XXIV EFMC International Symposium on Medicinal Chemistry Effects of novel acyl hydrazones derived from 4-quinolone on the acetylcholinesterase activity and Aβ42 peptide fibrils formation.
Gisele Silvestre IQ-Unicamp Manchester, UK - September 1, 2016
Alzheimer’s Disease (AD) AD is a neurodegenerative, progressive and irreversible process. It presents many cognitive and behavioral symptoms.
Loss of memory and control Changes in behaviour
Learning and thinking is affected
Lose all ability of acting for himself
Figure 1. Symptoms of Alzheimer's disease Currently, there are around 46 million people suffering from dementia. it will be a global epidemic in 2050. Alzheimer’s Association, Alzheimer’s Disease Facts and Figures, Alzheimer’s & Dementia, 2016;12(4), 8
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Pathological hallmarks of AD Amyloid plaques
Cholinergic deficit
ACh Amiloid β-peptide Aβ40/42 A
B
Figure 2. AD Markers A) amyloid plaques and Neurofibrillary tangles; B) Reduction in levels of acetylcholine (ACh).
Amyloid hypothesis: The overproduction of Amyloid β-peptide is a critical starter that trigger the progression of AD via accumulation and aggregation. http://www.alz.org/braintour
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Objectives Evaluate the effect of acyl hydrazones derived of quinolones against acetylcholinesterase (AChE) and beta-Amyloid (Aβ) peptide 1. Preparation of a series of acyl hydrazones 2. Evaluate the effect on the AChE activity and Aβ42 peptide fibrils formation AChE Aß-42
Acyl hydrazones Figure 3. Biological targets focused on this study IC50 = 70µM (AChE) Aβ peptide anti-fibrillization Huber, P.C. et al., 2015. Journal of Chemical and Pharmaceutical Research, 7(8), 993–1000.
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Materials and methods 1. Chemistry: Preparation of acyl hydrazones
Hydrazone
2. Molecular docking analysis
• Docking simulation in AutoDock Vina - using as a ligand donepezil • AChE enzyme 1EVE obtained from Protein Data Bank . 3. Biology AChE activity
Aβ42 fibrillization
• Ellman’s colorimetric Essay • AChE of Electrophorus electricus • Control : Tacrine
• Thioflavin-T fluorescence emission
Huber, P.C. et al., 2013. Polyhedron, 57, pp.14–19. Ellman, G.L. et al., 1961. Biochemical Pharmacology, 7, 88–95. O. Trott et al., 2010. Journal of Computational Chemistry, 31, 455-461.
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Chemical Results Preparation of the acyl hydrazones
R = -C6H5, 4-Cl-C6H4 , 3-Cl-C6H4 , 2,3-Cl-C6H4 , 4-NO2-C6H4 , 2-NO2-C6H4 4-SO2CH3- C6H4 , 4-(2’-thiophenyl),4-OMe-C6H4
Reagents and conditions: a) diethyl(ethoxymethylene)malonate , Δ, 1h; then, diphenyl ether addition, 24 h;
b) NH2NH2.2H2O, MeOH; c) aromatic aldehyde, ethanol, piperidine; Δ, 12–18 h; 63–90% yields.
Figure 4. Preparation of the acyl hydrazones • 10 acyl hydrazone (4-13) were prepared successfully with 60 – 90 % yields • All compounds were characterized by a combination of NMR (1H, 13C and 15N NMR) and infrared spectroscopy da Silva, G.S. et al., 2016. Journal of Enzyme Inhibition and Medicinal Chemistry, 6366, 1–7.
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Biological test results Biological Essay
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Aß-42
1 AChE
• •
Thioflavin T-Based Fluorometric essay
In Vitro Inhibition of AChE : Ellman’s method
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In Vitro Inhibition of AChE Table 1. In vitro IC50 values of acyl hydrazones Compound
R
IC50 (μM) ± SD 4.1 ± 0.1
4
Phenyl
5
4-chlorophenyl
13.6 ± 0.2
6
3-chlorophenyl
1.3 (*)
7
2,3-chlorophenyl
17.4 ± 0.1
8
4-nitrophenyl
10.2 ± 0.1
9
2-nitrophenyl
1.2 (*)
10
4-(methylsulfonyl)phenyl
11
4-methylthiophenyl
12
4-(2-thiophenyl)
3.4 (*)
13
4-methoxyphenyl
6.1 ± 0.1
nd 8.0 ± 0.1
Experiments were perfomed in triplicate. (*) SD < 0.02; nd: not determined
• The compounds 6 and 9 exhibit the best inhibitory effect on AChE • IC50 : Quinolone -70 μM > Acyl hydrazone 9 - 1.2 μΜ • We were able to improve the ability of inhibition of AChE in 98 %
Molecular docking Drawn of acyl hydrazones: Two conformers and two isomers for each acyl hydrazone
• So, four structures of acyl hydrazones were drawn: Anti-E, Anti-Z, Syn-E, Syn-Z. • Energy of affinity – Donepezil = 10.7 Kcal/mol • All acyl hydrazones interact with Trp84 - Indepedent of the conformation and/or stereochemistry
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Molecular docking Study on the Interaction between 6 -Anti-E and AChE Affinity Energy = -10.8 Kcal/mol Catalytic site – Trp84
Figure 5. Pose of compound 6 interacting with residues Tyr121 and Trp84 in the catalytic site of AChE • • • •
Hydrogen bonding between NH quinolone and the indole side chain (2.7-3.3 Å) Hydrogen bonding between CN imine of 6 and OH –Tyrosine Tyr121 – belong to the peripheral anionic site of acetylcholinesterase Competitive inhibition by Kinetic Studies: Bind to the same place of Acetylcholine10
Thioflavin-T Fluorometric Assay Tht-T+ Fibrils
Thioflavin-T (Tht-T)
+
=
Control: Aß-42 + DMSO + HCl; Sample: Aß-42 + DMSO + HCl+ Acyl hydrazone (0,2mM) Two Measurements of the sample
T0 T24 T0 = Immediately after mix of the sample T24 = After incubation at 37 °C by 24 hours 11
Thioflavin-T Fluorometric Assay 30000 25000 20000 15000 10000 5000
0 Control
4
5
6 t=0
7
8
9 t = 24 h
10
11
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Figure 6. ThT fluorescence emission in the presence of acyl hydrazones 9 - 18 in Aβ fibrils formation conditions (Control: Aß-42 + DMSO + HCl; Sample: Aß-42 + DMSO + HCl+ Acyl hydrazone)
• All acyl hydrazones present ability of inhibit fibrils formation • The emission of fluorescence in acylhydrazones decreased at 49% - 67%. • Maximum inhibitory effect was promoted by acylhydrazone 9. 12
Concluding Remarks • We were able to improve the inhibitory activity of quinolone on AChE • 9 acyl hydrazones presented inhibitory activity on AChE and the formation of Aβ42 fibrils. • Molecular docking studies suggested that all acyl hydrazones interact with catalytic site. • Additionally, kinetic studies were performed in our research group and the majority of acyl hydrazones are competitive inhibitor. • Next step: IC50 determination for the Aβ-fibrils formation • The results indicated that some acyl hydrazones offer potential prototype applications as inhibitors of β-Amyloid fibrillization and AChE. 13
Acknowledgements
Email:
[email protected] Science blog: Quimikinha https://www.blogs.unicamp.br/pt_BR/blog/category/quimikinha/
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