Identification of Fungal Bioactive Compounds Targeting MMP-9 for Endometriosis- An In silico Approach

Background: Endometriosis is a chronic inflammatory disease of the female reproductive system characterized by the presence of endometrial tissue outside the uterus that affects 5 to 10% of women of reproductive age, which is approximately 176 million women in the world. The women suffering from endometriosis have been reported to have high levels of matrix metalloproteinase (especially MMP-9) which regulates the inflammatory process. Thus, the aim of this study is to investigate the naturally available anti-inflammatory fungal compounds that can target the MMP-9 by various in silico approaches. Methodology: A wide variety of anti-inflammatory bioactive compounds were screened and five compounds were further selected based on ‘Lipinski’s rule of five’ using the PubChem database. The bioavailability, pharmacokinetics, ADMET properties and biological activity of these compounds were predicted computationally using databases such as SWISS-ADME, PubChem, pkCSM and PASS. The target 1L6J (Crystal structure of human matrix metalloproteinase MMP-9) structure was retrieved from the PDB database. Comparative analysis of the bioactive compounds Original Research Article Nabiya et al.; JPRI, 33(34B): 75-88, 2021; Article no.JPRI.70304 76 with the target was performed by AutoDock 4.2.6 and further visualisation of the target residues interacting with the compounds was performed using LigPlot v.2. 2. tool. Results: Based on the docking results, the compounds namely, Ergosterol peroxide, Lovastatin, Javanicin, Asperlin and Ergothioneine exhibited binding energy value of -10.25 kcal/mol, -8.4 kcal/mol, -7.64 kcal/mol, -7.07 kcal/mol and -6.19 kcal/mol respectively whereas Elagolix (control drug) exhibited binding energy value of -4.88 kcal/mol, thus, indicating that the selected bioactive compounds were seen to have better binding energy comparative to the control drug. Conclusion: Ergosterol peroxide derived from edible mushroom might act as a potential lead compound for designing a therapeutic drug for treating endometriosis and this compound can further be explored to evaluate its level of toxicity and efficacy in the wet laboratory studies by in vitro and in vivo methods.


INTRODUCTION
Endometriosis is a chronic inflammatory disease which occurs due to the presence of endometrial tissue outside the uterine cavity that affects 5 to 10% of women of reproductive age (between the ages of 15 to 49), which is approximately 176 million women in the world. The Endometriosis Society of India had estimated that 26 million Indian women are suffering from endometriosis.
According to the American Society for Reproductive Medicine (r-ASRM) classification [7], endometriosis can be categorized into four stages (Table 1) based upon the location, extent, depth of the endometriosis implants, severity of scar tissue and size of endometrial implants in the ovaries.
On the basis of the location of endometrial tissue, endometriosis can be classified [8]

Molecular Mechanism of Endometriosis
Though the exact cause of endometriosis remains unclear, several theories and molecular mechanisms have been presented to understand its development. Matrix Metalloproteinases (MMPs) is one such protein involved in the inflammatory process contributes to the progression of endometriosis matrix remodelling process such as reflux, adhesion, proteolysis, proliferation, angiogenesis, and scarring [9]. Among the different types of MMPs, MMP-9 also called as Gelatinase B (GELB) or 92 kDa gelatinase belong to the zinc-metalloproteinase family, which play an important role in tumour formation, angiogenesis-associated diseases and also is known to be involved in the degradation of the extracellular matrix. The MMP-9 levels are reported to vary with different types of endometriosis and between patients without endometriosis [10]. Therefore, MMP-9 is considered an important biomarker for endometriosis (Fig. 1).
Protein Tyrosine Kinase 7 (PTK7) has been known to activate the mitogen-activated protein kinase (MAPK) and NF-kappaB pathway which have been involved in the upregulation of MMP-9 as seen in the Fig. 1. MAP3K further activates ERK and JNK pathways which in turn activate the AP-1 complex components c-Fos and c-Jun. NF-κB and AP-1 bind to the corresponding binding sites and transactivates the MMP-9 which enhances invasive phenotype of the cells.

Current treatment options available in endometriosis
The treatment options available for endometriosis include progestin therapy, surgery and using medications such as gonadotropinreleasing hormone (Gn-RH) agonists and aromatase inhibitors. Women who are suffering from endometriosis usually undergo laparoscopic surgery to remove the endometrial tissue. However, there are cases where the endometriosis tissue returns even after surgery, recurrent lesions can occur within a few years [11]. Hence, because of these reasons pain medications are a preferred choice of treatment to reduce the pain and provide relief to women suffering from endometriosis. Elagolix (sold under the brand name Orilissa), a gonadotropinreleasing hormone (GnRH) antagonist was approved for medical use for endometriosis treatment in July 2018 [12] but later was discontinued due to side effects by 5 to 10% of women in clinical trials.

Role of bioactive compounds in endometriosis
Though endometriosis can be treated effectively with these drugs, most of the treatments are not suitable for long-term use due to their side effects [13]. Currently, research needs to be done to find a suitable drug in the treatment of endometriosis which can provide a relief from pain without possessing any side effects. Hence, the natural bioactive compounds can be explored for endometriosis as the bioactive compounds from natural sources are considered to be much safer and non-toxic when compared to the synthetic drugs.

Current Approach of In silico Study
The in silico methodologies have been a crucial part in drug development process as it is considered to be a preliminary step in screening the drug compound with least side effects that can aid in the treatment of endometriosis when compared to the conventional methods which involves more cost and time.
Fungal bioactive compounds were screened by literature review, out of which five bioactive compounds such as Ergosterol peroxide, Lovastatin, Javanicin, Asperlin and Ergothioneine were selected.

Preparation of Protein Structure
Protein structure of human matrix metalloproteinase MMP-9 (gelatinase B) with UniProt ID P14780 and PDB ID 1L6J having resolution of 2.5 Å, R-Value Free of 0.230 and R-Value Work of 0.187 was selected from the Protein Data Bank. The 3D structure of the MMP-9 was visualized by Biovia Discovery Studio Visualizer ( Table 2). The active sites were predicted by Computed Atlas of Surface Topography of Proteins (CAST-P) (http://sts.bioe.uic.edu/castp/index.html?3igg) onl ine tool. The heteroatoms (water molecules) were removed from the 1L6J structure. Before grid preparation, the polar hydrogen atoms and Kollman charge (which was -6.248) were added.

Ligand
Five bioactive compounds were selected based on literature review. PubChem database (https://pubchem.ncbi.nlm.nih.gov/) was used to extract the information such as PubChem ID, molecular formula and canonical SMILES. The molecular properties such as molecular weight, xlog P, Hydrogen Bond Donor, Hydrogen Bond Acceptor, Rotatable Bond Count, Topological Polar Surface Area were also predicted computationally using this database. The ligand structure was downloaded in 3D sdf format (.mol). By using the Open Babel software, the ligand file was converted from sdf (.mol) to PDB (.pdb) format and further saved for docking. The molecular properties were predicted computationally by this database. The ADMET, bioavailability and bioactivity of each compound was predicted computationally using the pkCSM (http://biosig.unimelb.edu.au/pkcsm/prediction), SWISSADME (http://www.swissadme.ch/) and Prediction of Activity Spectra for Substances (PASS)(http://www.pharmaexpert.ru/passonline/i ndex.php) tool respectively.

Molecular Docking and Visualization
Molecular docking is a structure-based drug design approach that identifies the essential amino acid interaction between the target and ligand. Docking of selected bioactive compounds with the target MMP-9 was carried out using AutoDock 4.2.6. The procedure for docking in AutoDock 4.2.6 consisted of six stages, namely protein preparation, ligand preparation, grid preparation, running the grid, docking file preparation, running AutoDock and analysing docking conformations. It consists of two main programs: autodock which performs the docking of the ligand to a set of grids describing the target and autogrid which pre-calculates these grids. The binding energy, hydrogen bond, Vander Waal's forces and intermolecular energy were noted. The docking interactions formed via the hydrogen bonds between the receptor and the compound can be analysed using LigPlot v2.2 in a 2D manner from standard pdb file input. It provides the information about the receptorligand interaction (hydrogen and hydrophobic interactions) and hydrogen bonds formed. Hydrogen bonds are indicated by dashed lines between the atoms involved, while hydrophobic contacts are represented by an arc with spikes radiating towards the ligand atoms they contact.

Selection of the ligand compounds based on literature review
During the clinical trial, women suffering from endometriosis were prescribed Elagolix which is a gonadotropin-releasing hormone (GnRH) antagonist [27], was approved for medical use in July 2018 [12], but serious adverse effects were observed such as appendicitis (0.3%), abdominal pain (0.2%), back pain (0.2%), and also other effects such as amenorrhea, decreased bone mineral density (BMD), changes in the blood lipid profile miscarriage, suicidality, and elevated liver enzymes. Because of this reason, Elagolix was discontinued by 5 to 10% of women in clinical trials and thus the natural bioactive compounds could be explored which is of natural origin with less side effects. This compound was able to suppress LPS inflammatory responses through inhibition of NF kappaB and C/EBPß transcriptional activity, and phosphorylation of MAPKs and of cyclooxygenase related with the expression of many inflammatory mediators [28] used to treat gynaecological cancer compound, Lovastatin derived from the same source Pleurotus ostreatus-oyster mushroom had shown to inhibit the mechanism of cell proliferation and angiogenesis in an experimental model for the development of endometriosis tissue [30]. In addition, it also showed the relevance in treatment of endometriosis Javanicin, an endophyte derived from species had shown to possess antibacterial activity and antifungal activity [32] fungi marine-derived from Aspergillus

80
Ergosterol peroxide derived from edible Pleurotus ostreatus (oyster mushroom) possess therapeutic properties such inflammatory and anticancer. This compound was able to suppress LPS-induced inflammatory responses through inhibition of NF-kappaB and C/EBPß transcriptional activity, and APKs and of cyclooxygenase related with the expression of [28]; also was used to treat gynaecological cancer [29]. Another derived from the same oyster mushroom had shown to inhibit the mechanism of cell proliferation and angiogenesis in an experimental model for the development of endometriosis-like . In addition, it also showed the clinical relevance in treatment of endometriosis [31]. Javanicin, an endophyte derived from Chloridium species had shown to possess antibacterial [32]. Asperlin, a Aspergillus species had shown to possess antifungal, anti inflammatory and anti-atherosclerotic activities [33]. High quantity of ergothioneine is present in different types of edible mushrooms and has been found to have role in cardiovascular disease [34], preeclampsia [35], vascular and neurological disorders.

Structure retrieval of the ligand compounds
The 2D structures of the fungal bioactive compounds and control drug-Elagolix (Fig. 2) were retrieved from the PubChem

Prediction of properties compounds
The PUBCHEM CID, molecular formula and Canonical SMILES of the selected bioactive compounds have been mentioned in the Table 3.

2D Structure of the Ligand Compounds Retrieved from PubChem (A) Ergosterol Peroxide, (B) Lovastatin, (C) Javanicin, (D) Asperlin, (E) Ergothioneine and (F) Elagolix
; Article no.JPRI.70304 had shown to possess antifungal, antiosclerotic activities ergothioneine is present in different types of edible mushrooms and has been found to have role in cardiovascular , vascular and

of the ligand
The 2D structures of the fungal bioactive Elagolix (Fig. 2) PubChem database.

Prediction of properties-ligand
The PUBCHEM CID, molecular formula and ILES of the selected bioactive compounds have been mentioned in the

Chemical properties of the ligand
The bioactive compounds for docking were selected on the basis of whether the compound followed the 'Lipinski rule of five' as depicted in the Fig. 3, which is considered as a very important criterion in evaluating the efficacy of the drug. Log P commonly used to measure the liphophilicity. The hydrogen bond donor and acceptor are an important criteria to determine the ligand's specificity towards the receptor Number of Rotatable Bonds (nrotb) is an important parameter to measure the molecular flexibility and oral bioavailability of drugs. An ideal compound should have nrotb less than 10; if it is less than 6, it means that the drug has good bioavailability. Topological Polar Surface Area (TPSA) is the sum of all polar atoms present in the molecule which ca determining the drugs ability to penetrate the

properties of the ligand
The bioactive compounds for docking were selected on the basis of whether the compound followed the 'Lipinski rule of five' as depicted in the Fig. 3, which is considered as a very important criterion in evaluating the efficacy of used to measure the The hydrogen bond donor and acceptor are an important criteria to determine the ligand's specificity towards the receptor. Number of Rotatable Bonds (nrotb) is an important parameter to measure the molecular and oral bioavailability of drugs. An ideal compound should have nrotb less than 10; if it is less than 6, it means that the drug has good bioavailability. Topological Polar Surface Area (TPSA) is the sum of all polar atoms present in the molecule which can help in determining the drugs ability to penetrate the cells. Molecules with TPSA greater than 140 are poor at penetrating the membrane. said to be active if there are not more than one violation seen in the Lipinski's rule.
All the compounds selected for the present study adhered to the Lipinski rule of five bioavailability value more than 0.55, TPSA was less than 140 Å², violation of only one rule maximum was observed in the bioactive compounds. Whereas, the control drug which had a molecular weight of 631.6 g/mol. Log P value of 6, Hydrogen Bond Acceptor (HBA) value was more than 10, bioavailability was low also had two violations seen in Lipinski's rule, indicating that it is not an ideal drug of choice and accentuates the search for novel drugs in treatment of endometriosis.

PubChem CID Molecular Formula
Canonical SMILES C 28  cells. Molecules with TPSA greater than 140 Å² are poor at penetrating the membrane. A drug is said to be active if there are not more than one violation seen in the Lipinski's rule.
ds selected for the present study Lipinski rule of five and showed bioavailability value more than 0.55, TPSA was ², violation of only one rule maximum was observed in the bioactive Whereas, the control drug Elagolix, which had a molecular weight of 631.6 g/mol. Log P value of 6, Hydrogen Bond Acceptor (HBA) value was more than 10, bioavailability was low also had two violations seen in Lipinski's rule, indicating that it is not an choice and accentuates the search for novel drugs in treatment of  Table 4.

ADME properties
The Absorption, Distribution, Metabolism, Excretion and Toxicity (ADMET) describes the pharmacokinetics and pharmacodynamics properties of the drug that helps to know the drug likeliness of the bioactive compound with medicinal properties and these properties were predicted using pkCSM (Table 5) [36].

Absorption
Parameters such Caco-2 permeability and human intestine absorption were tabulated.
Intestinal absorption percentage predicts the proportion of the compounds that were absorbed through the human intestine. If the absorbance is more than 80%, it means that the molecule is well absorbed while if the absorbance value of the molecule is less than 30%, it means that it is poorly absorbed. Most of the compounds showed higher absorption value than compared to the control drug and the compound, Asperlin showed 100% which means it can be highly absorbed. Caco-2 permeability has been used to predict the absorption of orally administered drugs. Most of the compounds showed higher Caco-2 permeability whereas control drug such as Elagolix showed -0.228cm/s.

Distribution
The distribution parameter consists of the volume of distribution (Vd). If the volume of distribution (Vd) value is below -0.15 log L/kg, it is considered low and considered to be high if Vd is more than 0.45 log L/kg. the Elagolix had the lowest Vd value whereas Ergosterol peroxide had the highest Vd value of 0.552 log L/kg.

Metabolism
The cytochrome CYP450 is an important enzyme present in the liver which is responsible for the metabolism of many drugs. There are two isoforms of this enzyme, namely, CYP2D6 and CYP3A4.

Excretion
Total clearance of the drugs refers to the bioavailability to determine the dosing rates to achieve steady state concentrations.

Toxicity
AMES test determines whether the bioactive compound is mutagenic or not. Asperlin was the only compound that had mutagenic properties.

Bioactivity prediction of biocompounds-PASS database
Pa (probability "to be active") helps in predicting the bioactivity of the compounds. The activities that were having Pa value above 0.7 were tabulated which indicates that the compound is highly active to perform the specific activity. The results based on PASS indicated that all the bioactive compounds were showing various activities which had Pa above 0.7, whereas the control drug-Elagolix exhibited no activity when Pa value was above 0.7 (Table 6).

Docking
Docking was performed with the Crystal structure of human matrix metalloproteinase MMP-9 (gelatinase B) [PDB 1L6J [37]] and bioactive compounds.
Molecular docking and comparative analysis of the selected fungal bioactive compounds and the control drug was performed using the software AutoDock 4.2.6. Prior to docking, the solvent molecules were removed from the structure of the target and the active sites residues in the target were retrieved using CASTp.
Each compound was docked individually and the results obtained from each docking information on the following parameters such as the Binding energy, Ligand efficiency, Intermolecular energy, Vander Waal's dissolved energy, Electrostatic energy and Torsional energy (Table 7).

Analysis of docking results and visualization
The selected bioactive compounds and control drug were individually docked against the MMP 9. All the selected bioactive compounds were shown to have binding energy more than the control drug-Elagolix, indicating that more research work is needed to study the efficacy of the drug and bring it to the clinical trial stage for the treatment of endometriosis. The selected bioactive compounds and control drug were individually docked against the MMP-9. All the selected bioactive compounds were shown to have binding energy more than the Elagolix, indicating that more study the efficacy of the drug and bring it to the clinical trial stage for
The hydrophobic interactions are the most common type of interaction seen in the protein ligand complex and are said to be stronger than hydrogen bond, Vander Waal's forces and intermolecular energy. The MMP residues interacting with the bioactive metabolites and the control drugs has been visualized ( Fig. 4)

Visualization by LigPlot + v.2.2 of MMP-9 with (A) Ergosterol Peroxide, (B) Lovastatin, (C) Javanicin, (D) Asperlin, (E) Ergothioneine and (F) Elagolix
; Article no.JPRI.70304 The bioactive compounds such as Ergosterol Lovastatin, Javanicin, Asperlin exhibited binding energy 8.4 kcal/mol, -7.64 -6.19 kcal/mol Elagolix (control exhibited binding energy value of 4.88 kcal/mol, which indicates that the selected compounds were seen to have better binding energy comparative to the control The hydrophobic interactions are the most common type of interaction seen in the proteinligand complex and are said to be stronger than drogen bond, Vander Waal's forces and intermolecular energy. The MMP-9 target residues interacting with the bioactive metabolites and the control drugs has been 9 with (A) Ergosterol Peroxide, (B) Lovastatin,

CONCLUSION AND FUTURE PERSPECTIVE
Based on the properties prediction, it can be concluded that all the selected bioactive compounds did not violate more than one Lipinski's rule, only the control drug was seen to have more than one violations. When computationally predicted using PASS database it was shown that the bioactive compounds exhibited several activities when the Pa value was above 0.7, while the control drug exhibited no activity. The bioavailability of the compounds was predicted by SWISSADME and it was seen that the bioavailability of control drug was very low, which indicated that the control drug has poor absorption where all the bioactive compounds showed bioavailability higher than 0.55.
The molecular docking was performed using AutoDock 4.2.6 and visualization was done using LigPlot v2.2. Based on the results Ergosterol peroxide, Lovastatin, Javanicin, Asperlin and Ergothioneine exhibited binding energy value of -10.25 kcal/mol, -8.4 kcal/mol, -7.64 kcal/mol, -7.07 kcal/mol and -6.19 kcal/mol respectively whereas Elagolix (control drug) exhibited binding energy value of -4.88 kcal/mol. Collectively, these results encourage further research of new drug from the fungal source which will effectively act against the MMP-9 receptor with the ultimate goal in the treatment of endometriosis and Ergosterol peroxide (sourceedible mushroom) can be further investigated for its role in the wet laboratory studies by in vitro and in vivo study.

DISCLAIMER
The products used for this research are commonly and predominantly use products in our area of research and country. There is absolutely no conflict of interest between the authors and producers of the products because we do not intend to use these products as an avenue for any litigation but for the advancement of knowledge. Also, the research was not funded by the producing company rather it was funded by personal efforts of the authors.

CONSENT
It is not applicable.

ETHICAL APPROVAL
It is not applicable.