Role of Persistent Organic Pollutants in Breast Cancer Progression and Identification of Estrogen Receptor Alpha Inhibitors Using In-Silico Mining and Drug-Drug Interaction Network Approaches

Simple Summary The role of persistent organic pollutants (POPs) in breast cancer progression and their bioaccumulation in adipose tissue has been reported. We used a computational approach to study molecular interactions of POPs with breast cancer proteins and identified natural and synthetic compounds to inhibit these interactions. Moreover, for comparative analysis, standard drugs and screened compounds were also docked against estrogen receptor alpha (ERα) and identification of the finest inhibitor was performed using in-silico mining and drug-drug interaction (DDI) network approaches. Based on scoring values, short-chained chlorinated paraffins demonstrated strong interactions with ERα compared to organo-chlorines and PCBs. Synthetic and natural compounds demonstrating strong associations with the active site of the ERα protein could be potential candidates to treat breast cancer specifically caused by POPs and other organic toxins and can be used as an alternative to standard drugs. Abstract The strong association between POPs and breast cancer in humans has been suggested in various epidemiological studies. However, the interaction of POPs with the ERα protein of breast cancer, and identification of natural and synthetic compounds to inhibit this interaction, is mysterious yet. Consequently, the present study aimed to explore the interaction between POPs and ERα using the molecular operating environment (MOE) tool and to identify natural and synthetic compounds to inhibit this association through a cluster-based approach. To validate whether our approach could distinguish between active and inactive compounds, a virtual screen (VS) was performed using actives (627 compounds) as positive control and decoys (20,818 compounds) as a negative dataset obtained from DUD-E. Comparatively, short-chain chlorinated paraffins (SCCPs), hexabromocyclododecane (HBCD), and perfluorooctanesulfonyl fluoride (PFOSF) depicted strong interactions with the ERα protein based on the lowest-scoring values of −31.946, −18.916, −17.581 kcal/mol, respectively. Out of 7856 retrieved natural and synthetic compounds, sixty were selected on modularity bases and subsequently docked with ERα. Based on the lowest-scoring values, ZINC08441573, ZINC00664754, ZINC00702695, ZINC00627464, and ZINC08440501 (synthetic compounds), and capsaicin, flavopiridol tectorgenin, and ellagic acid (natural compounds) showed incredible interactions with the active sites of ERα, even more convening and resilient than standard breast cancer drugs Tamoxifen, Arimidex and Letrozole. Our findings confirm the role of POPs in breast cancer progression and suggest that natural and synthetic compounds with high binding affinity could be more efficient and appropriate candidates to treat breast cancer after validation through in vitro and in vivo studies.

protein following the method of Rose et al. [22]. Protein Bank RCSB (https://www.rcsb.org/ (accessed on 21 November 2020)). was used to get the 3-D structure of the ERα (ERα/pdb id: 5W9D) protein of breast cancer ( Figure S1). The protein selection was entirely based on mutated genes and the MOE tool was used to prepare the protein file while removing water molecules and attached ligands while hydrogen atoms were added. Afterward, a discovery studio was used to visualize the protein structure.

Validation of Virtual Screening (VS) Protocol
To validate whether our approach can distinguish between active and inactive compounds, we have performed a virtual screen (VS) experiment using actives (627 ERα inhibitors, i.e. binders) as positive control and decoys (20,818 compounds, i.e. non-binders) as a negative dataset obtained from the database of Useful Decoys: Enhanced (DUD-E). All the dataset compounds were docked into the binding site of ERα (PDB ID: 5W9D). The ligand enzyme complexes with the lowest binding energy were analyzed by the MOE ligand interaction module. Finally, Discovery Studio Visualizer was used for the 3-D interaction plot.

Screening and Toxicity Detection of Pollutants
POPs, whose emissions and/or output can be eliminated, or at least reduced substantially, were screened from the list as demonstrated in the Stockholm Convention in 2001. The online server 'admetSAR' containing 27 predictive models [23] was used to check the toxicity of screened POPs, and all were lying under toxic classes I, II, and III.

Preparation of Ligand and Molecular Docking
PubChem offers free access to information and biological functions about chemical substances. The database contains chemical information from individual PubChem data providers and the integrated database contains a distinction between chemical structures and the database of substances [24]. PubChem (https://pubchem.ncbi.nlm.nih.gov/ (accessed on 12 June 2021)) was used to extract 3-D structures of screened ligands (POPs) while adopting the previously reported procedure [24]. Afterward, ligands were prepared using the molecular operating environment (MOE) tool.
The molecular docking (MD) technique for the identification and optimization of drug candidates was used to analyze and simulate molecular interactions between the ligand and targeted macromolecules as reported formerly [25]. The MOE tool was used to evaluate the mechanism of molecular interactions between ligands including POPs, approved drugs (positive control), progesterone and testosterone (negative control), and drug candidates (natural and synthetic compounds) with an ERα receptor protein, while Discovery Studio software (DS 4.1) was used to visualize the 3-D interactions following the method as reported earlier [26].

Collection and Mining of Natural and Synthetic Compounds
The ZINC database (zinc.docking.org/ (accessed on 12 July 2021)) was used for the collection of natural and synthetic compounds along with their chemical properties, including Zinc ID, molecular weight, hydrogen bond donors log p, polar dissociation, rotatable bonds, a-polar dissociation, and hydrogen bond acceptors. Lipinski's rule of five was applied to the collected drug dataset for mining the natural and synthetic compounds as described earlier [15].

Cluster Formation
Weka, a platform for clustering, association, pre-processing, regression, classification, and screening of data [27], was used for clustering of the drug dataset based on a k-means algorithm (k-mean) clustering system. According to the properties of drugs, this method tracks a modest and quick way to categorize a particular record "x 1 , x 2 , x 3 ... x n " to numbers of k clusters (k ≤ n), where k represents clusters and the row is denoted by n.

Drug-Drug Interaction (DDI) Network
Gephi, a primary platform for data analysis and the fastest graphical visualization of large networks [28], was used to create DDI networks from k-means clustering to find a strong connection between drug networks within each cluster. DDI networks were generated based on statistical parameters such as modularity (Ml), path lengths (PL), average degree (AD), average weighted degree (AWD), degree distribution (DD), and graph density (GD). Each network has borders E, vertical V, average path length L and node D, as well as network density and modularity classes. Most strongly-associated natural and synthetic compounds were docked against the targeted ERα protein to identify scoring values/binding energies. Standard breast cancer drugs i.e., Tamoxifen, Arimidex, Letrozole were used as a control to compare the scoring value of screened drugs.

Validation of VS and Reliability of MD
To validate whether our approach can distinguish between active and inactive compounds, a virtual screen (VS) experiment was performed using actives (627 ERα inhibitors i.e. binders) as positive control and decoys (20,818 compounds i.e. non-binders) as negative dataset obtained from the database of Useful Decoys: Enhanced (DUD-E) [29]. All the dataset compounds were docked into the binding site of ERα (PDB ID: 5W9D). Computed binding energy values of the active compound dataset were in the range of 28.6573 to −355.9801 kcal/mol and chemical structures of the most active binders are given in Figure S2. The binding energy values of the decoy set were in the range of −1.0988 to −3.0371 kcal/mol. Therefore, these findings suggest that our VS protocol can distinguish between active and inactive compounds.
The reliability of docking accuracy was assessed in two steps. In the first step, redocking of the native ligand was performed (Table S1). In the second step, a cross-docking experiment was carried out (Table S2). Three-dimensional structures of five estrogen receptor-alpha (PDB accession codes = 1A52, 3ERT, 1GWQ, 1UOM and 5W9D) were retrieved from PDB. In self-docking experiments, all the native ligands were extracted from receptors and root means square deviation (RMSD) was calculated for each re-docked and experimental native ligand [30]. Docking was carried out using the Triangle matcher algorithm (placement stage) and scored by the London dG scoring function [31]. Subsequently, best-scored poses were submitted to a rigid receptor protocol (refinement stage). Throughout the validation of docking protocol, the best performance in terms of computed RMSD value, conformation, binding energy, position, and pose (orientation) was obtained with the Triangle matcher London dG scoring function. The final score was calculated with the ASE scoring function. The whole validation process is presented in Supporting Information (Tables S1 and S2).

Interactions between POPs and ERα
Carcinogenesis is not a simple process; it involves initiation, promotion, and progression [32] of malignancy. The ERα gene is more likely to be involved in cell proliferation and is considered the most popular target to treat breast cancer. As per previous reports, POPs may not directly cause cancer, but act as co-carcinogenic agents [33]. It has been reported that organo-chlorines such as dichlorodiphenyltrichloroethane (DDT), hexachlorocyclohexane (HCH), aldrin, dieldrin, and polychlorinated biphenyls (PCBs) have the potential to stimulate breast cancer cell proliferation through the estrogenic pathway [34]. The association between organochlorine and PCBs exposure and risk of breast cancer has been reported. In the present study, substantial data retrieved from the molecular operating environment (MOE) tool, including scoring values, root-mean-square distance, and (RMSD) values, are given in Table 1. As reported earlier [35], the more negative the free binding affinity/scoring values are, the better the bond stability it forms between the ligand and the receptor protein [35]. In this context, strong relations between POPs and ERα protein of breast cancer were assessed based on scoring values ranging from −31.94 to −8.650 kcal/mol. Out of 27 POPs, short-chained chlorinated paraffins (SCCPs), hexabromocyclododecane (HBCD), and perfluorooctanesulfonyl fluoride (PFOSF) showed the strongest molecular interactions with the ERα protein based on their lowest-scoring values of −31.94, −18.91, −17.58 kcal/mol, respectively (Table 1). These findings revealed that SCCPs, HBCD, and PFOSF could be potentially involved in breast cancer prevailing compared to PCBs and organochlorine as reported in previous studies [34]. These POPs are more suspected to cause breast cancer and are widely used pesticides in developing countries of Asia due to their low cost and utility against various pests. The key nonoccupational exposure routes of these high-potency contaminants include ingestion, both directly and by tainted food, and dermal interaction with the substance [36]. Above mentioned results revealed that based on scoring values, short-chain chlorinated paraffin (SCCPs) have the strongest interactions with the ERα protein ( Figure 1A). SCCPs are commonly used in metalworking fluids, paints, sealants, adhesives, leather manufacturing chemicals, plastics, rubber, and as a plastic agent and flame retardant [37]. In addition, these pollutants have also been isolated from kidneys, adipose tissue, and breast milk of Inuit women [38]. As previously reported, SCCPs (C10-C13) belong to the third class of carcinogens and are highly toxic to aquatic organisms [39]. Moreover, previous reports have provided convincing evidence on the disruptive effect of SCCPs on thyroid hormones and glucocorticoids [40], as well as their role in the regulation of different signaling pathways and physiological mechanisms [41]. However, there are few studies on the health effects of SCCPs in humans, especially their contribution to endocrine disruption. In this context, the aforementioned results revealed the strong association of SCCPs with the ERα protein of breast cancer based on least binding energy or scoring value (−31.946 kcal/mol). This indicates that the strong binding capacity of SCCPs with ERα protein may be involved in the spread of breast cancer in women. Hexabromocyclododecane (HBCD) has also demonstrated strong interactions with the ERα protein (Figure 1B). Therefore, HBCD could also be one of the main contributors to breast cancer. Above mentioned results revealed that based on scoring values, short-chain chlorinated paraffin (SCCPs) have the strongest interactions with the ERα protein ( Figure 1A). SCCPs are commonly used in metalworking fluids, paints, sealants, adhesives, leather manufacturing chemicals, plastics, rubber, and as a plastic agent and flame retardant [37]. In addition, these pollutants have also been isolated from kidneys, adipose tissue, and breast milk of Inuit women [38]. As previously reported, SCCPs (C10-C13) belong to the third class of carcinogens and are highly toxic to aquatic organisms [39]. Moreover, previous reports have provided convincing evidence on the disruptive effect of SCCPs on thyroid hormones and glucocorticoids [40], as well as their role in the regulation of different signaling pathways and physiological mechanisms [41]. However, there are few studies on the health effects of SCCPs in humans, especially their contribution to endocrine disruption. In this context, the aforementioned results revealed the strong association of SCCPs with the ERα protein of breast cancer based on least binding energy or scoring value (−31.946 kcal/mol). This indicates that the strong binding capacity of SCCPs with ERα protein may be involved in the spread of breast cancer in women. Hexabromocyclododecane (HBCD) has also demonstrated strong interactions with the ERα protein (Figure 1B). Therefore, HBCD could also be one of the main contributors to breast cancer. Above mentioned results revealed that based on scoring values, short-chain chlorinated paraffin (SCCPs) have the strongest interactions with the ERα protein ( Figure 1A). SCCPs are commonly used in metalworking fluids, paints, sealants, adhesives, leather manufacturing chemicals, plastics, rubber, and as a plastic agent and flame retardant [37]. In addition, these pollutants have also been isolated from kidneys, adipose tissue, and breast milk of Inuit women [38]. As previously reported, SCCPs (C10-C13) belong to the third class of carcinogens and are highly toxic to aquatic organisms [39]. Moreover, previous reports have provided convincing evidence on the disruptive effect of SCCPs on thyroid hormones and glucocorticoids [40], as well as their role in the regulation of different signaling pathways and physiological mechanisms [41]. However, there are few studies on the health effects of SCCPs in humans, especially their contribution to endocrine disruption. In this context, the aforementioned results revealed the strong association of SCCPs with the ERα protein of breast cancer based on least binding energy or scoring value (−31.946 kcal/mol). This indicates that the strong binding capacity of SCCPs with ERα protein may be involved in the spread of breast cancer in women. Hexabromocyclododecane (HBCD) has also demonstrated strong interactions with the ERα protein (Figure 1B). Therefore, HBCD could also be one of the main contributors to breast cancer. and by tainted food, and dermal interaction with the substance [36]. Above mentioned results revealed that based on scoring values, short-chain chlorinated paraffin (SCCPs) have the strongest interactions with the ERα protein ( Figure 1A). SCCPs are commonly used in metalworking fluids, paints, sealants, adhesives, leather manufacturing chemicals, plastics, rubber, and as a plastic agent and flame retardant [37]. In addition, these pollutants have also been isolated from kidneys, adipose tissue, and breast milk of Inuit women [38]. As previously reported, SCCPs (C10-C13) belong to the third class of carcinogens and are highly toxic to aquatic organisms [39]. Moreover, previous reports have provided convincing evidence on the disruptive effect of SCCPs on thyroid hormones and glucocorticoids [40], as well as their role in the regulation of different signaling pathways and physiological mechanisms [41]. However, there are few studies on the health effects of SCCPs in humans, especially their contribution to endocrine disruption. In this context, the aforementioned results revealed the strong association of SCCPs with the ERα protein of breast cancer based on least binding energy or scoring value (−31.946 kcal/mol). This indicates that the strong binding capacity of SCCPs with ERα protein may be involved in the spread of breast cancer in women. Hexabromocyclododecane (HBCD) has also demonstrated strong interactions with the ERα protein (Figure 1B). Therefore, HBCD could also be one of the main contributors to breast cancer. and by tainted food, and dermal interaction with the substance [36]. Above mentioned results revealed that based on scoring values, short-chain chlorinated paraffin (SCCPs) have the strongest interactions with the ERα protein ( Figure 1A). SCCPs are commonly used in metalworking fluids, paints, sealants, adhesives, leather manufacturing chemicals, plastics, rubber, and as a plastic agent and flame retardant [37]. In addition, these pollutants have also been isolated from kidneys, adipose tissue, and breast milk of Inuit women [38]. As previously reported, SCCPs (C10-C13) belong to the third class of carcinogens and are highly toxic to aquatic organisms [39]. Moreover, previous reports have provided convincing evidence on the disruptive effect of SCCPs on thyroid hormones and glucocorticoids [40], as well as their role in the regulation of different signaling pathways and physiological mechanisms [41]. However, there are few studies on the health effects of SCCPs in humans, especially their contribution to endocrine disruption. In this context, the aforementioned results revealed the strong association of SCCPs with the ERα protein of breast cancer based on least binding energy or scoring value (−31.946 kcal/mol). This indicates that the strong binding capacity of SCCPs with ERα protein may be involved in the spread of breast cancer in women. Hexabromocyclododecane (HBCD) has also demonstrated strong interactions with the ERα protein (Figure 1B). Therefore, HBCD could also be one of the main contributors to breast cancer. and by tainted food, and dermal interaction with the substance [36]. Above mentioned results revealed that based on scoring values, short-chain chlorinated paraffin (SCCPs) have the strongest interactions with the ERα protein ( Figure 1A). SCCPs are commonly used in metalworking fluids, paints, sealants, adhesives, leather manufacturing chemicals, plastics, rubber, and as a plastic agent and flame retardant [37]. In addition, these pollutants have also been isolated from kidneys, adipose tissue, and breast milk of Inuit women [38]. As previously reported, SCCPs (C10-C13) belong to the third class of carcinogens and are highly toxic to aquatic organisms [39]. Moreover, previous reports have provided convincing evidence on the disruptive effect of SCCPs on thyroid hormones and glucocorticoids [40], as well as their role in the regulation of different signaling pathways and physiological mechanisms [41]. However, there are few studies on the health effects of SCCPs in humans, especially their contribution to endocrine disruption. In this context, the aforementioned results revealed the strong association of SCCPs with the ERα protein of breast cancer based on least binding energy or scoring value (−31.946 kcal/mol). This indicates that the strong binding capacity of SCCPs with ERα protein may be involved in the spread of breast cancer in women. Hexabromocyclododecane (HBCD) has also demonstrated strong interactions with the ERα protein (Figure 1B). Therefore, HBCD could also be one of the main contributors to breast cancer. and by tainted food, and dermal interaction with the substance [36]. Above mentioned results revealed that based on scoring values, short-chain chlorinated paraffin (SCCPs) have the strongest interactions with the ERα protein ( Figure 1A). SCCPs are commonly used in metalworking fluids, paints, sealants, adhesives, leather manufacturing chemicals, plastics, rubber, and as a plastic agent and flame retardant [37]. In addition, these pollutants have also been isolated from kidneys, adipose tissue, and breast milk of Inuit women [38]. As previously reported, SCCPs (C10-C13) belong to the third class of carcinogens and are highly toxic to aquatic organisms [39]. Moreover, previous reports have provided convincing evidence on the disruptive effect of SCCPs on thyroid hormones and glucocorticoids [40], as well as their role in the regulation of different signaling pathways and physiological mechanisms [41]. However, there are few studies on the health effects of SCCPs in humans, especially their contribution to endocrine disruption. In this context, the aforementioned results revealed the strong association of SCCPs with the ERα protein of breast cancer based on least binding energy or scoring value (−31.946 kcal/mol). This indicates that the strong binding capacity of SCCPs with ERα protein may be involved in the spread of breast cancer in women. Hexabromocyclododecane (HBCD) has also demonstrated strong interactions with the ERα protein (Figure 1B). Therefore, HBCD could also be one of the main contributors to breast cancer. These POPs are extensively used in flame retardants, as a neurotoxin, and in xenobiotic chemicals. HBCD acts as a nuclear receptor agonist, is hepatotoxic, and is an endocrine disruptor that induces developmental neurotoxicity in animals [42]. HBCD concentration in human breast milk, blood serum, and the umbilical cord has already been investigated. The concentrations in human breast milk raise questions about possible lactation and prenatal uptake during important developmental stages of the fetus [43]. Likewise, several fluorinated compounds, specifically perfluorooctanesulfonyl fluoride (PFOSF), also showed strong interactions with the ERα protein, based on a low scoring value (−17.58 kcal/mol), which allows it to easily form a stable complex ( Figure 1C). These compounds are used as impregnating and grading agents and corrosion inhibitors, like insecticides and flame retardants, in cosmetics, paper coatings, and surfactants. The possible association of PFOSF with hormone disorders, genotoxic potential, and tumor for- These POPs are extensively used in flame retardants, as a neurotoxin, and in xenobiotic chemicals. HBCD acts as a nuclear receptor agonist, is hepatotoxic, and is an endocrine disruptor that induces developmental neurotoxicity in animals [42]. HBCD concentration in human breast milk, blood serum, and the umbilical cord has already been investigated. The concentrations in human breast milk raise questions about possible lactation and prenatal uptake during important developmental stages of the fetus [43]. Likewise, several fluorinated compounds, specifically perfluorooctanesulfonyl fluoride (PFOSF), also showed strong interactions with the ERα protein, based on a low scoring value (−17.58 kcal/mol), which allows it to easily form a stable complex ( Figure 1C). These compounds are used as impregnating and grading agents and corrosion inhibitors, like insecticides and flame retardants, in cosmetics, paper coatings, and surfactants. The possible association of PFOSF with hormone disorders, genotoxic potential, and tumor for- These POPs are extensively used in flame retardants, as a neurotoxin, and in xenobiotic chemicals. HBCD acts as a nuclear receptor agonist, is hepatotoxic, and is an endocrine disruptor that induces developmental neurotoxicity in animals [42]. HBCD concentration in human breast milk, blood serum, and the umbilical cord has already been investigated. The concentrations in human breast milk raise questions about possible lactation and prenatal uptake during important developmental stages of the fetus [43]. Likewise, several fluorinated compounds, specifically perfluorooctanesulfonyl fluoride (PFOSF), also showed strong interactions with the ERα protein, based on a low scoring value (−17.58 kcal/mol), which allows it to easily form a stable complex ( Figure 1C). These compounds are used as impregnating and grading agents and corrosion inhibitors, like insecticides and flame retardants, in cosmetics, paper coatings, and surfactants. The possible association of PFOSF with hormone disorders, genotoxic potential, and tumor for- These POPs are extensively used in flame retardants, as a neurotoxin, and in xenobiotic chemicals. HBCD acts as a nuclear receptor agonist, is hepatotoxic, and is an endocrine disruptor that induces developmental neurotoxicity in animals [42]. HBCD concentration in human breast milk, blood serum, and the umbilical cord has already been investigated. The concentrations in human breast milk raise questions about possible lactation and prenatal uptake during important developmental stages of the fetus [43]. Likewise, several fluorinated compounds, specifically perfluorooctanesulfonyl fluoride (PFOSF), also showed strong interactions with the ERα protein, based on a low scoring value (−17.58 kcal/mol), which allows it to easily form a stable complex ( Figure 1C). These compounds are used as impregnating and grading agents and corrosion inhibitors, like insecticides and flame retardants, in cosmetics, paper coatings, and surfactants. The possible association of PFOSF with hormone disorders, genotoxic potential, and tumor for- These POPs are extensively used in flame retardants, as a neurotoxin, and in xenobiotic chemicals. HBCD acts as a nuclear receptor agonist, is hepatotoxic, and is an endocrine disruptor that induces developmental neurotoxicity in animals [42]. HBCD concentration in human breast milk, blood serum, and the umbilical cord has already been investigated. The concentrations in human breast milk raise questions about possible lactation and prenatal uptake during important developmental stages of the fetus [43]. Likewise, several fluorinated compounds, specifically perfluorooctanesulfonyl fluoride (PFOSF), also showed strong interactions with the ERα protein, based on a low scoring value (−17.58 kcal/mol), which allows it to easily form a stable complex ( Figure 1C). These compounds are used as impregnating and grading agents and corrosion inhibitors, like insecticides and flame retardants, in cosmetics, paper coatings, and surfactants. The possible association of PFOSF with hormone disorders, genotoxic potential, and tumor for- These POPs are extensively used in flame retardants, as a neurotoxin, and in xenobiotic chemicals. HBCD acts as a nuclear receptor agonist, is hepatotoxic, and is an endocrine disruptor that induces developmental neurotoxicity in animals [42]. HBCD concentration in human breast milk, blood serum, and the umbilical cord has already been investigated. The concentrations in human breast milk raise questions about possible lactation and prenatal uptake during important developmental stages of the fetus [43]. Likewise, several fluorinated compounds, specifically perfluorooctanesulfonyl fluoride (PFOSF), also showed strong interactions with the ERα protein, based on a low scoring value (−17.58 kcal/mol), which allows it to easily form a stable complex ( Figure 1C). These compounds are used as impregnating and grading agents and corrosion inhibitors, like insecticides and flame retardants, in cosmetics, paper coatings, and surfactants. The possible association of PFOSF with hormone disorders, genotoxic potential, and tumor for- These POPs are extensively used in flame retardants, as a neurotoxin, and in xenobiotic chemicals. HBCD acts as a nuclear receptor agonist, is hepatotoxic, and is an endocrine disruptor that induces developmental neurotoxicity in animals [42]. HBCD concentration in human breast milk, blood serum, and the umbilical cord has already been investigated. The concentrations in human breast milk raise questions about possible lactation and prenatal uptake during important developmental stages of the fetus [43]. Likewise, several fluorinated compounds, specifically perfluorooctanesulfonyl fluoride (PFOSF), also showed strong interactions with the ERα protein, based on a low scoring value (−17.58 kcal/mol), which allows it to easily form a stable complex ( Figure 1C). These compounds are used as impregnating and grading agents and corrosion inhibitors, like insecticides and flame retardants, in cosmetics, paper coatings, and surfactants. The possible association of PFOSF with hormone disorders, genotoxic potential, and tumor for- These POPs are extensively used in flame retardants, as a neurotoxin, and in xenobiotic chemicals. HBCD acts as a nuclear receptor agonist, is hepatotoxic, and is an endocrine disruptor that induces developmental neurotoxicity in animals [42]. HBCD concentration in human breast milk, blood serum, and the umbilical cord has already been investigated. The concentrations in human breast milk raise questions about possible lactation and prenatal uptake during important developmental stages of the fetus [43]. Likewise, several fluorinated compounds, specifically perfluorooctanesulfonyl fluoride (PFOSF), also showed strong interactions with the ERα protein, based on a low scoring value (−17.58 kcal/mol), which allows it to easily form a stable complex ( Figure 1C). These compounds are used as impregnating and grading agents and corrosion inhibitors, like insecticides and flame retardants, in cosmetics, paper coatings, and surfactants. The possible association of PFOSF with hormone disorders, genotoxic potential, and tumor for- These POPs are extensively used in flame retardants, as a neurotoxin, and in xenobiotic chemicals. HBCD acts as a nuclear receptor agonist, is hepatotoxic, and is an endocrine disruptor that induces developmental neurotoxicity in animals [42]. HBCD concentration in human breast milk, blood serum, and the umbilical cord has already been investigated. The concentrations in human breast milk raise questions about possible lactation and prenatal uptake during important developmental stages of the fetus [43]. Likewise, several fluorinated compounds, specifically perfluorooctanesulfonyl fluoride (PFOSF), also showed strong interactions with the ERα protein, based on a low scoring value (−17.58 kcal/mol), which allows it to easily form a stable complex ( Figure 1C). These compounds are used as impregnating and grading agents and corrosion inhibitors, like insecticides and flame retardants, in cosmetics, paper coatings, and surfactants. The possible association of PFOSF with hormone disorders, genotoxic potential, and tumor for- Above mentioned results revealed that based on scoring values, short-chain chlorinated paraffin (SCCPs) have the strongest interactions with the ERα protein ( Figure 1A). SCCPs are commonly used in metalworking fluids, paints, sealants, adhesives, leather manufacturing chemicals, plastics, rubber, and as a plastic agent and flame retardant [37]. In addition, these pollutants have also been isolated from kidneys, adipose tissue, and breast milk of Inuit women [38]. As previously reported, SCCPs (C10-C13) belong to the third class of carcinogens and are highly toxic to aquatic organisms [39]. Moreover, previous reports have provided convincing evidence on the disruptive effect of SCCPs on thyroid hormones and glucocorticoids [40], as well as their role in the regulation of different signaling pathways and physiological mechanisms [41]. However, there are few studies on the health effects of SCCPs in humans, especially their contribution to endocrine disruption. In this context, the aforementioned results revealed the strong association of SCCPs with the ERα protein of breast cancer based on least binding energy or scoring value (−31.946 kcal/mol). This indicates that the strong binding capacity of SCCPs with ERα protein may be involved in the spread of breast cancer in women. Hexabromocyclododecane (HBCD) has also demonstrated strong interactions with the ERα protein ( Figure 1B). Therefore, HBCD could also be one of the main contributors to breast cancer.
These POPs are extensively used in flame retardants, as a neurotoxin, and in xenobiotic chemicals. HBCD acts as a nuclear receptor agonist, is hepatotoxic, and is an endocrine disruptor that induces developmental neurotoxicity in animals [42]. HBCD concentration in human breast milk, blood serum, and the umbilical cord has already been investigated. The concentrations in human breast milk raise questions about possible lactation and prenatal uptake during important developmental stages of the fetus [43]. Likewise, several fluorinated compounds, specifically perfluorooctanesulfonyl fluoride (PFOSF), also showed strong interactions with the ERα protein, based on a low scoring value (−17.58 kcal/mol), which allows it to easily form a stable complex ( Figure 1C). These compounds are used as impregnating and grading agents and corrosion inhibitors, like insecticides and flame retardants, in cosmetics, paper coatings, and surfactants. The possible association of PFOSF with hormone disorders, genotoxic potential, and tumor formation in rodents has been suggested previously [44]. Hormones can be indirectly imitated by endocrine disorders or hormone disorders. Strong associations of SCCPs, HBCD, and PFOSF with the ERα protein revealed their possible role in breast cancer; therefore the role of these POPs should be further investigated in detail.
Biology 2021, 10, x FOR PEER REVIEW 8 of 16 mation in rodents has been suggested previously [44]. Hormones can be indirectly imitated by endocrine disorders or hormone disorders. Strong associations of SCCPs, HBCD, and PFOSF with the ERα protein revealed their possible role in breast cancer; therefore the role of these POPs should be further investigated in detail.

Natural and Synthetic Compounds Collection, Mining, and Clustering
Approximately 7856 natural and synthetic compounds were collected with their structures and properties using the ZINC database (zinc.docking.org/ (accessed on 12 July 2020)). While applying the Lipinski rule of five on the drug dataset, 2390 compounds were chosen for further processing and the rest were discarded after mining. Afterward, 12 clusters were generated for natural and synthetic compounds based on the k-means clustering system using the Weka tool. The use of k-means clustering avoids the repetition of compounds, so natural and synthetic compounds exhibiting similar properties were placed in one group. Clusters possessing similar properties, including molecular weight, hydrogen bond donors log P, polar dissociation, rotatable bonds, a-polar dissociation, and hydrogen bond acceptors (HBA) are shown in a plot matrix with their attributes utilizing different color representations ( Figure S3).

DDI Network
In total, 12 networks were generated using the k-means clustering algorithm and Gephi tool ( Figure S4) based on statistical parameters as mentioned in Table 2. A strongly interacted network as illustrated in Figure 2 was generated from 457 compounds having

Natural and Synthetic Compounds Collection, Mining, and Clustering
Approximately 7856 natural and synthetic compounds were collected with their structures and properties using the ZINC database (zinc.docking.org/ (accessed on 12 July 2020)). While applying the Lipinski rule of five on the drug dataset, 2390 compounds were chosen for further processing and the rest were discarded after mining. Afterward, 12 clusters were generated for natural and synthetic compounds based on the k-means clustering system using the Weka tool. The use of k-means clustering avoids the repetition of compounds, so natural and synthetic compounds exhibiting similar properties were placed in one group. Clusters possessing similar properties, including molecular weight, hydrogen bond donors log P, polar dissociation, rotatable bonds, a-polar dissociation, and hydrogen bond acceptors (HBA) are shown in a plot matrix with their attributes utilizing different color representations ( Figure S3).

DDI Network
In total, 12 networks were generated using the k-means clustering algorithm and Gephi tool ( Figure S4) based on statistical parameters as mentioned in Table 2. A strongly Biology 2021, 10, 681 9 of 16 interacted network as illustrated in Figure 2 was generated from 457 compounds having higher modularity class in 12 networks based on the aforementioned parameters. Repulsion strength was set to 10,000, and Force Atlas and Fruchterman rein gold layouts were used to display and visualize the networks. higher modularity class in 12 networks based on the aforementioned parameters. Repulsion strength was set to 10,000, and Force Atlas and Fruchterman rein gold layouts were used to display and visualize the networks. Nodes and edges in each network represent compounds and interactions between them, respectively. Darker color and larger size nodes show the strength of drug IDs/ZINC IDs. As shown in Table 2  Nodes and edges in each network represent compounds and interactions between them, respectively. Darker color and larger size nodes show the strength of drug IDs/ZINC IDs. As shown in Table 2 (Table 2).

Validation of Natural and Synthetic Compounds
ERα, the most common and effective target for breast cancer treatment, was docked against the screened natural and synthetic compounds [35]. For docking, the active site of the ERα protein was identified using standard drugs used to treat breast cancer i.e., Tamoxifen, Arimidex, and Letrozole as a positive control group, while progesterone and testosterone were utilized as a negative control group. As shown in Table 3, scoring values of Tamoxifen, Arimidex and Letrozole ranged from −31.26 to −20.97 kcal/mol. Tamoxifen showed pi-sulfur and pi-alkyl interactions; Arimidex also showed pi-sulfur and pi-alkyl interactions, while Letrozole showed conventional hydrogen bond and pi-sulfur interactions with the active site of ERα, as shown in Figure 3a-c.

Validation of Natural and Synthetic Compounds
ERα, the most common and effective target for breast cancer treatment, was docked against the screened natural and synthetic compounds [35]. For docking, the active site of the ERα protein was identified using standard drugs used to treat breast cancer i.e., Tamoxifen, Arimidex, and Letrozole as a positive control group, while progesterone and testosterone were utilized as a negative control group. As shown in Table 3, scoring values of Tamoxifen, Arimidex and Letrozole ranged from −31.26 to −20.97 kcal/mol. Tamoxifen showed pi-sulfur and pi-alkyl interactions; Arimidex also showed pi-sulfur and pi-alkyl interactions, while Letrozole showed conventional hydrogen bond and pi-sulfur interactions with the active site of ERα, as shown in Figure 3a-c.

Validation of Natural and Synthetic Compounds
ERα, the most common and effective target for breast cancer treatment, was docked against the screened natural and synthetic compounds [35]. For docking, the active site of the ERα protein was identified using standard drugs used to treat breast cancer i.e., Tamoxifen, Arimidex, and Letrozole as a positive control group, while progesterone and testosterone were utilized as a negative control group. As shown in Table 3, scoring values of Tamoxifen, Arimidex and Letrozole ranged from −31.26 to −20.97 kcal/mol. Tamoxifen showed pi-sulfur and pi-alkyl interactions; Arimidex also showed pi-sulfur and pi-alkyl interactions, while Letrozole showed conventional hydrogen bond and pi-sulfur interactions with the active site of ERα, as shown in Figure 3a-c.

Validation of Natural and Synthetic Compounds
ERα, the most common and effective target for breast cancer treatment, was docked against the screened natural and synthetic compounds [35]. For docking, the active site of the ERα protein was identified using standard drugs used to treat breast cancer i.e., Tamoxifen, Arimidex, and Letrozole as a positive control group, while progesterone and testosterone were utilized as a negative control group. As shown in Table 3, scoring values of Tamoxifen, Arimidex and Letrozole ranged from −31.26 to −20.97 kcal/mol. Tamoxifen showed pi-sulfur and pi-alkyl interactions; Arimidex also showed pi-sulfur and pi-alkyl interactions, while Letrozole showed conventional hydrogen bond and pi-sulfur interactions with the active site of ERα, as shown in Figure 3a-c.

Validation of Natural and Synthetic Compounds
ERα, the most common and effective target for breast cancer treatment, was docked against the screened natural and synthetic compounds [35]. For docking, the active site of the ERα protein was identified using standard drugs used to treat breast cancer i.e., Tamoxifen, Arimidex, and Letrozole as a positive control group, while progesterone and testosterone were utilized as a negative control group. As shown in Table 3, scoring values of Tamoxifen, Arimidex and Letrozole ranged from −31.26 to −20.97 kcal/mol. Tamoxifen showed pi-sulfur and pi-alkyl interactions; Arimidex also showed pi-sulfur and pi-alkyl interactions, while Letrozole showed conventional hydrogen bond and pi-sulfur interactions with the active site of ERα, as shown in Figure 3a-c.

Validation of Natural and Synthetic Compounds
ERα, the most common and effective target for breast cancer treatment, was docked against the screened natural and synthetic compounds [35]. For docking, the active site of the ERα protein was identified using standard drugs used to treat breast cancer i.e., Tamoxifen, Arimidex, and Letrozole as a positive control group, while progesterone and testosterone were utilized as a negative control group. As shown in Table 3, scoring values of Tamoxifen, Arimidex and Letrozole ranged from −31.26 to −20.97 kcal/mol. Tamoxifen showed pi-sulfur and pi-alkyl interactions; Arimidex also showed pi-sulfur and pi-alkyl interactions, while Letrozole showed conventional hydrogen bond and pi-sulfur interactions with the active site of ERα, as shown in Figure 3a-c.

Validation of Natural and Synthetic Compounds
ERα, the most common and effective target for breast cancer treatment, was docked against the screened natural and synthetic compounds [35]. For docking, the active site of the ERα protein was identified using standard drugs used to treat breast cancer i.e., Tamoxifen, Arimidex, and Letrozole as a positive control group, while progesterone and testosterone were utilized as a negative control group. As shown in Table 3, scoring values of Tamoxifen, Arimidex and Letrozole ranged from −31.26 to −20.97 kcal/mol. Tamoxifen showed pi-sulfur and pi-alkyl interactions; Arimidex also showed pi-sulfur and pi-alkyl interactions, while Letrozole showed conventional hydrogen bond and pi-sulfur interactions with the active site of ERα, as shown in Figure 3a-c.

Validation of Natural and Synthetic Compounds
ERα, the most common and effective target for breast cancer treatment, was docked against the screened natural and synthetic compounds [35]. For docking, the active site of the ERα protein was identified using standard drugs used to treat breast cancer i.e., Tamoxifen, Arimidex, and Letrozole as a positive control group, while progesterone and testosterone were utilized as a negative control group. As shown in Table 3, scoring values of Tamoxifen, Arimidex and Letrozole ranged from −31.26 to −20.97 kcal/mol. Tamoxifen showed pi-sulfur and pi-alkyl interactions; Arimidex also showed pi-sulfur and pi-alkyl interactions, while Letrozole showed conventional hydrogen bond and pi-sulfur interactions with the active site of ERα, as shown in Figure 3a-c.

Validation of Natural and Synthetic Compounds
ERα, the most common and effective target for breast cancer treatment, was docked against the screened natural and synthetic compounds [35]. For docking, the active site of the ERα protein was identified using standard drugs used to treat breast cancer i.e., Tamoxifen, Arimidex, and Letrozole as a positive control group, while progesterone and testosterone were utilized as a negative control group. As shown in Table 3, scoring values of Tamoxifen, Arimidex and Letrozole ranged from −31.26 to −20.97 kcal/mol. Tamoxifen showed pi-sulfur and pi-alkyl interactions; Arimidex also showed pi-sulfur and pi-alkyl interactions, while Letrozole showed conventional hydrogen bond and pi-sulfur interactions with the active site of ERα, as shown in Figure 3a-c.     Progesterone and testosterone were used as a negative control group as their scoring values are −23.53 and −23.95 respectively, as shown in Table 3. Previous research reveals that adding an androgen to estrogen treatment lowers mammary epithelial proliferation and ER expression, implying that androgens may protect against breast cancer in the same way as progesterone does. In the past, androgens were used to treat breast cancer with moderate effectiveness [45]. There are mixed theories about these hormones, as some claim they down-regulate breast cancer progression, whereas some claims they up-regulate the process. However, according to our studies, both progesterone and testosterone have shown interactions with ER alpha protein. Furthermore, this study reveals that these hormones may up-regulate breast cancer development, as they interacted at the active site of ER-alpha with binding energies of −23.53 (progesterone) and −23.95 (testosterone). Upregulation of breast cancer due to an imbalance of these hormones has already been reported. It has been reported that the effects of sex hormones on breast cancer development are clear from the advantages of hormone withdrawal treatment, with particular evidence of a relationship between completion of hormone withdrawal and clinical benefit [46]. Progesterone and testosterone were used as a negative control group as their scoring values are −23.53 and −23.95 respectively, as shown in Table 3. Previous research reveals that adding an androgen to estrogen treatment lowers mammary epithelial proliferation and ER expression, implying that androgens may protect against breast cancer in the same way as progesterone does. In the past, androgens were used to treat breast cancer with moderate effectiveness [45]. There are mixed theories about these hormones, as some claim they down-regulate breast cancer progression, whereas some claims they up-regulate the process. However, according to our studies, both progesterone and testosterone have shown interactions with ER alpha protein. Furthermore, this study reveals that these hormones may up-regulate breast cancer development, as they interacted at the active site of ER-alpha with binding energies of −23.53 (progesterone) and −23.95 (testosterone). Upregulation of breast cancer due to an imbalance of these hormones has already been reported. It has been reported that the effects of sex hormones on breast cancer development are clear from the advantages of hormone withdrawal treatment, with particular evidence of a relationship between completion of hormone withdrawal and clinical benefit [46]. Progesterone and testosterone were used as a negative control group as their scoring values are −23.53 and −23.95 respectively, as shown in Table 3. Previous research reveals that adding an androgen to estrogen treatment lowers mammary epithelial proliferation and ER expression, implying that androgens may protect against breast cancer in the same way as progesterone does. In the past, androgens were used to treat breast cancer with moderate effectiveness [45]. There are mixed theories about these hormones, as some claim they down-regulate breast cancer progression, whereas some claims they up-regulate the process. However, according to our studies, both progesterone and testosterone have shown interactions with ER alpha protein. Furthermore, this study reveals that these hormones may up-regulate breast cancer development, as they interacted at the active site of ER-alpha with binding energies of −23.53 (progesterone) and −23.95 (testosterone). Upregulation of breast cancer due to an imbalance of these hormones has already been reported. It has been reported that the effects of sex hormones on breast cancer development are clear from the advantages of hormone withdrawal treatment, with particular evidence of a relationship between completion of hormone withdrawal and clinical benefit [46]. Progesterone and testosterone were used as a negative control group as their scoring values are −23.53 and −23.95 respectively, as shown in Table 3. Previous research reveals that adding an androgen to estrogen treatment lowers mammary epithelial proliferation and ER expression, implying that androgens may protect against breast cancer in the same way as progesterone does. In the past, androgens were used to treat breast cancer with moderate effectiveness [45]. There are mixed theories about these hormones, as some claim they down-regulate breast cancer progression, whereas some claims they up-regulate the process. However, according to our studies, both progesterone and testosterone have shown interactions with ER alpha protein. Furthermore, this study reveals that these hormones may up-regulate breast cancer development, as they interacted at the active site of ER-alpha with binding energies of −23.53 (progesterone) and −23.95 (testosterone). Upregulation of breast cancer due to an imbalance of these hormones has already been reported. It has been reported that the effects of sex hormones on breast cancer development are clear from the advantages of hormone withdrawal treatment, with particular evidence of a relationship between completion of hormone withdrawal and clinical benefit [46]. Progesterone and testosterone were used as a negative control group as their scoring values are −23.53 and −23.95 respectively, as shown in Table 3. Previous research reveals that adding an androgen to estrogen treatment lowers mammary epithelial proliferation and ER expression, implying that androgens may protect against breast cancer in the same way as progesterone does. In the past, androgens were used to treat breast cancer with moderate effectiveness [45]. There are mixed theories about these hormones, as some claim they down-regulate breast cancer progression, whereas some claims they up-regulate the process. However, according to our studies, both progesterone and testosterone have shown interactions with ER alpha protein. Furthermore, this study reveals that these hormones may up-regulate breast cancer development, as they interacted at the active site of ER-alpha with binding energies of −23.53 (progesterone) and −23.95 (testosterone). Upregulation of breast cancer due to an imbalance of these hormones has already been reported. It has been reported that the effects of sex hormones on breast cancer development are clear from the advantages of hormone withdrawal treatment, with particular evidence of a relationship between completion of hormone withdrawal and clinical benefit [46]. Progesterone and testosterone were used as a negative control group as their scoring values are −23.53 and −23.95 respectively, as shown in Table 3. Previous research reveals that adding an androgen to estrogen treatment lowers mammary epithelial proliferation and ER expression, implying that androgens may protect against breast cancer in the same way as progesterone does. In the past, androgens were used to treat breast cancer with moderate effectiveness [45]. There are mixed theories about these hormones, as some claim they down-regulate breast cancer progression, whereas some claims they up-regulate the process. However, according to our studies, both progesterone and testosterone have shown interactions with ER alpha protein. Furthermore, this study reveals that these hormones may up-regulate breast cancer development, as they interacted at the active site of ER-alpha with binding energies of −23.53 (progesterone) and −23.95 (testosterone). Upregulation of breast cancer due to an imbalance of these hormones has already been reported. It has been reported that the effects of sex hormones on breast cancer development are clear from the advantages of hormone withdrawal treatment, with particular evidence of a relationship between completion of hormone withdrawal and clinical benefit [46]. Progesterone and testosterone were used as a negative control group as their scoring values are −23.53 and −23.95 respectively, as shown in Table 3. Previous research reveals that adding an androgen to estrogen treatment lowers mammary epithelial proliferation and ER expression, implying that androgens may protect against breast cancer in the same way as progesterone does. In the past, androgens were used to treat breast cancer with moderate effectiveness [45]. There are mixed theories about these hormones, as some claim they down-regulate breast cancer progression, whereas some claims they up-regulate the process. However, according to our studies, both progesterone and testosterone have shown interactions with ER alpha protein. Furthermore, this study reveals that these hormones may up-regulate breast cancer development, as they interacted at the active site of ER-alpha with binding energies of −23.53 (progesterone) and −23.95 (testosterone). Up-regulation of breast cancer due to an imbalance of these hormones has already been reported. It has been reported that the effects of sex hormones on breast cancer development are clear from the advantages of hormone withdrawal treatment, with particular evidence of a relationship between completion of hormone withdrawal and clinical benefit [46].
The majority of clinical investigations that have utilized total testosterone as a measure of androgen exposure have found that greater total testosterone levels are related to an increased risk of breast cancer [47]. Progesterone metabolites may consequently be involved in the regulation of the generation of estradiol in the normal breast cell and so may be a multifaceted component in breast carcinogenesis [48]. Progesterone triggers normal human breast epithelial through paracrine mechanisms and is a risk factor for breast cancer because it promotes pre-neoplastic progression by stimulating cyclic proliferation of mammary stem cell pools or cell-initiating tumors in maturing breast epithelium. The development of cancer is therefore further promoted by progesterone signaling and a transition to autocrine proliferation regulation [49].
Natural and synthetic compounds possessing the highest binding affinity, RMSD less than 2 Å, and accurate binding sites were considered to form the most stable complexes. Data of 61 natural and synthetic compounds were collected from strong DDI networks and the top five synthetic (ZINC08441573, ZINC00664754, ZINC00702695, ZINC00627464, ZINC08440501) and four nature compounds (capsaicin, flavopiridol, tectorigenin, and ellagic acid) were docked with ERα (Table 3). Based on their scoring values and RMCD, both synthetic and natural compounds depicted strong binding capacity with the active site of the ERα protein.
Predicted scoring values of the top five synthetic compounds, ZINC08441573, ZINC00664754, ZINC00702695, ZINC00627464, and ZINC08440501, were −32.47, −31.38, −30.35, −30.31, and −29.350 kcal/mol, respectively. These findings were further confirmed by 3-D interactions with the active site of the ERα protein as shown in Figure 4a-e. The ZINC08441573 drug compound showed pi-pi, T shaped and pi-sigma interactions; ZINC00664754 exhibited conventional hydrogen bonding, pi-sigma, pi-sulphur and pi-alkyl bonding; ZINC00702695 had pi-sulfur, pi-lone pair and pi-alkyl associations; ZINC00627464 showed conventional hydrogen bonding, pi-pi T shaped, pi-lone pair and pi-alkyl associations; and ZINC08440501 had conventional hydrogen bond, Sulfur-X, pi-sulfur, and pi-alkyl interactions with the active sites of the ERα protein. Our findings suggest that, relatively, most of the synthetic compounds have scoring values even less than the standard breast cancer drugs, therefore showing strong interactions with the ERα protein. These compounds could be potential candidates to treat breast cancer. The inhibition potential of all synthetic compounds was greater than two standard breast cancer drugs i.e., Arimidex and letrozole. Two synthetic compounds including ZINC08441573 and ZINC00664754 exhibited a highly significant strong binding capacity (based on lowest scoring value) with the ERα protein. These two compounds possess the strong potential to inhibit interactions of all types of POPs (Table 1) with ERα protein and could be the best alternative to standard breast cancer drugs used currently.
Likewise, the binding capacity of the top four natural compounds from our dataset, namely capsaicin, flavopiridol, tectorigenin, and ellagic acid, with the ERα protein is demonstrated in Table 3. The scoring value of these compounds ranged from −25.30 to −16.36 kcal/mol, while their RMSD was between 0.905-1.875 Å. The association between these natural compounds and the ERα protein was further confirmed by 3-D networks as mentioned in Figure 5a-d. Capsaicin showed conventional hydrogen bonds and pilone pair interactions with ERα protein; flavopiridol had pi-sulfur, pi-sigma, and pialkylinteractions; tectorigenin exhibited conventional hydrogen bonding and pi-sulfur interactions; and ellagic acid showed conventional hydrogen bonding, pi-sulfur, and pi-alkyl interactions. Based on scoring values, it can be predicted that capsaicin and flavopiridol have a strong association with ERα protein, which is even stronger than standard drugs (Arimidex and letrozole). These natural compounds also have the potential to inhibit the binding of almost all POPs with ERα protein except short-chained chlorinated paraffins (Table 1). Based on scoring values and RMSD, the majority of the synthetic and natural compounds have the potential to inhibit various interactions between POPs and active sites of the ERα breast cancer protein. Such compounds could also be potential candidates for breast cancer drugs, and can also be useful as an alternative to standard drugs to treat breast cancer caused by POPs and other organic toxins.  Likewise, the binding capacity of the top four natural compounds from our dataset, namely capsaicin, flavopiridol, tectorigenin, and ellagic acid, with the ERα protein is demonstrated in Table 3. The scoring value of these compounds ranged from −25.30 to −16.36 kcal/mol, while their RMSD was between 0.905-1.875 Å. The association between these natural compounds and the ERα protein was further confirmed by 3-D networks as mentioned in Figure 5a-d. Capsaicin showed conventional hydrogen bonds and pi-lone pair interactions with ERα protein; flavopiridol had pi-sulfur, pi-sigma, and pi-alkylinteractions; tectorigenin exhibited conventional hydrogen bonding and pi-sulfur interactions; and ellagic acid showed conventional hydrogen bonding, pi-sulfur, and pi-alkyl interactions. Based on scoring values, it can be predicted that capsaicin and flavopiridol have a strong association with ERα protein, which is even stronger than standard drugs (Arimidex and letrozole). These natural compounds also have the potential to inhibit the binding of almost all POPs with ERα protein except short-chained chlorinated paraffins (Table 1). Based on scoring values and RMSD, the majority of the synthetic and natural compounds have the potential to inhibit various interactions between POPs and active sites of the ERα breast cancer protein. Such compounds could also be potential candidates for breast cancer drugs, and can also be useful as an alternative to standard drugs to treat breast cancer caused by POPs and other organic toxins.

Conclusions
The bioaccumulation of POPs in adipose tissue and their role in breast cancer development and/or progression was evaluated using the molecular interaction approach. Based on scoring values, short-chained chlorinated paraffins demonstrated strong inter-

Conclusions
The bioaccumulation of POPs in adipose tissue and their role in breast cancer development and/or progression was evaluated using the molecular interaction approach. Based on scoring values, short-chained chlorinated paraffins demonstrated strong interactions with the ERα breast cancer protein compared to organo-chlorines and PCBs. Both synthetic and natural compounds which demonstrated strong associations with the active site of the ERα protein could be potential candidates to treat breast cancer specifically caused by POPs and other organic toxins, and can be used as an alternative to standard drugs. Furthermore, our findings could be validated using in vitro and in vivo approaches.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/biology10070681/s1, Table S1: Results of re-docking of native inhibitors; Table S2: Crossdocking results for various PDB IDs from ERα; Figure S1: Structure of estrogen receptor alpha (ERα/pdb id = 5W9D) protein; Figure S2: Chemical structures and binding energies (B.E. in kcal/mol) of the active compounds obtained from CHEMBL; Figure S3. Plot matrix representation of drug compounds with their attributes; Figure S4. DDI networks generated using K means clustering algorithm and Gephi tool.