Computer-aided Analysis of Selective Phytochemicals as Potent Inhibitors of Parkin: Major Biological Target of Parkinson’s disease

Parkinson’s disease, caused by mutations in the Parkin that leads to loss of neuron is the second most widespread neurodegenerative disorder in the world. Phytochemicals are being considered due to their medicinal properties to cure many human diseases. The present study targets the inhibition of Parkin, a major biological target for Parkinson using 3150 phytochemicals from various medicinal plants. These plants are naturally growing in a local climate of Pakistan, India and China and being used for a long time for the medicinal purpose. A total of 3150 phytochemicals from various medicinal plants were collected for this in silico study. The pharmacological assessments prediction, molecular docking and density functional theory (DFT) based studies were done to fi nd out the latent inhibitory properties of these phytochemicals against Parkin. Out of 3150 phytochemicals, 175 showed human-suitable pharmacological properties and among those 175 compounds, 5 phytochemicals, i.e. Liquirtin, Shinfl avanone, Glabrone, GlycyrdioneB and IsoangustoneA to have potent inhibitory properties against Parkin and can be deliberated for additional in vitro and in vivo studies to evaluate their inhibitory eff ects against Parkin. They revealed binding affi nity greater than various previously reported inhibitors against Parkin. Additionally, DFT based analysis exhibited high reactivity for these fi ve phytochemicals in the binding pocket of Parkin, based on ELUMO, EHOMO and band energy gap. A total of 5 out of 175 phytochemicals are reported as highly potent inhibitors against Parkin which are liquirtin, Shinfl avanone, Glabrone, Glycyrdione B and IsoangustoneA from the same medicinal plant Glycyrrhiza glabra. However, these 5 phytochemicals can be considered for further in vivo and in vitro analysis for the clinical development of a drug against the world’s second most common brain disorder, the Parkinson’s disease. Graphical abstract Research Article Computer-aided Analysis of Selective Phytochemicals as Potent Inhibitors of Parkin: Major Biological Target of Parkinson’s disease Nadia Arif1, Andleeb Subhani1, Waqar Hussain2,3 and Nouman Rasool3* 1Department of Life Sciences, University of Management and Technology, Lahore-54770, Pakistan 2National Center of Artifi cial Intelligence, Punjab University College of Information Technology, University of the Punjab, Lahore-54000, Pakistan 3Center for Professional Studies, Lahore-54000, Pakistan Received: 02 February, 2020 Accepted: 09 June, 2020 Published: 10 June, 2020 *Corresponding author: Nouman Rasool, Center for Professional Studies, Lahore-54000, Pakistan, E-mail: noumanrasool@gmail.com


Introduction
Parkinson's disease (PD) is the second most common and prevalent neurodegenerative disorder. It is characterized by loss of dopaminergic neuron in the Substantia Nigra Pars Compacta (SNpc), the formation of Lewy bodies (LBs) and progressive deterioration of motor function. Several genes are known to be associated with the familial forms of PD [1], while Parkin mutations are the key source of early-onset PD [2]. It is categorized by two types of syndromes; motor symptoms and non-motor symptoms. The former type includes bradykinesia, hypokinesia, infl exibility, quiescent tremor, and postural unpredictability. Later type includes autonomic dysfunction, sleep irregularities, depression, anxiety and loss of memory (dementia) [3][4][5]. Dementia is a major cause of debility, and presently there is no effective indicative treatment of PD like the Alzheimer's disease (AD) and 47% of PD patients show sign of depression [6].
Since PD is described clinically by intracellular protein aggregates termed Lewy bodies, it is postulated that Parkin is used for promoting the UPS, which is activated by K48linked polyubiquitination of substrate proteins [11]. Mutation of Parkin damages the ubiquitin-proteasome pathway (UPS) of protein degradation and leads to the lethal accumulation of misfolded or aggregated proteins, Subsequently, the discovery that Parkin promotes mitophagy [12] has been proved through experiments that the ubiquitination of mitochondrial proteins by Parkin triggers the autophagic machinery through staffi ng of ubiquitin-binding adaptors, such as HDAC6 and p62/ SQSTM1. This is the actual mechanism that how Parkin involves in Parkinson's diseases [13][14][15].
Previous studies demonstrate that selective mutations in the Parkin have been reported in sporadic Parkinson's disease [16][17][18] . Thus, it would be interesting to discover potential inhibitors that inhibit the mechanism Parkin gene and that will surely provide a new perspective for the treatment of Parkinson's disease as many loss-of-function mutations in Parkin have been linked with hereditary PD [19,20].
Phytomedicines are generally planted derivative medicines formed by compounds termed as phytochemicals. These are naturally occurring compounds having copious medicinal properties. The effectiveness of phytochemicals against various chronic diseases has been reported in many studies [21] . The phytochemicals are produced during biosynthetic pathways of the plants and acts as secondary metabolites, and there is an enormous diversity of these compounds which are known to have potential antiviral, antibacterial, antifungal, anticancer, and other properties [22]. The phytochemicals found in different groups like alkaloids, fl avonoids, terpenoids and sesquiterpenes and each group of these disease-preventing phytochemicals consists of a number of different chemicals with different potential [23].
The in silico methodologies using computational approaches facilitate the drug discovery process since the screening of drugs using in vitro and in vivo analysis is becoming increasingly challenging, time-consuming and expensive due to a high number of compounds under investigation. Thus, computational techniques are making the analysis economical and resource effi ciency. Within the same time, far more drugs can be discovered using the computational biology and chemistry mechanisms with the least investment of money and time. Remunerations of using in silico methods can be subjugated the whole process of drug development, i.e. from the preclinical discovery phase to the late phase of clinical development. In silico drug discovery methods helps to evaluate the potent and most important medicinal compound with high effi ciency [24]. though, this study evaluated the inhibition potential of these phytochemicals against Parkin.

Structure retrieval
This study targeted the discovery of potential inhibitors against Parkin, crystal structure of the protein was required.
Structure of the targeted protein was available at RCSB PDB [26] and was downloaded in PDB format using PDB ID: 5C1Z.

ADMET Analysis and drug-likeness prediction
By using the PreADMET server [27], phytochemicals were screened out on the basis of following pharmaceutical properties which were solubility (ESOL), gastrointestinal (GI) absorption, blood-brain barrier (BBB) Permeability, Lipinski's rules violations, toxicity and carcinogenicity.
In this study the criteria set for initial screening was; Citation: Arif  Carcinogenicity and Toxicity = Zero/Nil [28].

Molecular docking and binding energy calculations
Molecular docking of Parkin with screened phytochemicals was accomplished using AutoDock Tools and AutoDock Vina [29,30]. AutoDock Tools were used to prepare Parkin by the addition of polar hydrogen bonds and removal of nonstandard residues which improved the interactions between phytochemicals and Parkin. A three-dimensional grid for Parkin was designed with a size of 16×14×18 Å 3 , to explicate the search space for phytochemicals to be docked against Parkin.
To calculate the binding affi nities of different phytochemicals with our target protein, AutoDock Vina [31] was run through command prompt to get binding affi nities (kcal/mol). K i values of the compounds were estimated by using this formula: After calculating binding affi nities, complexes of protein and phytochemicals were formed using Chimera 1.11.2rc. Discovery Studio 2.5 was used to get interaction plots.

Density functional theory analysis
To analyze the reactivity and effi ciency of the strongly docked phytochemicals against Parkin, a density functional theory (DFT)-based analysis was performed using highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) energy by relating the Becke, 3-parameter, Lee-Yang-Parr (B3LYP) correlation function of DFT [32]. The band energy gap (ΔE) was also measured using the expression E LUMO -E HOMO . The energy estimations were made using ORCA Program [33].  (Table 1).  (Table 2).    residues of Parkin protein respectively (Table 3). Glabrone

Analysis of reactivity using DFT
The  (Table 4).
There are many known causes reported for PD but the formation of Lewy bodies and loss of neuron function due to mutations in the Parkin gene are the key cause that contributes 60 to 70% of cases [35]. Parkin, an E3 ubiquitin ligase concerned in Parkinson's disease, triggers degradation of mal-functional mitochondria by autophagy. By proteomic and cellular tactics, it has been shown that upon translocation to mitochondria, Parkin activates the UPS for pervasive degradation of outer membrane proteins. Because of some genetic and environmental factors if any mutation occurs in Parkin then the whole process of autophagy and ubiquitination disturbed [36][37][38].
Bioactive phytochemicals or bio-nutrients are found in medicinal plants in huge amount. Throughout the past 2-3 decades, studies have shown that these phytochemicals have a crucial role in curbing with chronic diseases like cancer, diabetes and coronary heart disease [47]. Also, phytomedicine has long been used to cure neural diseases like mental disorders, including neurodegenerative diseases such as AD, PD and many other memory-related disorders. Currently, increasing evidence directs that neuroglia derived chronic infl ammatory responses play a compulsive role in the central nervous system. Phytomedicines that have anti-infl ammatory properties and its constituents are being ascertained to be a potent neuroprotector against various brain disorders [48,49].
There are few ADMET properties that are to be passed by a compound to act like a drug. One of them is BBB permeability. BBB has become the bottleneck in brain drug development as it is the single most important factor limiting the future growth of neurotherapeutics [50]. Especially in case of neural disorders, it is a most complex phenomenon that either a drug can cross BBB or not as it is reported in the previous literature that a drug can be more effective if it would not cross that barrier [51]. Conversely, in many brain pathologies like AD, it is necessary for a drug to be BBB permeant to enhance effi ciency [52,53]. So from the neuropathic literature it is cleared that a drug must be BBB permeant in case of cognitive disorders [54].  Other properties include Lipinski's rule of fi ve which determine either a compound can be orally used or not. This rule deals with the suitable number of hydrogen bonds of donor and acceptor, molecular weight and log P of the compound to assure its drug likeliness [55]. Furthermore, it was seen that the phytochemicals having property of BBB permeability also showed high GI absorption which is linked with epithelial cells.
Additionally, the phytochemicals showing optimum (high and moderate) solubility were only selected for analysis, which is known as an effective parameter in the drug discovery process [56].
Computer-aided protocols of drug discovery [57][58][59][60]  However, these phytochemicals should be further assessed by in vivo and in vitro analysis for the development of a potent phyto-drug against Parkinson' disease.