Characterization of neurochemical activities through inhibition of CDK5 or AChE by dimers of tacrine and neostigmine respectively

Abstract

Background: Alzheimer's disease (AD), as the most serious degenerative disease in both clinical and social aspects of view point, has currently no approved disease-modifying strategies but only limited symptom-releasing treatment options. To design and develop novel disease-modifying drugs to cure AD, dimerization drug development strategy has been shown as a powerful drug design strategy, which demonstrates its multiple advantages in increasing drug potency, decreasing drug's side effects, and making non-effective pharmacophore become effective. Methodology: For the studies on tacrine-based homo-dimers' novel anti-AD targets mainly focused on bis(3)-tacrine (B3C) and monomeric tacrine, known as cyclin-dependent kinase 5 (CDK5) and its involved cell signaling pathways, a series of in vitro biochemical methods have been employed in the experimental set-ups for such investigation. The drugs' direct enzymatic inhibitory effects were measured by experiment kit designed base on enzyme-linked immunosorbent assay with chemo-luminescence. The protein expression level changes in the drug-treated rat cerebellar granule neuronal cells (CGN) were detected by the cellular compartment purification and following west blotting with corresponding specific antibodies, as well as the immune-histochemistical staining technology. In silico methods were also applied to primarily assess and molecularly explain the enzymatic CDK5 inhibitory effects of proposed chemicals. Various in vitro biochemical methods have been employed in the experimental set-ups for the investigation of the pharmacological characters of neostigmine-demecarium monomer-dimer drug chemical pair targeting enzyme acetylcholinesterase (AChE) and enzyme butylcholinesterase (BChE). Based on Ellman's method, different experimental modifications were made to assess the IC50 value, inhibition manners, inhibition kinetics, and AChE / BChE selective factors of neostigmine and demecarium. MTT assay was also involved in the drug safety assessments of mentioned chemicals, which were carried on L02 cell lines, PC12 cell lines and primarily cultured rat CGN cells, while aspartate transaminase / glutamic-oxaloacetic transaminase (AST / GOT) enzyme content activity assay was specially performed on L02 liver cell line. In silico computer-aid protein-drug interaction docking methods were also applied to describe the molecular-level interaction modes of such chemicals to enzyme AChE and BChE. Results: It was clearly demonstrated that B3C at concentration of 10 μM could almost fully reverse the inhibition of myocyte enhancer factor 2 (MEF2D) caused by glutamate-induced cytotoxicity in primarily cultured rat CGN cells in a dose-dependent manner. It was also showed that protein kinase A (PKA) inhibitor H89 was not able to abolish the neuroprotective effects of B3C, suggesting that the MEF2D stimulation effects carried by B3C was not associated with the PKA pathways. Exposure of primary cultured rat CGN cells to glutamate increased the conversion of protein regulating fragment p35 to p25, applied as a bio-marker for CDK5's abnormal hyper-activation, while B3C demonstrated the ability to dose-dependently reverse the decreasing of p35 levels as well as the increasing of p25 fragment level caused by such glutamate-induced neuronal cytotoxicity. B3C inhibited MEF2D's hyper-phosphorylation at the CDK5's modification recognition site of Ser-444. Biochemical and enzymatic assays showed that B3C could dose-dependently inhibit the enzymatic function of CDK5 directly. In silico results also suggested that within the tacrine-based dimeric drug series, dimerization process made the non-effective tacrine pharmacophore become effective for the direct enzymatic inhibitory effect to CDK5s. Comprehensive consideration of results clearly suggested that B3C represented its neuroprotective effects against the glutamate-induced cytotoxicity in primarily cultured rat CGN cells via the MEF2D cell signaling transduction pathway through the direct enzymatic inhibitory effect of CDK5 and its corresponding up-regulation at MEF2D's specific CDK5 recognition site Ser-444. The dimeric demecarium was determined as an AChE / BChE selective, competitive, reversible and non-time-dependent AChE inhibitory chemical with the IC50 value of 2.6 nM and Ki value of 1.2 nM, while its monomeric form neostigmine was indicated as a non-AChE / BChE selective, competitive, reversible and non-time-dependent AChE inhibitory chemical with the IC50 value of 106 nM and Ki value of 21 nM. Both neostigmine and demecarium had no cellular-level structural nor functional toxicities to L02 human liver cell and PC12 rat sub-cultured cell line at any high concentrations the experimental set-ups could reach. While, demecarium had the neuronal cytotoxicity to primarily cultured rat CGN cells at absolute high concentration over 100 μM, where is significantly far from its possible therapeutic concentration which is predicted based on its IC50 value and Ki value to AChE. In silico protein-drug docking results, together with inhibition manner studies, suggested that both monomeric neostigmine and dimeric demecarium shared the similar interaction mode to AChE at the similar amino acid residue sites. Conclusion: Dimerization showed its power to transfer non-effective tacrine pharmacophore into effective B3C and B7C dimers for new drug development targeting CDK5 and its involved cell signaling pathways including MEF2D for the treatment of AD and other types of central nervous system degenerative disease. Tacrine-based homo-dimer drug series including B3C had the potentials to be developed as the leading compounds for further novel CDK5 inhibitory anti-AD drug developments. In addition, dimerization made demecarium become a more potent AChE inhibitor with the similar side effects and the interaction mode compared to its monomeric neostigmine. Based on series of parameters collected from this project and other researcher's achievements, dimerization drug design strategy could be regarded as a powerful evidence-based novel drug development tool in different pharmacological research fields with multiple advantages.