Molecular Docking of Fisetin as a Multi-target drug in the treatment of Alzheimer’s disease

 

Malathi R, Vailina Dsouza, Puja, Rithika R, Sneha P

Department of Life Sciences, Kristu Jayanti College, Autonomous K. Narayanapura,

Kothanur, Bengaluru, Karnataka 560077.

 

ABSTRACT:

Alzheimer’s disease is a slow deadly form of dementia occurring in almost 70% of the older generation. Throughout the world, there are approximately 47 million people affected. Countries that are mostly affected by Alzheimer’s disease with the highest are Turkey and Lebanon by 57% and 56% respectively. The lowest rates include India, Cambodia, Georgia, and Singapore. This includes symptoms such as disorientation, mood swings, behavioral issues, etc. ultimately leading to death. The primitive appearance of the alpha-beta plaques and neurofibrillary tangles in the different regions of the brain leads to the cause of AD progression. In this present study, Fisetin which is a plant flavonoid having neurotropic and neuroprotective properties is docked with the drug targets of Alzheimer’s disease. The study was focused on analysing the molecular interaction of Fisetin with potential drug targets of Alzheimer’s disease. The docking was performed using AutoDock 4.2. The minimum binding energy studies explain the efficiency of the ligand binding with the therapeutic target proteins. Proteins play a significant role in Alzheimer’s disease as it is responsible for various functions which also are the major attributes of Alzheimer's disease namely amyloid-β production, tau phosphorylation, synaptic function, neurogenesis, and memory which all are influenced by dysregulation of this enzyme. Four proteins were selected based on the action and function they play in the progression of Alzheimer’s disease namely FYN tyrosine kinase, Beta Secretase (BACE 1), Gamma secretase, and Glycogen synthase kinase 3-beta (GSK3-β). The minimum binding energy scores for the following 3D molecular structures were FYN tyrosine kinase = -8.2 kcal/mol, BACE1= -10.67kcal/mol, Gamma secretase = - 10.03kcal/mol, GSK3 Beta = -10.47kcal/mol; No. of hydrogen bonds: 12, 10, 10 and 21 respectively. From the 4 potential Alzheimer’s drug targets, Glycogen synthase kinase 3-beta (GSK3-β) and Beta Secretase, had the best interaction with Fisetin with the lowest binding energy. Along with this Fisetin was analyzed for its molecular properties, drug-likeness, biological activity, and toxicity using the Way2drug bio tool.

 

 

 

INTRODUCTION: 

Alzheimer’s disease (AD) is one of the more frequent causes of dementia in the world, and normalcy continues to rise in the aging global community. The disease was named after its discoverer Dr. Alois Alzheimer in the year 1906 when he observed changes in the cerebral tissue of a woman by the name of Auguste Deter who passed away from an unusual mental illness.

 

This process of neurodegenerative disease stands out with two major pathologies, one of which is β-amyloid plaque deposition and the other is hyperphosphorylated tau neurofibrillary entanglement. An estimated 60 % to 70% of the world’s 50 million people with dementia suffer from Alzheimer’s disease^1–4. Thediagnosis is based on a clinical submission that meets several criteria, including fluid and imaging biomarkers.

 

Treatment is currently targeted towards symptomatic therapy, aiming to reduce the production of pathology within the brain involves the targeting of neurofibrillary entanglements and sensitive plaques (Aβ). In recent years, a great deal of attention has been given to plant-based bioactive compounds for the treatment of neurodegenerative diseases such as Alzheimer's disease. Among them, the flavonoid chemical class is known to be most bio-active. Fisetin assists with learning and memory reduce neuronal cell mortality and reduce oxidative stress^5,6.

 

β secretase which is also known as β-site amyloid precursor protein cleaving enzyme 1 (BACE1) plays a significant role in the early-stage development of Alzheimer’s disease by initiating the production of the toxic amyloid β (Aβ). An amyloid-beta peptide which is also known as Aβ is the product of the last cleavage step of APP (Amyloid Precursor Protein)⁷. βsecretase is the first enzyme to cleave and form a membrane-bound C-terminal fragment called the C99 which is the fragment of APP. In Alzheimer’s BACE1 is a main curative target to reduce the cerebral Aβ concentrations. BACE1 inhibitors are currently used to prevent AD as they have the potential to lower cerebral Aβ concentrations. 

 

A second enzyme called γ secretase also participates in the cleavage C99 then to release Aβ. This is a multi-protein complex located inside the membrane, having an important role in neuronal function⁸.

 

Fyn tyrosine kinase is one more promising target for AD which gets activated due to the binding of amyloid-beta to prion protein which is a high-affinity receptor for Aβ this activation, in turn, causes synaptotoxicity and tau pathology^9–11.

 

Fyn also plays an important role involved in causing abnormal hyperphosphorylation of Tau¹². It has been found that there are deposits of neurofibrillary tangles in the AD patients' brain along with Aβ plaques, which are composed of this hyperphosphorylated Tau¹³. Saracatini an inhibitor has been checked for its safety, tolerability, and cerebrospinal fluid penetration in AD patients for targeting fyn kinase¹⁴.

 

Glycogen synthase kinase 3-beta (GSK3-β) enzyme also plays a crucial role in AD.

 

Activation of GSK3-β directly causes tau hyper-phosphorylation which results in neurodegeneration and accumulation of amyloid which leads to neuronal death in AD.  The generation of neurons in the hippocampus region is also one of the functions of GSK3-β which aids learning and memory. Synaptic plasticity is also found to be supported by GSK3-β. The hippocampal LTP (long-term potentiation) is found to be inhibited when glycogen synthase kinase 3- beta gets overactivated.

 

The involvement of GSK3-β signaling in the development of AD, its inhibition, and therapeutic potential is highly investigated in clinical studies¹⁵.

The objective of this study was to analyze the inhibitory potential of fisetin based on its minimum binding energy with the enzymes beta-secretase (BACE 1),Gama secretase, Glycogen synthase 3 beta (GSK3 beta), and Fyntyrosine kinase through molecular dockingstudies.

 

MATERIALS AND METHODS:

Ligand Generation:

The 2D structure of FISTEIN (a potential drug to target Alzheimer’s), was procured from the ZINC database (https://zinc.docking.org/substances/ZINC000000039111/), 3 ringed structured containing 6 Hetero Atoms, Molecular formula C15h10O6. The ligand was downloaded and saved in the spatial data file (SDF) format. This was then converted to a PDB file format using the Open Babel toolbox.

 

Assortment and constructionof therapeutic target proteins:

The 3D crystal structure of four proteins was eradicated from (ww.rscb.org) in a Protein Data bank format (PDB), these include the following set of therapeutic target proteins– (PDB Id: 1G83) FYN, (PDB Id: 5MXD) BACE–1, (PDB Id:1GNG) GSK3 BETA, PDB Id:5FN5- Gamma secretase. These have an atom count of each 2642, 9015, 6227, and 9645 and along with their molecular weight 37.82 kDa,145.17kda, 95.85kda and 172.25kda subsequently.

 

Molecular docking studies and visualization:

Docking studies on the above-mentioned therapeutic target proteins were performed using AutoDock 4.2 10, a free and easily accessible molecular docking tool. It is especially effective for protein-ligand docking.

 

The docking calculations were performed by using two algorithms namely Genetic and Lamarckian. These calculations predicted the binding affinity allying fisetin and the therapeutic target proteins. AutoGrid an auxiliary program precalculated grid maps, one for each atom type present in the ligand being docked. After the therapeutic target proteins were ready ligand was docked by surface docking it onto the protein a process that is used to dock the ligand into a binding site in a protein-based on the complementarity of the intermolecular atomic contacts which is based on atomic contact surface area and the chemical properties of the contacting atoms. The obtained algorithm was then made to run on Cygwin and converted to PDB format. The ligand and protein interaction was then visualized through UCSF CHIMERA. Drug-Likeness and molecular property of fisetin was predicted using molsoft. The biological activity and toxicity of the ligand fisetin were determined using Way2Drug.

 

 

RESULTS:

The continuous and rapid progress of computing techniques in the field of science has resulted in a variety of discoveries. It is completely based on predicting the bond affinities of the target molecule with crystallographic X-ray structures along with possible drugs used for analysis via docking.

 

Surface Grid-based docking method was used to scrutinize different binding of the drug to the amino acids in the active site of the therapeutic target proteins, we have subjugated docking analysis of Fisetin to the active site of the therapeutic target proteins (PDB Id: 1G83) FYN, (PDB Id: 5MXD) BACE – 1,(PDB Id:1GNG) GSK3 BETA, PDB Id:5FN5- Gamma secretase.

 

Results of the following docking containingthe five best minimum binding energy of the ligand (fisetin) with the 4 therapeutic target proteins arewere described in Tables 1 and 2 respectively.

 

Table 1: Minimum Binding Energy of fisetin with the 4 drug targets of the top hits

1G83 - FYN SH3-SH2			1GNG - GSK3 BETA
S. No	Run	Minimum Binding Energy (kcal/mol)	S.no	Run	Minimum binding energy (kcal/mol)
1	2	-8.2	1	9	-10.47
2	5	-7.99	2	7	-10.47
3	8	-7.77	3	10	-10.47
4	7	-7.77	4	3	-10.46
5	3	-7.77	5	4	-10.44
5MXD - BACE-1			5FN5 – gamma-secretase
S.NO	Run	minimum binding  energy (kcal/mol)	S.no	Run	Minimum binding energy (kcal/mol)
1	9	-10.67	1	7	-10.03
2	4	-9.2	2	4	-8.61
3	1	-9.19	3	8	-8.58
4	10	-9.13	4	2	-8.58
5	5	-8.93	5	10	-8.58

 

The following figures show the lowest binding energy conformation of the ligand Fisetin with the four therapeutic target proteins (PDB Id: 1G83) FYN, (PDB Id: 5MXD) BACE – 1, (PDB Id:1GNG) GSK3 BETA, PDB Id:5FN5- Gamma secretase. Visualization of these structures was done using the chimera 11- program used for interactive visualization along with the analysis of the various molecular structures and their related data. Glycogen synthase kinase 3-beta (GSK3-β) and Beta Secretase, had the best interaction with Fisetin with the lowest binding energy with No. of hydrogen bonds: 12, 10, 10, and 21 respectively.

 

 

Fig 1: 5FN5 Gamma Secretase

 

The red color signifies the oxygen atom in the ligand fisetin. The blue and orange color lines signify the interaction between the ligand and the protein molecule denoting the hydrogen bonds. No. of hydrogen bond = 10.

 

 

Fig 2: G83 FYN tyrosine kinase.

 

The red color signifies the oxygen atom in the ligand fisetin. The blue and orange color lines signify the interaction between the ligand and the protein molecule denoting the hydrogen bonds. No of hydrogen bond = 12.

 

 

Table 2: Lowest binding energy of fisetin with the 4 drug targets of the top hits

S. No.	Protein	Final Intermolecular Energy (vdW + Hbond + desolv Energy+Electrostatic Energy)  (kcal/mol)	Final Total Internal Energy  (kcal/mol)	Torsional Free Energy  (kcal/mol)	Unbound System's Energy  (kcal/mol)
1	BACE - 1	-11.07	-0.41	0.3	-0.51
2	GSK3 BETA	-10.64	-0.53	0.3	-0.4
3	FYN	-10.3	-0.54	0.3	-0.52
4	GAMMA SECRETASE	-8.48	-0.054	0.3	-0.52

 

 

Fig 3: 5MXD Beta secretase .

 

The red color signifies the oxygen atom in the ligand fisetin. The blue and orange color lines signify the interaction between the ligand and the protein molecule denoting the hydrogen bonds. No. of hydrogen bond =10.

 

 

Fig 4: 1GNG Glycogen synthase kinase 3 beta

 

The red color signifies the oxygen atom in the ligand fisetin. The blue and orange color lines signify the interaction between the ligand and the protein molecule denoting the hydrogen bonds. No. of hydrogen bond = 21.

 

Molecular Properties of fisetin was analyzed using molsoft:

 

Fig 5: Molecular properties anddrug-likeness

Fig 5 shows that the selected compound has very good drug likeliness which was analyzed using Molsoft software.

 

Analysis of the score for Fisetin:

Log P: 1.35

HBD: 4

HBA: 6

 

The following analysis meets the criteria of Lipinski's benchmark rule of five (RO5)5 which defines desirable drug candidate physicochemical property space as MW < 500 Da, log P < 5, HBD < 5, and HBA <10.

 

The Biological Activity and Toxicity of the ligand fisetin were analysed by the Way2Drug Bio tool.

Table 3: Biological Activity

SI. No	Pa	Biological activity
1	0.978	Chlordeconereductase inhibitor
2	0.892	Beta carotene 15,15’ monooxygenase inhibitor
3	0.833	JAK2 expression inhibitor
4	0.817	Iodide peroxidase inhibitor
5	0.768	CYP3 A inducer
6	0.753	Free radical scavenger
7	0.727	Antineoplastic

 

Table 4: Toxicity

SI. No	Pa	Toxicity
1	0.823	shivering
2	0.716	withdrawal

 

Way 2 drug Bio-tool was used to find the biological activity, and toxicity of Fisetin, where pa [pharmacologically active] correlates to the probability of the activity. Therefore If pa >0.7 – the molecule indicates to be pharmacologically active, hence from Tables 3 and 4 we can conclude that Fisetin is having properties that can be effective against Alzheimer’s disease.

 

CONCLUSION:

In the present study, the efficacy of fisetin interaction with the target proteins of Alzheimer’s disease was evaluated. This paves the effective treatment of Alzheimer’s disease.

 

Fisetin showed significant binding docking studies to all the 4 therapeutic target proteins (PDB Id: 1G83) FYN, (PDB Id: 5MXD) BACE–1, (PDB Id:1I09) GSK3 BETA, PDB Id:5FN5- Gamma secretase. No. of hydrogen bonds: 12, 10, 21, and 10 respectively.

 

The following results from this study would be helpful and applicable in further understanding the neuroprotective mechanism effect of fisetin.

 

Amyloid-Beta Clusters (Plaques) and twisted tangles of Tau protein that lead to neuronal death are the main elemental causes of Alzheimer’s disease, as distinguished by Alois Alzheimer in 1906. The inhibition of the proteins responsible for the formation of causative factors and other known targets can help control Alzheimer’s disease.

 

Since fisetin can interact with the multi-target therapeutic proteins which are involved in the development of Alzheimer’s disease, fisetin can be one of the effective drugs candidates to manage Alzheimer’s disease.

 

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

 

ACKNOWLEDGMENTS:

The authors would like to thank the support rendered by Kristu Jayanti College, Autonomous.

 

REFERENCES:

1.      Schachter AS, Davis KL. Alzheimer’s disease. Dialogues Clin Neurosci. 2000; 2(2): 91. doi:10.31887/DCNS.2000.2.2/ASSCHACHTER

2.      Alzheimer’s disease facts and figures. Alzheimer’s Dement. 2017; 13(4): 325-373. doi:10.1016/J.JALZ.2017.02.001

3.      Bondi MW, Edmonds EC, Salmon DP. Alzheimer’s Disease: Past, Present, and Future. J Int Neuropsychol Soc. 2017; 23(9-10): 818-831. doi:10.1017/S135561771700100X

4.      Weller J, Budson A. Current understanding of Alzheimer’s disease diagnosis and treatment. F1000 Research. 2018;7. doi:10.12688/F1000RESEARCH.14506.1

5.      Crous-Bou M, Minguillón C, Gramunt N, Molinuevo JL. Alzheimer’s disease prevention: from risk factors to early intervention. Alzheimer's Res Ther. 2017; 9(1). doi:10.1186/S13195-017-0297-Z

6.      Cummings JL, Goldman DP, Simmons-Stern NR, Ponton E. The costs of developing treatments for Alzheimer’s disease: A retrospective exploration. Alzheimer’s Dement. Published online 2021. doi:10.1002/ALZ.12450

7.      Yan R, Vassar R. Targeting the β secretase BACE1 for Alzheimer’s disease therapy. Lancet Neurol. 2014; 13(3): 319. doi:10.1016/S1474-4422(13)70276-X

8.      C. GL, M. P, A. L. gamma-Secretase as a therapeutic target in Alzheimer’s disease. Curr Drug Targets. 2010; 11(4): 506-517. doi:10.2174/138945010790980349

9.      Chin J, Palop JJ, Puoliväli J, et al. Fyn Kinase Induces Synaptic and Cognitive Impairments in a Transgenic Mouse Model of Alzheimer’s Disease. J Neurosci. 2005; 25(42): 9694. doi:10.1523/JNEUROSCI.2980-05.2005

10.   Nygaard HB. Targeting Fyn Kinase in Alzheimer’s Disease. Biol Psychiatry. 2018; 83(4): 369-376. doi:10.1016/J.BIOPSYCH.2017.06.004

11.   Targeting Fyn Kinase in Alzheimer’s Disease: Another Failed Clinical Trial,  2019-08-14 , Relias Media - Continuing Medical Education Publishing. Accessed March 22, 2022. https://www.reliasmedia.com/articles/144947-targeting-fyn-kinase-in-alzheimers-disease-another-failed-clinical-trial

12.   Wegmann S, Biernat J, Mandelkow E. A current view on Tau protein phosphorylation in Alzheimer’s disease. Curr Opin Neurobiol. 2021; 69:131-138. doi:10.1016/J.CONB.2021.03.003

13.   Lee G, Thangavel R, Sharma VM, et al. Phosphorylation of tau by fyn: implications for Alzheimer’s disease. J Neurosci. 2004; 24(9): 2304-2312. doi:10.1523/JNEUROSCI.4162-03.2004

14.   Nygaard HB, Van Dyck CH, Strittmatter SM. Fyn kinase inhibition as a novel therapy for Alzheimer’s disease. Alzheimers Res Ther. 2014; 6(1): 8. doi:10.1186/ALZRT238

15.   Lauretti E, Dincer O, Praticò D. Glycogen synthase kinase-3 signaling in Alzheimer’s disease. Biochim Biophys acta Mol cell Res. 2020; 1867(5). doi:10.1016/J.BBAMCR.2020.118664

 

 

 

 

Accepted on 10.08.2023           © RJPT All right reserved

Research J. Pharm. and Tech 2023; 16(12):5813-5817.