Putative Molecular Mechanisms of Neuroprotective Cerebrosides and Their Docking Studies on Acetyl Cholinesterase Enzyme Inhibition for the Treatment of Alzheimer’s Disease

The Dementia disease is characterised by neuropsychiatric disturbances due to lack of proper synaptic communication between neurons causing the cognitive behavioural problems. The Alzheimer’s disease (AD) in elderly population is one of the several forms of Dementia. Recent data by World Health Organisation indicates that nearly 10 million people are getting dementia every year, of which 60-70% accounts for AD. The etiology of AD involves the formation of amyloid-β plaques and neurobrillary Tau tangles in the brain resulting in the death of neural cells. There is no permanent solution for AD treatment, except the FDA approved drugs like galantamine, donepezil, rivastigmine and memantine that are normally associated with side effects. At this juncture, cerebrosides, the natural secondary metabolites identied from different taxa with potential neuroprotective effects offer a promising scope for the treatment of AD. In this paper, cerebrosides reported from all taxa are pooled up along with their functions and listed. The review of literature revealed that Cerebrosides can increase the cognitive functions by regulating or interacting with the N-methyl-d-aspartate (NMDA) calcium ion (Ca 2+ ) channels at postsynaptic receptor; nitric oxide (NO); Bcl2, Bax, amyloid precursor (APP) and Tau proteins; brain-derived neurotrophic factor (BDNF) and cAMP- response element-binding proteins (CREB).This indicates that the Cerebrosides could be potential therapeutic agents for the protection of neurons involved in neurodegenerative disease like Alzheimer’s disease. The current neuroprotective drugs are AChE inhibitors; hence, in the present investigation, in silico molecular docking study on cerebrosides for the inhibition of AChE was assessed to nd out their capacity to interact with an active catalytic site of AChE. The results of present investigation revealed that all 22 cerebrosides selected for this work interacted with catalytic active site of AChE measured in terms of Gibbs free binding energy. Of all the cerebrosides assessed, compound 6 exhibited strong interaction, followed by 15. This is the rst report of molecular docking study on cerebrosides for AChE enzyme inhibition


Introduction
The effective neuronal communication in healthy neurons is made possible by chemical or neurotransmitter from presynaptic axon terminals to postsynaptic dendrites or sometimes neuronal bodies. Ineffective neuronal communication leads to neuronal dysfunction causing the death of neurons (Lovinger 2008) (Lepeta et al. 2016). The synaptic communication initiates calcium-dependent signaling events that regulate the expression of a group of genes in the CNS, like c-Fos and BDNF (Cohen and Greenberg 2008). These genes are important in neuronal development and plasticity, and various other aspects of neuronal function of cells to response to extracellular stimuli. . Every neuron has capillary structure, and the human brain capillary network is of 650 km in length. In Alzheimer's disease, the length of capillaries is reduced with the formation of amyloid plaque and neuro brillary tangles (Silva et al. 2019). In AD, neurotoxic Aβ 1-42 peptide residues trigger a series of pathological neurotoxic events of neurodegeneration and, the hyperphosphorylation of tau (p-Tau) protein leads to the formation of neuro brillary tangles causing neuronal death. Acetylcholine (ACh) is a neurotransmitter that sends signals to other cells. The choline acetyltransferase (ChAT) enzyme combines the choline and acetyl-CoA into acetylcholine and CoA in the presynaptic nerve. Whereas, acetylcholinesterase (AChE) catalyse the hydrolysis of ACh into choline and acetic acid in the synaptic cleft. AChE is a membrane dependant enzyme consisting of multiple subunits and is present in muscles, cholinergic neurons and brain to terminate the nerve conduction in the cholinergic synapses of the somatic and central nervous systems (Işık et al. 2020). Inhibition of AChE increases the levels of ACh in the synapse and ultimately improves the healthy neural communications. The cerebrosides are sphingolipids abundantly present in the human brain and other living genera and work as secondary metabolites. The cerebrosides possess antifungal, anticancer, anti-HIV-1, antinociceptive, and neuritogenic activities. The neuroprotective cerebrosides were reported from eukaryotes but not from prokaryotes (Tan and Chen 2003).

Etiology Of Alzheimer's Disease (Ad)
Amyloid precursor protein (APP) is an integral membrane glycoprotein present in many tissues and concentrated in the synapse of neurons. APP is cleaved by three precursor enzymes viz., α, β and γ secretases. The enzyme, α-secretase dissociates APP and generates neurotrophic C-terminus fragment with C83 amino acids and soluble neuroprotective APP (Pamela 2013). β-secretase hydrolyses APP to Cterminal fragment with C99 fragment amino acids in the membrane that act as a substrate for γsecretase. The γ-secretase cleaves APP to amyloid β (Aβ) peptide containing 37-49 amino acid residues into extra cellular space and APP intracellular domain. Among these Aβ extracellular peptides, Aβ 1-40 and Aβ 1-42 are found to be neurotoxic (Narlawar et al. 2008). The longer form, Aβ 42 with high expression than Aβ 40 is highly brillogenic and triggers a series of neurotoxic events leading to neurodegeneration by the accumulation of Aβ fragments outside of the neural cell between the neurons.
These Aβ fragments then join with other molecules and non-nerve cells to form insoluble plaques which cause neuronal in ammation and damage other neurons leading to loss of neuronal communication, memory loss and nally develop Alzheimer disease (AD).
In healthy neurons, microtubules act like tracks guiding nutrients and molecules to all parts of the neurons, from the body of the cell to axon and back. Tau protein binds the microtubule network for stabilisation. Tau and microtubule binding strength is regulated by phosphorylation (Okuda et al. 2016) events that are in turn regulated by cyclin dependent kinase-5 (CDK5), glycogen synthase kinase-3β (GSK-3β), protein phosphatase-1 (PP-1) and protein phosphatase-2A (PP-2A). The imbalance between kinase and phosphatase activities is called hyper-phosphorylation. The hyperphosphorylation of tau at Ser 413 (of p-Tau) leads to the synaptic dysfunction and loss of neuronal signalling (Lasagna-Reeves et al. 2011) which form laments in the neurons called neuro brillary tangles (NFT) that cause the death of cells. NFT is the major pathological symptom of AD along with an elevated Aβ 1-42 senile plaques (Okuda et al. 2016) (Ali et al. 2015). The abnormal tau hyperphosphorylation activates the GSK3β in the primary hippocampal region that favour the deposition of Aβ (Wang et al. 2016). Hence, the inhibition of GSK-3β pathway helps to reduce the Aβ bril deposition (Mairet-Coello et al. 2013).

Prevention And Treatment
Amyloid precursor protein (APP), presenilin-1 and presenilin-2 are early-onset of deterministic genes (Williamson et al. 2009), changing of life-style habits and solving health related issues could minimise or post-pone the occurrence of AD (Silva et al. 2019). The older brains are more likely to build-up Amyloid plaques, neuro brillary (or tau) tangles and, other additional proteins, making research and treatment more complicated (van der Flier 2005). The current attempt to treat Alzheimer's disease by targeting the amyloid plaques has been unsuccessful in clinical trials because of accumulation of several other toxic products (Huang et al. 2020). In the healthy brain, AChE is the most important enzyme regulating the level of ACh. Irreversible AChE inactivation leads to acetylcholine accumulation, hyperstimulation of ACh receptors (nicotinic and muscarinic receptors) disrupting neurotransmission, but reversible inactivation of this enzyme in the synaptic junction is considered to be a promising strategy for the reduction of neurological disorders such as Alzheimer's disease (AD), senile dementia, ataxia, and myasthenia gravis (Tripathi and Vibha 2009). Presently, galantamine, donepezil, rivastigmine and memantine are the only FDA approved drugs for the management of Alzheimer's disease, which are also AChE enzyme inhibitors with many side-effects (Kaushik et al. 2018). Other than this, regular physical activity, healthy and balanced diet, exposure to sun light (for vitamin D) and hormone replacement therapy (estrogen or combination therapy) may aid in AD treatment (Silva et al. 2019).

Nature Of Cerebrosides
Structurally diverse cerebrosides from living organisms and synthetic sources have been collected and listed with an aim to study their potential for treatment of AD (Tan and Chen 2003). The ceramides are lipids formed by the combination of sphingosine and 2-hydroxy (un)saturated fatty acids, which are soluble in nonpolar solvents. The C1-hydoxyl of ceramide linked with O-glycosidic saccharide head group is called cerebroside. The cerebrosides generally consists of 3 parts, viz, shpingoid base chain, N-acyl side chain and carbohydrate group. In plants, the most common sphingoid base is trans-4-sphingenine, d18:2 4t ), where, 'd' represents number of hydroxyl groups, '18' represents carbon chain length in sphingoid base chain, '2' represents number of double bonds and 'superscript 4t' represents the position of transdouble bond. This enables plant cerebrosides to easily cross the blood-brain barrier (BBB) (Eguchi et al. 2020). The mammals contains sphinganine (d18:0) and phytosphingosine (t18:0), whereas, the fungi and marine organisms produce structurally distinct sphingoid bases such as 9-methyl sphingadienine  Table 1).

Molecular Docking Study
Molecular docking is one of the most frequently used methods in structure-based drug design, due to its ability to predict the binding conformations of small molecule ligand to the appropriate target binding site. It aims to achieve an optimised conformation for both the protein and the ligand such that free energies of the overall system is minimised. The most suitable method of evaluating the accuracy of a docking procedure is to determine how closely the lowest energy poses predicted by the scoring function is matching to that one determined by XRD experimental binding mode. The root mean square deviations (RMSD) between the predicted conformation and the observed X-ray crystallographic conformation of native protein compounds were comparable.
The X-ray crystal structure of AChE has two main binding sites, the catalytic active site (CAS), located near the bottom of gorge, and peripheral anionic site (PAS) present in the middle of gorge and looks like a bottleneck narrow gorge (Xu et al. 2008). The molecules with an elongated conformational dual binding site were known to t correctly at the site of AChE lengthy space. The drug with dual binding properties i.e., extensive orientation form near the bottom of Trp86 of catalytic active site and at Trp286 of peripheral anionic site like that of donepezil active site, can inhibit AChE activity and block amyloid plaque formation (Junaid et al. 2019). The X-ray structure of human AChE co-crystallised with donepezil (PDB: 4EY7) having a resolution of 2.35 Å showed the best binding poses, and the same was selected for the current study due to exible conformations of CAS and PAS amino acids that are necessary to accommodate bulky inhibitors (Makhaeva et al. 2020). In the present investigation, the docking studies were carried out on fungal and plant cerebrosides but not on animal cerebrosides due to lack of con rmed structures.

Docking Study
The three-dimensional X-ray crystal structure of human AChE (PDB: 4EY7, resolution: 2.35 Å) was retrieved in pdb format from the protein data bank. 4EY7 has two chains, A and B, with the presence of donepezil ligand. Protein preparation was done by AutoDock Tools 1.5.6 by removing water molecules, adding polar hydrogen and minimising the receptor structure by applying force elds. The co-crystallised ligand (donepezil) was removed from the protein using Biovia Discovery Studio Visualiser. All the ligand molecules were drawn using ChemSketch and for the 3D structures of the ligands, the addition of charges and the energy minimisation was done by using AutoDock Tools 1.5.6. The grid box in AutoDockVina was kept at -14.624, -40.551 and 22.645 for X, Y and Z centers, respectively. The molecular docking was performed using AutoDockVina and the binding a nities were observed in negative score of kcal/mole. The highest negative value is considered as highest binding a nity at the active site of the protein. The results of protein-ligand binding interactions of 3D docked poses were analysed from Biovia Discovery Studio Visualiser. The hydrogen bonding, hydrophobic and π-alkyl group interactions (Table 2) were considered for the prediction analysis.

Review of neuroprotective cerebrosides
The present review-cum-technical paper is concerned with the screening and identi cation of potential neuroprotective cerebrosides, their reported mechanistic details followed Molecular Docking studies to validate their role in neuroprotection to aged people with AD. Big potassium (BK Ca ) channels are voltagegated K + channels that deliver large amounts of K + across the cell membrane. These channels play an important role in the stabilisation of cell membrane at negative potentials to control the excitation of the nerve (Zhou et al. 2014). Cerebral ischemia causes excessive entry of Ca 2+ by NMDA receptors leading to neuronal death (Fig. 1). Hence, BK Ca channels could be potential therapeutic targets for treating ischemic stroke (Zhou et al. 2014). The BK Ca channel activation was induced by the cerebrosides 1-7 (Fig. 1, Table   1) isolated from mushroom, Termitomyces albuminosus. Termitomycesphins A-D (1-4) contains branched allylic alcohol system at C8 in 1 and 2, and C9 in 3 and 4, on sphingoid long chain nitrogen base. At lower concentrations, nitric oxide (released from eNOS and nNOS) confer neuroprotective and antiapoptotic effects, but at higher concentrations, particularly during oxidative stress (catalysed from iNOS), it exert neurotoxic and proapoptotic effects (Fig. 1) (Benarroch 2011). Hence, inhibition of higher concentration of NO or iNOS could help in protection against neurotoxicity (Dzoljic et al. 2015). A rare cerebroside, 1-O-β-d-glucopyranosyl-(2S,3S,4E,8Z)-2-[(2'R)-2'-hydroxy hexadecanoyl-amino]-hexadeca-4,8diene-1,3-diol or glaucerebroside (8) ( Table 1)

Neuroprotective Cerebrosides docking on Molecular Operating Environment
In the molecular docking study, the cerebrosides  were tted in the active target site of the important amino acids of the AChE enzyme by making hydrogen and hydrophobic interactions (Fig. 2). From the obtained results, the minimum negative values of the Gibbs free binding energy were taken as the top scoring modes with the root mean square deviation (RMSD) values <1Å. The binding a nities of fungal cerebrosides ranged from -7.1 kcal/mol (4) to -8.2 kcal/mol (6), and for plant cerebrosides, from -7.3 kcal/mol (10) to -8.0 kcal/mol (21) (Fig. 3) ( Table 1). The AChE co-crystallised donepezil and its analogues having key residues in the receptor-ligand interactions at Trp86, Tyr337, Phe330, Tyr341, Tyr124, Trp286 were considered as an active site amino acid residues and the same may be required for strong binding with the ligand (Junaid et al. 2019). All the cerebrosides  in the current study have shown the strong interactions with these amino acid residues but with the exception of the presence of Phe338 in place of Phe330 (Supplementary data). Among all the cerebrosides, 6 have shown the highest binding a nity with the active site. It has displayed strong conventional hydrogen bonding interactions (Fig. 4) with Tyr72 (O…HO, 2.34Å), Tyr124 (O…HO, 2.86Å), Trp186 (O…HO, 2.54Å), Gln291 (O…HO, 2.38Å) and Trp286 (O…HO, 2.46Å).

Structure Activity Relationship (SAR)
All the cerebrosides from plantae are having absence of methyl branch at C-9 and hydroxyl at C-8 or C-9. Structurally, cerebroside 5 is dehydroderivative of 7. All fungal compounds (1-7) possess d-erythrosubstitution at C2/C3 in the sphingoid base chain and, the absolute con guration at the chiral atoms were determined as 2S, 3R and 2'R (Qi et al. 2000) (Qi et al. 2001) (Li et al. 2012). The fungal cerebrosides consists of either hydroxyl group (1,2,6,7) or unsaturation (3)(4)(5) at C-8, and, additional alkenyl (1,2) or methyl (3)(4)(5)(6)(7) side chain at C-9. The formation of unsaturation at C8 could be the result of elimation of hydroxyl group at C-8 with a proton from C-7 or C-9. An extra hydroxyl group in the middle of long chain base played an important role (Qi et al. 2000) for the enhancement of the activity. The higher neuroprotective PC12 cell differentiation activity was observed in less carbon numbered C16 sphingoid chain of 1,3 and 6 than C18-sphingoid chain of 2,4,5 and 7 compounds (Qi et al. 2000) (Qi et al. 2001) (Li et al. 2012) and this was further supported by molecular docking studies. The SAR of the docking studies revealed the hydroxyl group at C-8 with lower number of carbons at N-acyl side chain showed higher activity in the compounds of 1 and 6 than, the respective lower number of carbons in 2 and 7, whereas, the additional hydroxyl group at C-9 showed poor activity in 4 than 5 with the same number of carbons in N-acyl side chain. Hence the result is in agreement with the neuroprotective activity of fungal cerebrosides depends on the additional hydroxyl groups present in sphingoid base chain (Qi et al. 2001).
From Fig 3 and Table 1, it is clearly observed the cis con guration (10-15) having higher activity than the trans con guration (16-21) with the proportional increase in the carbon number of the sphingoid base chain. These results are also re ecting in the neuroprotection of glutamate injured PC12 cells half maximal effective concentration (EC 50 ) values and also in binding a nity values of molecular docking study. The compounds 14 and 22 are having same structural arrangement with the exception of additional C4-hydroxyl group in sphingoid base chain of 22, but both the compounds neither displayed the signi cant binding a nity with the protein in the docking study nor their neuroprotection towards the glutamate injured PC12 neural cells. Among all the compounds 1-22, the compound 8 is only having lesser number of carbons in the sphingoid base chain. The compounds 8 structurally similar with 10 but the displayed higher activity of 8 could be due to a smaller number of terminal methylene groups in the sphingoid base chain. The unsaturation at C8 either in cis or trans forms was observed in all plant cerebrosides but not in fungal cerebrosides.
Among the two binding sites, CAS and PAS of AChE, the Trp286 residue of PAS plays a signi cant role in ligand recognition and also in the allosterism. The hydrophobic indole Trp286 aromatic interaction was observed in all the compounds with both the aliphatic shpingoid base chain and N-sphingoid base chains, hence this could modulate the AChE inhibition (Sussman et al. 1991). All these amino acid residues having large π-cation interaction sites needed for the stabilisation of the substrates at the binding site are listed.
In summary, in design of ideal cerebrosides for the neuroprotection of Alzheimer's disease an additional hydroxy group at C-8 with methyl side chain at C-9 in the N-acyl side chain (6), a smaller number of carbon atoms in sphingoid base chain (8), and cis-con guration with higher number of carbon atoms in N-acyl side chain (15) topographies could be considered.

Conclusion
The current study suggests the cerebroside secondary metabolites from fungi, plants  and animals are neuroprotective with different molecular targets involving nitric oxide, PI3K/Akt and BDNF/CREB pathways. Our Molecular Docking Studies revealed that the binding a nities of cerebrosides are -7.1 to 8.2 kcal/mol. The key residues in the receptor-ligand interactions at Trp86, Tyr337, Phe330, Tyr341, Tyr124, and Trp286 are considered as active site amino acid residues and may be required for strong binding with the ligand. All the cerebrosides  in the current study have shown the strong interactions with these amino acid residues. The increased binding a nity due to the reduced number of sphingoid base carbons was observed. The hydrophobic indole Trp286 aromatic interaction was observed with both the aliphatic sphingoid base chain, and N-sphingoid base chains, hence this could modulate the AChE inhibition. Further studies are required in the line of drug development using selective cerebrosides for neuroprotective effects.      Cerebroside 6 with strong binding interactions at the active site.

Supplementary Files
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