Computational Assessment and Pharmacological Property Breakdown of Eight Patented and Candidate Drugs against Four Intended Targets in Alzheimer’s Disease

Alzheimer’s Disease (AD) is the most prevalent age-related dementia. AD can be caused by abnormal processing of amyloid precursor protein (APP) or by oxidative stress or may be due to the actions of kinases or the degeneration and loss of functions of neurons in the brain. Although various treatments have already gained success in the in vitro studies, however, till now not a single satisfactory drug has been proven that can cure this disease perma-nently till now. In this study, the best possible drug has been determined from a group of drug molecules using methods of molecular docking. Molecular docking is a computational approach which helps to determine the best molecule from a group of molecules which may bind with the highest affinity with the intended target by mimicking the original biological environment in a computer. The tested drug molecules in this experiment are the disease modifying agents, capable of inhibiting a particular protein involving in the AD pathway. Eight drug molecules (ligands)-memantine (−4.075 Kcal/mol), hymenialdisine (−8.079 Kcal/mol), tideglusib (−6.445 Kcal/mol), kenpaullone this experiment which are intended targets in current AD treatment approaches. Investigation of docking results, druglikeness properties and ADME/T testing results suggest that the best findings of this experiment are memantine, hymenialdisine, dihydrospiro[dibenzo[a,d][7]annulene-5,4’-imi-dazol] and harmol, that could be the best possible drugs for the treatment of AD.

have already gained success in the in vitro studies, however, till now not a single satisfactory drug has been proven that can cure this disease permanently till now. In this study, the best possible drug has been determined from a group of drug molecules using methods of molecular docking. Molecular docking is a computational approach which helps to determine the best molecule from a group of molecules which may bind with the highest affinity with the intended target by mimicking the original biological environment in a computer. The tested drug molecules in this experiment are the disease modifying agents, capable of inhibiting a particular protein involving in the AD pathway. Eight drug molecules (ligands)-memantine (−4.075 Kcal/mol), hymenialdisine (−8.079 Kcal/mol), tideglusib (−6.445 Kcal/mol), kenpaullone (−7.545 Kcal/mol), dihydrospiro[dibenzo[a,d] [7]annulene-5,4'-imidazol] (−4.742 Kcal/mol), harmine (−7.57 Kcal/mol), harmol (−6.583 Kcal/mol) and 1-Methyl-4-Phenylpyridinium (−5.214 Kcal/mol), have been docked successfully against four targets (proteins)-N-Methyl-D-Aspartate Receptor (NMDAR), glycogen synthase kinase-3β (GSK-3β), beta-secretase (β-secretase) and dual specificity tyrosine (Y)-phosphorylation-regulated kinase 1A (DYR-K1A) in

Introduction
Alois Alzheimer first described Alzheimer's Disease (AD) in 1907. It is the most prevalent age-related dementia in the world [1]. AD is a common type of age-related dementia that is increasing its numbers day by day [2]. The common symptoms of AD include functional and intellectual morbidity, hallucinations, delusions, psychomotor dysregulation etc. [3]. Genetic causes are also involved in the familial cases of AD [4]. However, there are many reasons that lead to the onset of AD development. Many hypotheses shed light on several reasons. One such hypothesis is the "amyloid cascade hypothesis". According to this hypothesis, the deposition of β-amyloid plaques in the brain is the main reason of AD development. These plaques are generated by abnormal processing of amyloid precursor protein (APP) by β-secretase enzyme. These plaques interfere with the normal activities and functions of the brain [5]. Moreover, there is another hypothesis called "oxidative stress hypothesis". According to this hypothesis, increased amount of iron and mercury in the brain is capable of generating free radicals, thus increasing lipid peroxidation and protein and DNA oxidation in the brain and thus producing stresses on the brain. And these stresses produced by oxidation in the brain are mainly responsible for AD development [6]. According to another hypothesis called "cholinergic hypothesis", the degeneration and loss of functions of cholinergic neurons and cholinergic neurotransmission in the brain, cause AD [7]. Although there is no permanent treatment to cure AD, scientists are working on various disease modifying approaches that target various enzymes that take part in the regulatory pathways which may lead to the onset of AD [8].
Various compounds can be used as disease modifying agents to treat AD. The main concept of disease modifying treatment is to modify the protein or enzymes involved in the AD pathway. Most of such modifying agents are not commercially available yet. Memantine can be used to treat abnormal N-methyl-D-aspartate (NMDA) pathway by inhibiting the NMDA receptors (NMDARs) [9]. Hymenialdisine, tideglusib and kenpaullone have gained success in inhibiting glycogen synthase kinase-3β, a major enzyme involved in AD [10] [11] [12].
In this study, we have conducted experiments to determine which one of the above mentioned ligand molecules could be the best option to treat AD by interfering specified target proteins involved in the AD pathway.

N-Methyl-D-Aspartate Receptor (NMDAR) (Receptor) and Memantine (Ligand)
In the mammalian central nervous system (CNS), a potential neurotransmitter, glutamate plays very important roles and it is the main excitatory neurotransmitter in the CNS. Glutamate mediates its effects by many families of receptors such as ionotropic glutamate receptors (iGluRs) and metabotrophic glutamate receptors (mGluRs). The iGluRs family contains many types of receptors.
Among them, N-methyl-D-aspartate receptors or NMDARs are the receptors that are mainly responsible for learning and memory [15]. Therefore, any disruption in the normal signalling pathway of the NMDARs may lead to the damage of the CNS that may cause the AD to develop.
N-methyl-D-aspartate (NMDA) selectively mediates NMDARs. The NMDARs are encoded by human genes GRIN1, GRIN2A, GRIN2B, GRIN2C and GRIN2D [16]. The NMDARs can be divided into two groups: synaptic and extrasynaptic NMDARs. The activation of synaptic NMDARs leads to synaptic plasticity and cell survival ( Figure 1) [17]. However, inappropriate NMDAR signalling leads to injuries in the neuronal system.   Figure 2) [18]. Therefore, the entry of excessive levels of ions leads to the toxic condition in the cell and causes neuronal cell death. This leads to the onset of AD. On the other hand, the β amyloid plaques, formed due to AD, selectively activates extrasynaptic NMDARs. The extrasynaptic NMDARs cause the deleterious effects like tau protein phosphorylation and induction of apoptosis by activating caspase-3, which also leads to the onset of AD [19].
Administration of memantine can block the activity of NMDARs by binding with those receptors and thus mediate its therapeutic properties in inhibition of AD [20]. In this experiment, memantine (PubChem CID: 4054) was used to dock against GluN2D (PDB ID: 3OEM), which is a NMDAR or ionotropic glutamate receptor [21]. In normal condition (a), the glutamate is secreted and binds to NMDAR, thus activates the receptor and mediates the calcium ion transport across the neuron cell. In abnormal condition (b), inappropriate stimulation of glutamate production causes the over-activation of NMDARs, which causes excessive entry of ions into the neuron cells, causing the disruption of cellular functions.

Glycogen Synthase Kinase-3β (Receptor) and Hymenialdisine, Tideglusib and Kenpaullone (Ligands)
Glycogen synthase kinase-3β (GSK-3β) is an enzyme kinase that plays important role in the development of AD by phosphorylating the tau protein [22]. Aβ is caused by defective proteolytic processing of amyloid precursor protein (APP). This defection leads to the production and deposition of 42 amino acids long neurotoxic forms of β-amyloid (Aβ) peptides. Three enzymes determine whether the neurotoxic forms of β-amyloid will be formed or not. β-secretase and γ-secretase cleave APP sequentially at the N-terminus and C-terminus, respectively. These cleavages lead to the beta amyloid production and when α-secretase cleaves APP, the possibility of formation of Aβ minimizes [23] [24] [25]. APP is a surface membrane protein that can be processed by two major pathways: non-amyloidogenic pathway and amyloidogenic pathway. In the non-amyloidogenic pathway, the α-secretase and γ-secretase enzymes cleave the transmembrane domain of APP, sequentially. These cleavages give rise to the fragments that are easily degradable [26]. However, in amyloidogenic pathway, the APP is cut by β-secretase and γ-secretase, which form β-amyloid (Aβ) peptides and the Aβ peptides tend to aggregate and form plaques [27]. Microtubule associated protein (MAP) tau is a protein that is found in the neuron cells and their main function is to stabilize the microtubules. They are phosphorylated in lesser extent in the normal adult brain. However, in the AD patients, they are found to be highly phosphorylated. The abnormally phosphorylated tau acquires the shape of paired helical filaments (PHFs) and forms neuro fibrillary tangles (NFTs) with other abnormally phosphorylated tau proteins. These NFTs are insoluble tangles that appear to be accumulated as tangled mass in the brain. NFTs interfere with the normal functions of the neurons by destabilizing the microtubules [28]. One of the proteins responsible for the tau phosphorylation is GSK-3β. The GSK-3 is a serine/threonine kinase enzyme. In the brain, the GSK-3β is responsible for the tau phosphorylation [29]. GSK-3β phosphorylates 36 sites on the tau protein [30]. There is evidence that, Aβ is responsible for the tau phosphorylation [31]. Aβ activates and causes over production of GSK-3β signaling by inhibiting the inhibitory phosphorylation mechanism of this enzyme. Therefore, the formation of Aβ directly causes the over-activation of GSK-3 which in turn hyper-phosphorylate the tau protein and form NFTs. NFTs ultimately result the AD development. Moreover, the formation of NFTs later leads to the apoptosis of the neuron ( Figure 3) [32]. A potent inhibitor of GSK-3β is hymenialdisine [10]. Studies have found that another compound named tideglusib can also be used as GSK-3β inhibitor [11]. Moreover, kenpaullone is another compound that has GSK-3β inhibitory activity [12]. Molecular docking has already been performed successfully against the GSK-3β (PDB ID: 1Q5K) using 1,3-disubstituted-1H-pyrazol-5-ols as ligands [33]. In the experiment, docking was performed using hymenialdisine (PubChem CID: 11313622), tideglusib (PubChem CID: 135413546) and kenpaullone (PubChem CID: 3820) as ligands against the GSK-3β (PDB CID: 1Q5K).

β-Secretase (Receptor) and Dihydrospiro[Dibenzo[a,d][7]Annulene-5,4'-Imidazol] (Ligand)
The pathway of β-secretase enzyme involves the abnormal proteolytic processing of APP. The cleavage of the APP protein by β-secretase leads to the formation of β-amyloid (Aβ) plaques [34]. The formation of Aβ is decided by the activities of three enzymes: α-, βand γ-secretases. The APP protein can be cleaved by two major pathways: nonamyloidogenic pathway and amyloidogenic pathway. In non-amyloidogenic pathway, the transmembrane portion of APP protein is cleaved sequentially by αand γ-secretases. These cleavages don't lead to the formation of Aβ. Since α-secretase cleave within the Aβ region, the Aβ formation never occurs [26].
However, in the amyloidogenic pathway, the abnormal cleavage of APP is carried out sequentially by βand γ-secretases and the β-secretase cuts the APP protein at a site 99 amino acids away from the C-terminus, leaving the C-terminal portion of the protein in the membrane, called C99. This newly generated C99 fragment contains the first amino acid of the Aβ plaque, at the newly generated N-terminus. Then γ-secretase cuts the C99 between 38th and 43th amino acids and liberates the Aβ peptides, which later aggregate together with other Aβ peptides and form plaques. This Aβ plaque formation is one of the main reasons behind the AD onset ( Figure 3) [35].

DYRK1A Enzyme (Receptor) and Harmine, Harmol, 1-Methyl-4-Phenylpyridinium (Ligands)
Abnormal phosphorylation of tau protein is one of the main reasons of AD development [37]. Many enzymes are responsible for such type of phosphorylation, for example, glycogen synthase kinase 3 (GSK-3), cyclin-dependent kinase 5 (CDK-5), cAMP-dependent protein kinase A etc. The DYRK1A (dual specificity tyrosine (Y)-phosphorylation-regulated kinase 1A) is a recently discovered enzyme that is also responsible for the abnormal phosphorylation of tau protein.
This enzyme is expressed from the DYRK1A gene of 21 st chromosome. This enzyme exhibits dual specificity. First, the enzyme autophosphorylates itself on the tyrosine 321 residue for activation and then phosphorylation of the target protein occurs [38]. The abnormally phosphorylated tau acquires the shape of paired helical filaments (PHFs) and forms NFTs that are insoluble and appear to be accumulated as tangled mass in the brain ( Figure 3) [28].

In Silico Docking Study and ADME/T-Test
Due to the advancements of various computer softwares, it is now possible to NMDAR, glycogen synthase kinase-3 (GSK-3), β-secretase and dual specificity tyrosine (Y)-phosphorylation-regulated kinase 1A (DYRK1A), respectively, to study their potential interaction in a search for the best possible drug compound.  atom RMSD (root-mean-square-deviation) to 30 Å and any remaining water less than 3 H bonds to non water was again deleted during the minimization step.

Ligand Preparation
The

Receptor Grid Generation
Grid usually confines the active site to shortened specific area of the receptor protein for the ligand to dock specifically. In Glide, a grid was generated using default van der Waals radius scaling factor 1.0 and charge cutoff 0.25 which was then subjected to OPLS_2005 force field. A cubic box was generated around the active site (reference ligand active site). Then the grid box volume was adjusted to 15 × 15 × 15 for docking test.

Ligand Based Drug Likeness Property and ADME/Toxicity Prediction
The molecular structures of every ligands were analyzed using SWISSADME

Binding Energy
All the selected ligand molecules were docked successfully against NMDAR, GSK-3β, β-secretase and DYRK1A.    Toxicity  III  III  III  III  III  III (Table   1 and Figure 8).

Druglikeness Property
Lipinski's rule of five demonstrates that the acceptable ranges of the best drug molecule for all the five parameters are: molecular weight: ≤500, number of hy-  [46]. All the ligand molecules followed the Lipinski's rule of five without any violation. The results are listed in Table 2.
None it did not exhibit any of the deleterious effects. Kenpaullone showed druglikeness score of 1.08, solubility score of −5.14 and drug score of 0.33, however, its reproductive effectivity was quite high, although it didn't have any irritant, tumorigenic and

ADME/T-Test
The results of ADME/T test of selected ligand molecules are listed in Table 3.
All the ligand molecules showed the ability to cross the Blood-Brain Barrier (BBB) and gave positive results in human intestinal absorption (HIA). Only memantine and 1-methyl-4-phenylpyridinium showed Caco-2 permeability. All the molecules were proved to be P-glycoprotein Inhibitors.
All the molecules were non-substrate of CYP450 2C9 and CYP450 2D6. Only harmine and harmol showed AMES-toxicity. Though all the ligand molecules were non-carcinogenic, all of them were not readily biodegradable and all of them showed level-III oral acute toxicity.

Discussion
Molecular docking demonstrates the best possible pose of a ligand molecule within the binding site of the receptor molecule and calculates a score of binding energy. This score is also known as the "docking score". The lower the binding energy, the higher the affinity of binding and vice versa [47]. Hymenialdisine ADME/T-tests examine the pharmacological and pharmacodynamic properties of a candidate drug molecule inside a biological system. Therefore, it is a crucial determinant of the success of a drug discovery approach. BBB is the most crucial element for those drugs that target primarily the brain cells. Oral delivery system is the most commonly used route of drug administration. Therefore, it would be appreciable that the drug is highly absorbed in intestinal tissue. Since P-glycoprotein in the cell membrane facilitates the transport of many drugs, therefore, its inhibition may affect the drug transport. In vitro study of drug permeability test utilizes Caco-2 cell line and its permeability reflects that the drug is easily absorbed in the intestine. Orally absorbed drugs travel through the blood circulation and deposit back to liver where it is degraded by group of enzymes of Cytochrome P450 family and excreted as bile or urine. Therefore, inhi- given in Table 2.
The synthetic accessibility (SA) score estimates how easily a target compound can be synthesized. The score 1 represents very easy to synthesize and the score 10 represents very hard to synthesize [56]. The synthetic accessibility scores are listed in Table 2. The bioavailability score gives the insight of permeability and bioavailability properties of a compound [57]. All the ligands showed similar bioavailability score of 0.55.
All the ligand molecules have been docked successfully against their target proteins. This indicates that all of them can inhibit their target proteins. Memantine showed the best result among all the ligands in the ADME/T test, how-

Conclusion
Eight drug molecules were investigated to find out the best possible drug against their respective targets and thus the best possible treatment to cure AD. Many drugs are already available in the market and many more are still in pre-clinical and clinical trials. This experiment was focused to analyze eight drug molecules to select the best ones which can be directed against various specific targets However, other molecules could also be investigated as they also performed well in docking experiment. Hopefully, the results of this study should help the researchers to identify the best treatment process to treat AD.