Identification of potential neuroprotective compound from Ganoderma lucidum extract targeting microtubule affinity regulation kinase 4 involved in Alzheimer's disease through molecular dynamics simulation and MMGBSA

Abstract Objective Alzheimer's disease (AD) is one of the most prevalent neurological ailments, affecting around 50 million individuals globally. The condition is characterized by nerve cell damage due to the formation of amyloid‐beta plaques and neurofibrillary tangles. Only a few US Food and Drug Administration (FDA)‐approved medications are available in the market which are devoid of side effects, thus, making it imperative to investigate new alternatives for countering this disease. According to a recent study, microtubule affinity regulation kinase 4 (MARK4) is attributed as one of the most promising drug targets for AD, thus, being selected for this study. Compounds from Ganoderma lucidum (Reishi mushroom) extracts were selected to be used as ligands for this study. Methods In this study, the five most potent compounds from Ganoderma lucidum were selected and their absorption, distribution, metabolism, excretion, and toxicity (ADMET) analysis was performed, followed by molecular docking, and molecular dynamics simulation of each compound with MARK4 and supported by molecular mechanics generalized born surface area (MMGBSA) binding free energy calculations. Results The promising compounds were selected based on their ADMET profile and interactions with the active site residues of MARK4. Based on docking scores of −9.1 and −10.3 kcal/ mol, respectively, stability assessment by molecular dynamics simulation, and MMGBSA calculations, ganoderic acid A and ganoderenic acid B were found to be the most promising compounds against MARK4 which will require further in vitro and in vivo validations. Conclusion Through this study, it is suggested that ganoderic acid A and ganoderenic acid B might be a class of promising compounds against AD, based on computational research, and can be further studied for preclinical and clinical studies.


| INTRODUC TI ON
The Alzheimer's disease (AD) is one of the most common neurodegenerative disorders, with symptoms including memory loss, mood swings, and functional difficulties. 1 Dementia, which includes AD, affects 50 million individuals globally, most of whom are senile.
According to the World Health Organization (WHO), AD is the world's fifth leading cause of death, with the number of fatalities anticipated to triple by 2050 if current trends continue. 2 AD is diagnosed using brain imaging, physical and cognitive evaluations, laboratory tests, and medical history. 3 The risk factors for AD include apolipoprotein E4 (APOE4) genotype and traumatic brain injury, family history and age, obesity, diabetes, hypercholesterolemia, hypertension, and a lack of education. 4 Therefore, pharmaceutical and nonpharmacological treatments, such as physical, social, and cognitive activities, should be incorporated into the therapy regimen. Sodium valproate and lithium are the medications used to treat mild to moderate cognitive dysfunction. 5,6 Nonsteroidal antiinflammatory drugs (NSAIDs), such as naproxen and ibuprofen, reduce neuroinflammation and prevent neurodegeneration. 7 Headaches, nausea, vomiting, neuromuscular dysfunction, and breathing issues have all been reported as side effects of these medications. 5,9 Only five US Food and Drug Administration (FDA)approved medicines are currently available to treat AD symptoms.
Aducanumab was recently approved in the year 2021. Memantine (N-methyl-D-aspartate receptor [NMDAR] inhibitor), was approved 10 years ago. Rivastigmine, donepezil (both cholinesterase inhibitors [ChEls]), and galantamine (an NMDAR antagonist) are the remaining drugs. 3,10 In this study, microtubule affinity regulation kinase 4 (MARK4), a member of the serine/threonine kinase family, primarily involved in microtubule assembly and the cell cycle, was selected as a target protein. The crystal structure of MARK4 was first observed in a complex with the pyrazolopyrimidine inhibitor. MARK4 overexpression has been observed in the initiation of neurodegenerative diseases, such as AD and Parkinson's disease (PD). Overexpressed MARK4 is responsible for the phosphorylation of Tau protein at the Ser262 site, which is required for the binding of microtubules to tau proteins. Colocalization of phosphorylated MARK4 with phospho-tau Ser262 in GVDs accumulating in AD samples has been reported. Studies have shown an association of MARK4 expression with neurodegenerative diseases, such as AD and PD, with MARK4 being present in significantly higher expression levels in neurodegenerative disease cases. These results hypothesized a potent MARK4 inhibitor can be designed to be used as an inhibitor against neurodegenerative diseases. 11 In this computational study, Ganoderma lucidum, also known as "Reishi mushroom," which comes under the category of medicinal mushroom, was selected. Ganoderma lucidum has been known to possess immune-stimulating activities, anti-inflammatory, and antiallergenic properties. This study aims to find a promising compound from Ganoderma lucidum, which can be used for preclinical and clinical trials against neurodegenerative disorders. In this study, the five most promising molecules from the Reishi mushroom: ganoderic acid A, ganoderic acid D, ganoderic acid F, ganoderenic acid B, and ganoderenic acid D 12 were selected. Molecular docking for the five compounds mentioned above was performed, followed by molecular dynamics simulation for all five compounds. Out of these five, two compounds, namely, ganoderic acid A and ganoderenic acid B, were found to be most potent inhibitors against the MARK4 protein.
2 | ME THODS 2.1 | ADMET (absorption, distribution, metabolism, excretion and toxicity analysis SwissADME webserver was used to estimate the pharmacokinetic and pharmacological properties of the five compounds from Ganoderma lucidum.

| Molecular docking
Molecular docking was performed with AutoDock 4.2.6, using the standard procedures. The 3D coordinates of the MARK4 protein were retrieved from the RCSB database with the PDB ID 5ES1 and five compounds of Ganoderma lucidum from the PubChem database.
A grid box with x, y, and z directions was generated, keeping the grid spacing at 0.375 Å, and flexible multiple ligand docking was performed using a Perl script. The outputs were analyzed and visualized in Discovery Studio Visualizer. The NPT ensemble was used throughout the simulation, with a temperature of 300 K and a pressure of 1 atm.

| Molecular mechanics generalized born and surface area calculations
The binding free energies (∆G bind ) of the anchored complexes were calculated using the Molecular Mechanics Generalized Born Surface Area (MMGBSA) module (Schrodinger suite, LLC). Binding free energies were calculated using OPLS 2005 force field, VSGB solvent model, and rotamer search methods. 18 After performing Molecular Dynamics simulations (MDS), a period of 10 ns was used to select the MD orbital frame. The total free binding energy is calculated using the below-mentioned formula: where ∆G bind , binding free energy; ∆G complex , free energy of the complex; ∆G protein , free energy of the target protein, and ∆G ligand , free energy of the ligand.

| ADME absorption, distribution, metabolism, a excretion analysis
The absorption, distribution, metabolism, excretion, and toxicity (ADMET) analysis of the selected molecules was done. It describes the absorption, distribution, drug metabolism, and excretion of a substance or medication. 19,20 Solubility and permeability are critical characteristics that influence the oral absorption rate. 21,22 As soon as the medication is dissolved, it circulates throughout the body and is distributed throughout different systems. Drug distribution into tissues and organs is governed by pharmacokinetic characteristics, such as plasma protein binding, lipophilicity, and phospholipids, three essential variables to consider. 23,24 Drug metabolism is the process by which a drug molecule is broken down or altered enzymatically, followed by excretion. 25 The most important metric for determining pharmaceutical excretion in humans is renal clearance, which is essential for assessing pharmaceutical excretion in humans. 26 Lipinski's rule of five, also referred to as the Pfizer rule of five, requires that oral pharmaceuticals have a molecular weight of less than 500, no more than five hydrogen bond donors, and 10 hydrogen bond acceptors, and a distribution ratio of less than five to be evaluated for approval. 26

| Molecular docking
The selected five molecules from the Ganoderma lucidum were ΔG bind = ΔG complex − ΔG protein + ΔG ligand .

| Molecular dynamics simulation
The

| DISCUSS ION
In structure-based drug design, molecular docking is the most extensively utilized technique because it allows small compounds to interact with targets at the molecular level. Using this technique, we can better understand small molecule behavior in specific protein targets and predict the structure of ligand-receptor complexes, which is critical for drug development. In this study, five compounds of Ganoderma lucidum extract were selected, which include gano-   can be focused upon and can be further studied for preclinical studies. This computational study has shed a new light on the roles of ganoderenic acid B and ganoderic acid A as novel inhibitors of the MARK4 protein.

| CON CLUS ION
Ganoderma lucidum is known to exhibit cytotoxic, hepatoprotective, antioxidative, anticancer, and antinociceptive activities. This study predicted that Ganoderma lucidum could even be used to treat neurological disorders, like AD. The potential use of the best-identified molecules ganoderic acid A and ganoderenic acid B against AD can be studied further using in vitro and in vivo models, on the basis of this study's computational validations.

ACK N OWLED G EM ENTS
None.

FU N D I N G I N FO R M ATI O N
No funding was receieved for this study.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data generated during this study are included in this article.