead towards a lysine’s amino group surrogate. Notably, despite the presence of a polar aldehyde substituent, the covalent compound demonstrated to be able to passively permeate the BBB and the MTT assay resulted in a good cell viability showing also a potential neuroprotective action of the covalent derivative.