*2.1. Disease Selection*

Breast cancer (BC) was the target disease because of its prevalence around the globe. Currently, more than two million cases of breast cancer have been diagnosed in women [9], while in Pakistan BC is diagnosed in over 90,000 women annually, out of which 40,000 will not survive [20].

## *2.2. Identification of the Mutated Gene*

Gene identification was completely disease specific. The GeneCard (www.genecards. org/ (accessed on 20 November 2020)) was used along with a literature review to determine a list of mutated genes involved in breast cancer as reported earlier [21]. Based on GeneCard, the estrogen receptor gene (ERg) was identified as a mutated gene of breast cancer.

## *2.3. Selection and Preparation of Targeted Protein*

The Protein Data Bank (PDB), a global database providing the 3-D structure of biological molecules like proteins, DNA, and RNA, was used to select and prepare the targeted

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.

#### *2.4. 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.

#### *2.5. 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.

#### *2.6. 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].

#### *2.7. 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].

#### *2.8. 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 "x1, x2, x<sup>3</sup> ... x*n*" to numbers of k clusters (k ≤ *n*), where k represents clusters and the row is denoted by *n*.

#### *2.9. 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.

#### **3. Results and Discussion**

#### *3.1. 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).

#### *3.2. 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

*Biology* 

−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]. 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. 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 (Figmetalworking fluids, chemicals, and as plastic and flame 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 are toxic aquatic [39]. have provided convincing SCCPs on thyroid their role physiological [41]. ies 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 breast cancer based on least energy or kcal/mol). that the of SCCPs protein the breast cancer in (HBCD) has demonstrated interactions the ure 1B). Therefore, HBCD could also be one of the main contributors to breast cancer. 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 (Figthird 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 (Figof Inuit women [38]. As reported, (C10–C13) to of are toxic aquatic [39]. reports have provided disruptive effect SCCPs on [40], their role and physiological [41]. the SCCPs their contribution disruption. context, aforementioned association <sup>α</sup> breast cancer based least (kcal/mol). that strong of ERα protein may be involved in the spread of breast cancer in women. Hexabromocy-(HBCD) demonstrated with ERα protein 1B). Therefore, HBCD of 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 (Fig-


ure 1B). Therefore, HBCD could also be one of the main contributors to breast cancer.

ure 1B). Therefore, HBCD could also be one of the main contributors to breast cancer.

ure 1B). Therefore, HBCD could also be one of the main contributors to breast cancer.

*Biology* **2021**, *10*, x FOR PEER REVIEW 6 of 16

*Biology* **2021**, *10*, x FOR PEER REVIEW 6 of 16

*Biology* **2021**, *10*, x FOR PEER REVIEW 6 of 16

*Biology* **2021**, *10*, x FOR PEER REVIEW 6 of 16

*Biology* **2021**, *10*, x FOR PEER REVIEW 6 of 16

and by tainted food, and dermal interaction with the substance [36].

and by tainted food, and dermal interaction with the substance [36].

and by tainted food, and dermal interaction with the substance [36].

and by tainted food, and dermal interaction with the substance [36].

and by tainted food, and dermal interaction with the substance [36].

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 non-occupational exposure routes of these high-potency contaminants include ingestion, both directly

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 non-occupational exposure routes of these high-potency contaminants include ingestion, both directly

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 non-occupational exposure routes of these high-potency contaminants include ingestion, both directly

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 non-occupational exposure routes of these high-potency contaminants include ingestion, both directly

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 non-occupational exposure routes of these high-potency contaminants include ingestion, both directly

to stimulate pathway The association between organochlorine and PCBs exposure and risk of breast cancer has reported. present study, retrieved environment root-mean-square distance,

values, are given Table [35], negative affinity/scoring the the stability it the and and the receptor protein [35]. In this context, strong relations between POPs and ERα

REVIEW to cell pathway of breast study, retrieved environment root-mean-square distance, values, are Table As [35], the negative affinity/scoring the stability and receptor protein strong and ERprotein on ranging of POPs, short-chained paraffins (SCCPs), and (PFOSF) strongest molecular interactions with the ERα protein based on their lowest-scoring val-

protein of breast cancer were assessed based on scoring values ranging from −31.94 to

Out of POPs, short-chained paraffins (SCCPs), and (PFOSF) the on of −18.91, kcal/mol, respectively (Table These revealed that SCCPs, HBCD, and PFOSF could be potentially involved in breast cancer prevailing com-

of −18.91, kcal/mol, respectively (Table These findings revealed that SCCPs, HBCD, be potentially com-

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

more suspected breast cancer used developing tries Asia low and The routes high-potency contaminants and by tainted food, and dermal interaction with the substance [36]. Above mentioned results revealed that based on scoring values, short-chain chlorin-

ated interactions with (Figure

as previous [34]. suspected breast used developing tries Asia low and against The routes high-potency ingestion, tainted food, dermal interaction with substance [36]. revealed that ated (SCCPs) (Figure are used fluids, manufacturing chemicals, plastics, rubber, and as a plastic agent and flame retardant [37]. In addition, been

organochlorine [34].

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

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

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

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

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

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

These POPs are extensively used in flame retardants, as a neurotoxin, and in xenobichemicals. HBCD a is hepatotoxic, an endo-

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-

breast milk, and already been in human breast milk raise about possible tion and prenatal uptake during important developmental stages of the fetus [43]. Like-

wise, fluorinated compounds,

insecticides and flame retardants, in cosmetics, paper coatings, and surfactants. The possible association of PFOSF with hormone disorders, genotoxic potential, and tumor for-

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-

insecticides and flame retardants, in cosmetics, paper coatings, and surfactants. The possible association of PFOSF with hormone disorders, genotoxic potential, and tumor for-

(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

kcal/mol), to (Figure These used grading and inhibitors,

interactions protein, based

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-

20. Lindane −12.91 0.631

19. Heptachlor, −13.70 0.999

20. Lindane −12.91 0.631

20. Lindane −12.91 0.631

20. Lindane −12.91 0.631

20. Lindane −12.91 0.631

20. Lindane −12.91 0.631

20. Lindane −12.91 0.631

20. Lindane −12.91 0.631

20. Lindane −12.91 0.631

19. Heptachlor, −13.70 0.999

19. Heptachlor, −13.70 0.999

19. Heptachlor, −13.70 0.999

22. Mirex −12.39 1.508

21. Chlorinated\_naphthalenes −12.58 0.903

21. Chlorinated\_naphthalenes −12.58 0.903

21. Chlorinated\_naphthalenes −12.58 0.903

22. Mirex −12.39 1.508

22. Mirex −12.39 1.508

22. Mirex −12.39 1.508

22. Mirex −12.39 1.508

22. Mirex −12.39 1.508

22. Mirex −12.39 1.508

23. Chlordecone −11.87 1.207

22. Mirex −12.39 1.508

23. Chlordecone −11.87 1.207

23. Chlordecone −11.87 1.207

23. Chlordecone −11.87 1.207

23. Chlordecone −11.87 1.207

23. Chlordecone −11.87 1.207

23. Chlordecone −11.87 1.207

22. Mirex −12.39 1.508

22. Mirex −12.39 1.508

24. Pentachlorophenol −9.566 1.215

23. Chlordecone −11.87 1.207

24. Pentachlorophenol −9.566 1.215

24. Pentachlorophenol −9.566 1.215

24. Pentachlorophenol −9.566 1.215

24. Pentachlorophenol −9.566 1.215

24. Pentachlorophenol −9.566 1.215

24. Pentachlorophenol −9.566 1.215

23. Chlordecone −11.87 1.207

23. Chlordecone −11.87 1.207

25. Hexachlorobenzene −9.501 1.212

24. Pentachlorophenol −9.566 1.215

24. Pentachlorophenol −9.566 1.215

25. Hexachlorobenzene −9.501 1.212

25. Hexachlorobenzene −9.501 1.212

25. Hexachlorobenzene −9.501 1.212

25. Hexachlorobenzene −9.501 1.212

25. Hexachlorobenzene −9.501 1.212

25. Hexachlorobenzene −9.501 1.212

24. Pentachlorophenol −9.566 1.215

26. Pentachlorobenzen −9.500 1.596

25. Hexachlorobenzene −9.501 1.212

26. Pentachlorobenzen −9.500 1.596

26. Pentachlorobenzen −9.500 1.596

26. Pentachlorobenzen −9.500 1.596

26. Pentachlorobenzen −9.500 1.596

26. Pentachlorobenzen −9.500 1.596

25. Hexachlorobenzene −9.501 1.212

25. Hexachlorobenzene −9.501 1.212

27. Hexachlorobutadiene −8.650 1.090

26. Pentachlorobenzen −9.500 1.596

27. Hexachlorobutadiene −8.650 1.090

27. Hexachlorobutadiene −8.650 1.090

27. Hexachlorobutadiene −8.650 1.090

27. Hexachlorobutadiene −8.650 1.090

27. Hexachlorobutadiene −8.650 1.090

27. Hexachlorobutadiene −8.650 1.090

27. Hexachlorobutadiene −8.650 1.090

27. Hexachlorobutadiene −8.650 1.090

27. Hexachlorobutadiene −8.650 1.090

26. Pentachlorobenzen −9.500 1.596

26. Pentachlorobenzen −9.500 1.596

26. Pentachlorobenzen −9.500 1.596

B.E. Binding energy, RMSD. Root-mean-square distance, S. #. Serial number

B.E. Binding energy, RMSD. Root-mean-square distance, S. #. Serial number

B.E. Binding energy, RMSD. Root-mean-square distance, S. #. Serial number

B.E. Binding energy, RMSD. Root-mean-square distance, S. #. Serial number

B.E. Binding energy, RMSD. Root-mean-square distance, S. #. Serial number

B.E. Binding energy, RMSD. Root-mean-square distance, S. #. Serial number

B.E. Binding energy, RMSD. Root-mean-square distance, S. #. Serial number

B.E. Binding energy, RMSD. Root-mean-square distance, S. #. Serial number

B.E. Binding energy, RMSD. Root-mean-square distance, S. #. Serial number

B.E. Binding energy, RMSD. Root-mean-square distance, S. #. Serial number

B.E. Binding energy, RMSD. Root-mean-square distance, S. #. Serial number

*Biology* 


**Table 1.** *Cont.* 17. PCBs (Polychlorinated biphenyls) −14.16 1.233 17. PCBs (Polychlorinated biphenyls) −14.16 1.233 17. PCBs (Polychlorinated biphenyls) −14.16 1.233 17. PCBs (Polychlorinated biphenyls) −14.16 1.233 17. PCBs (Polychlorinated biphenyls) −14.16 1.233 17. PCBs (Polychlorinated biphenyls) −14.16 1.233 17. PCBs (Polychlorinated biphenyls) −14.16 1.233

1.233

*Biology* **2021**, *10*, x FOR PEER REVIEW 7 of 16

*Biology* **2021**, *10*, x FOR PEER REVIEW 7 of 16

*Biology* **2021**, *10*, x FOR PEER REVIEW 7 of 16

*Biology* **2021**, *10*, x FOR PEER REVIEW 7 of 16

*Biology* **2021**, *10*, x FOR PEER REVIEW 7 of 16

*Biology* **2021**, *10*, x FOR PEER REVIEW 7 of 16

*Biology* **2021**, *10*, x FOR PEER REVIEW 7 of 16

Aldrin 1.312

Hexa bromodiphenyl ethers 1.099

Hexabromobiphenyl 1.541

FOR PEER

8. Aldrin −16.19 1.312

8. Aldrin −16.19 1.312

8. Aldrin −16.19 1.312

8. Aldrin −16.19 1.312

8. Aldrin −16.19 1.312

8. Aldrin −16.19 1.312

8. Aldrin −16.19 1.312

8. Aldrin −16.19 1.312

9. Hexa bromodiphenyl ethers −15.72 1.099

9. Hexa bromodiphenyl ethers −15.72 1.099

9. Hexa bromodiphenyl ethers −15.72 1.099

9. Hexa bromodiphenyl ethers −15.72 1.099

9. Hexa bromodiphenyl ethers −15.72 1.099

9. Hexa bromodiphenyl ethers −15.72 1.099

9. Hexa bromodiphenyl ethers −15.72 1.099

10. Hexabromobiphenyl −15.67 1.541

10. Hexabromobiphenyl −15.67 1.541

9. Hexa bromodiphenyl ethers −15.72 1.099

10. Hexabromobiphenyl −15.67 1.541

10. Hexabromobiphenyl −15.67 1.541

10. Hexabromobiphenyl −15.67 1.541

10. Hexabromobiphenyl −15.67 1.541

10. Hexabromobiphenyl −15.67 1.541

11. Penta-bromodiphenyl ethers −15.39 1.261

11. Penta-bromodiphenyl ethers −15.39 1.261

10. Hexabromobiphenyl −15.67 1.541

11. Penta-bromodiphenyl ethers −15.39 1.261

11. Penta-bromodiphenyl ethers −15.39 1.261

11. Penta-bromodiphenyl ethers −15.39 1.261

11. Penta-bromodiphenyl ethers −15.39 1.261

11. Penta-bromodiphenyl ethers −15.39 1.261

12. PCDDs (Polychlorinated dibenzodioxins) −15.01 1.345

12. PCDDs (Polychlorinated dibenzodioxins) −15.01 1.345

11. Penta-bromodiphenyl ethers −15.39 1.261

12. PCDDs (Polychlorinated dibenzodioxins) −15.01 1.345

12. PCDDs (Polychlorinated dibenzodioxins) −15.01 1.345

12. PCDDs (Polychlorinated dibenzodioxins) −15.01 1.345

12. PCDDs (Polychlorinated dibenzodioxins) −15.01 1.345

13. Chlordane −14.85 0.937

13. Chlordane −14.85 0.937

12. PCDDs (Polychlorinated dibenzodioxins) −15.01 1.345

12. PCDDs (Polychlorinated dibenzodioxins) −15.01 1.345 13. Chlordane −14.85 0.937 14. Toxaphene −14.55 0.818

13. Chlordane −14.85 0.937

13. Chlordane −14.85 0.937

13. Chlordane −14.85 0.937

13. Chlordane −14.85 0.937

13. Chlordane −14.85 0.937

14. Toxaphene −14.55 0.818

14. Toxaphene −14.55 0.818

14. Toxaphene −14.55 0.818

14. Toxaphene −14.55 0.818

14. Toxaphene −14.55 0.818

14. Toxaphene −14.55 0.818

14. Toxaphene −14.55 0.818

15. PCDFs(polychlorinated dibenzofurans) −14.39 1.885

16. Beta endosulfans −14.26 1.791

16. Beta endosulfans −14.26 1.791

16. Beta endosulfans −14.26 1.791

16. Beta endosulfans −14.26 1.791

16. Beta endosulfans −14.26 1.791

16. Beta endosulfans −14.26 1.791

16. Beta endosulfans −14.26 1.791

otic 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 otic chemicals. HBCD acts as a nuclear receptor agonist, is hepatotoxic, and is an endocrine disruptor neurotoxicity in breast milk, and the umbilical cord already been in human breast milk raise questions about possible tion 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 kcal/mol), it to (Figure 1C). These used impregnating grading agents and corrosion inhibitors, like insecticides and flame retardants, in cosmetics, paper coatings, and surfactants. The posotic 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 otic chemicals. HBCD acts as a nuclear receptor agonist, is hepatotoxic, and is an endocrine disruptor that induces developmental neurotoxicity in animals [42]. HBCD concenhuman breast milk, serum, the umbilical cord already vestigated. The breast milk questions about possible tion 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 used impregnating and grading agents corrosion like otic 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 otic 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 otic 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. tated 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.

mation in rodents has been suggested previously [44]. Hormones can be indirectly imi-

*Biology* **2021**, *10*, x FOR PEER REVIEW 8 of 16

**Figure 1.** Two dimensional interactions of short chain chlorinated paraffins (**A**), Hexabromocyclododecane (**B**) and Perfluorooctanesulfonyl fluoride (**C**) with ERα protein of Breast Cancer. **Figure 1.** Two dimensional interactions of short chain chlorinated paraffins (**A**), Hexabromocyclododecane (**B**) and Perfluorooctanesulfonyl fluoride (**C**) with ERα protein of Breast Cancer.

#### *3.3. Natural and Synthetic Compounds Collection, Mining, and Clustering 3.3. 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 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).

#### different color representations (Figure S3). *3.4. DDI Network*

*3.4. 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

In total, 12 networks were generated using the k-means clustering algorithm and

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.

**Table 2.** Statistical parameters used to predict network interactions of various natural and synthetic compounds.


AD. Average degree, AWD. Average weighted degree, ND. Network diameter, GD. Graph density, Ml. modularity, APL. Average path length, N. nodes, E. edges, FSIN. Final strongly interacted network. sion strength was set to 10,000, and Force Atlas and Fruchterman rein gold layouts were used to display and visualize the networks.

**Figure 2.** Final strongly drug-drug interaction network. **Figure 2.** Final strongly drug-drug interaction network.

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, network 1 comprises of 507.0 nodes and 1169 edges with modularity of 0.518; the second network consists of 266 nodes and 564 edges with 0.524 modularity. Network 3 showed 185.0 nodes and 383.0 edges with 0.538 modularity. Network 4 comprises 64.00 nodes and 98.00 edges with 0.613 modularity. Network 5 contains 923.0 nodes and 2146 edges with a modularity of 0.509. Network 6 consists of 188 nodes and 395.0 edges with a modularity of 0.571. Network 7 comprises 469.0 nodes and 1067 edges and possesses a modularity of 0.525. Network 8 consists of 107.0 nodes and 181.0 edges, and 0.001 graph density with a modularity of 0.552. Network 9 comprises 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, network 1 comprises of 507.0 nodes and 1169 edges with modularity of 0.518; the second network consists of 266 nodes and 564 edges with 0.524 modularity. Network 3 showed 185.0 nodes and 383.0 edges with 0.538 modularity. Network 4 comprises 64.00 nodes and 98.00 edges with 0.613 modularity. Network 5 contains 923.0 nodes and 2146 edges with a modularity of 0.509. Network 6 consists of 188 nodes and 395.0 edges with a modularity of 0.571. Network 7 comprises 469.0 nodes and 1067 edges and possesses a modularity of 0.525. Network 8 consists of 107.0 nodes and 181.0 edges, and 0.001 graph density with a modularity of 0.552. Network 9 comprises 369.0 nodes and 870.0 edges

369.0 nodes and 870.0 edges with modularity of 0.500. Network 10 consists of 541.0 nodes

modularity. Likewise, the finally generated strong network contains 923.0 nodes and 2166

**Table 2.** Statistical parameters used to predict network interactions of various natural and synthetic compounds. **Networks AD AWD ND GD Ml APL N E**  1. 2.306 2.860 1.000 0.005 0.518 1.000 507.0 1169 2. 2.120 2.519 1.000 0.008 0.524 1.000 266.0 564.0 3. 2.070 2.649 1.000 0.011 0.538 1.000 185.0 383.0 4. 1.531 2.266 1.000 0.024 0.613 1.000 64.00 98.00 5. 2.325 2.476 1.000 0.003 0.509 1.000 923.0 2146 6. 2.101 3.005 1.000 0.011 0.571 1.000 188.0 395.0 7. 2.275 3.028 1.000 0.005 0.525 1.000 469.0 1067 8. 1.629 2.056 1.000 0.001 0.552 1.000 107.0 181.0 9. 2.371 2.792 1.000 0.006 0.500 1.000 367.0 870.0 10. 2.155 2.662 1.000 0.004 0.501 1.000 541.0 1166 11. 1.518 1.789 1.000 0.007 0.528 1.000 218.0 331.0

edges with 0.503 modularity (Table 2).

4

4

4

4

4

4

4

4

nyl]carbamoyl]cyclohex-3-ene-1-carboxylic acid.

nyl]carbamoyl]cyclohex-3-ene-1-carboxylic acid.

nyl]carbamoyl]cyclohex-3-ene-1-carboxylic acid.

nyl]carbamoyl]cyclohex-3-ene-1-carboxylic acid.

nyl]carbamoyl]cyclohex-3-ene-1-carboxylic acid.

nyl]carbamoyl]cyclohex-3-ene-1-carboxylic acid.

nyl]carbamoyl]cyclohex-3-ene-1-carboxylic acid.

nyl]carbamoyl]cyclohex-3-ene-1-carboxylic acid.

(1*R*,6*S*)-6-[[2-[4-(4-methylphenyl) piperazine-1-carbonyl]phe-

(1*R*,6*S*)-6-[[2-[4-(4-methylphenyl) piperazine-1-carbonyl]phe-

(1*R*,6*S*)-6-[[2-[4-(4-methylphenyl) piperazine-1-carbonyl]phe-

(1*R*,6*S*)-6-[[2-[4-(4-methylphenyl) piperazine-1-carbonyl]phe-

(1*R*,6*S*)-6-[[2-[4-(4-methylphenyl) piperazine-1-carbonyl]phe-

(1*R*,6*S*)-6-[[2-[4-(4-methylphenyl) piperazine-1-carbonyl]phe-

(1*R*,6*S*)-6-[[2-[4-(4-methylphenyl) piperazine-1-carbonyl]phe-

[ZINC00627464]

[ZINC00627464]

[ZINC00627464]

[ZINC00627464]

[ZINC00627464]

[ZINC00627464]

[ZINC00627464]

[ZINC00627464]

with modularity of 0.500. Network 10 consists of 541.0 nodes and 1166 edges with a 0.501 modularity. Network 11 comprises 218.0 nodes and 331.0 edges and 0.528 modularity, while network 12 contains 300.0 nodes, 666.0 edges, and 0.500 modularity. Likewise, the finally generated strong network contains 923.0 nodes and 2166 edges with 0.503 modularity (Table 2). 12. 2.220 2.967 1.000 0.007 0.500 1.000 300.0 666.0 FSIN 2.325 2.476 1.000 0.003 0.503 1.000 923.0 2146 AD. Average degree, AWD. Average weighted degree, ND. Network diameter, GD. Graph density, Ml. Modularity, APL. Average path length, N. Nodes, E. Edges, FSIN. Final strongly interacted network.3.5 12. 2.220 2.967 1.000 0.007 0.500 1.000 300.0 666.0 FSIN 2.325 2.476 1.000 0.003 0.503 1.000 923.0 2146 AD. Average degree, AWD. Average weighted degree, ND. Network diameter, GD. Graph density, Ml. Modularity, APL. Average path length, N. Nodes, E. Edges, FSIN. Final strongly interacted network.3.5 12. 2.220 2.967 1.000 0.007 0.500 1.000 300.0 666.0 FSIN 2.325 2.476 1.000 0.003 0.503 1.000 923.0 2146 AD. Average degree, AWD. Average weighted degree, ND. Network diameter, GD. Graph density, Ml. Modularity, APL. *Biology* **2021**, *10*, x FOR PEER REVIEW 10 of 16 12. 2.220 2.967 1.000 0.007 0.500 1.000 300.0 666.0 FSIN 2.325 2.476 1.000 0.003 0.503 1.000 923.0 2146 AD. Average degree, AWD. Average weighted degree, ND. Network diameter, GD. Graph density, Ml. Modularity, APL. 12. 2.220 2.967 1.000 0.007 0.500 1.000 300.0 666.0 FSIN 2.325 2.476 1.000 0.003 0.503 1.000 923.0 2146 AD. Average degree, AWD. Average weighted degree, ND. Network diameter, GD. Graph density, Ml. Modularity, APL. 12. 2.220 2.967 1.000 0.007 0.500 1.000 300.0 666.0 FSIN 2.325 2.476 1.000 0.003 0.503 1.000 923.0 2146 AD. Average degree, AWD. Average weighted degree, ND. Network diameter, GD. Graph density, Ml. Modularity, APL. Average path length, N. Nodes, E. Edges, FSIN. Final strongly interacted network.3.5 12. 2.220 2.967 1.000 0.007 0.500 1.000 300.0 666.0 FSIN 2.325 2.476 1.000 0.003 0.503 1.000 923.0 2146 AD. Average degree, AWD. Average weighted degree, ND. Network diameter, GD. Graph density, Ml. Modularity, APL. 12. 2.220 2.967 1.000 0.007 0.500 1.000 300.0 666.0 FSIN 2.325 2.476 1.000 0.003 0.503 1.000 923.0 2146 AD. Average degree, AWD. Average weighted degree, ND. Network diameter, GD. Graph density, Ml. Modularity, APL.

#### *3.5. Validation of Natural and Synthetic Compounds 3.5. Validation of Natural and Synthetic Compounds*  Average path length, N. Nodes, E. Edges, FSIN. Final strongly interacted network.3.5 Average path length, N. Nodes, E. Edges, FSIN. Final strongly interacted network.3.5 Average path length, N. Nodes, E. Edges, FSIN. Final strongly interacted network.3.5 Average path length, N. Nodes, E. Edges, FSIN. Final strongly interacted network.3.5

Average path length, N. Nodes, E. Edges, FSIN. Final strongly interacted network.3.5

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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. 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 interac-*3.5. 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 interac-*3.5. 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 interac-*3.5. 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 *3.5. 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 interac-*3.5. 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 interac-*3.5. 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 interac-*3.5. 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 interac-

interactions, while Letrozole showed conventional hydrogen bond and pi-sulfur interac-

**Table 3.** Docking results of positive control, negative control, synthetic and natural compounds with ERα protein. tions with the active site of ERα, as shown in Figure 3a–c. tions with the active site of ERα, as shown in Figure 3a–c. tions with the active site of ERα, as shown in Figure 3a–c. tions with the active site of ERα, as shown in Figure 3a–c. tions with the active site of ERα, as shown in Figure 3a–c. tions with the active site of ERα, as shown in Figure 3a–c. tions with the active site of ERα, as shown in Figure 3a–c. tions with the active site of ERα, as shown in Figure 3a–c.


−30.31 1.650

−30.31 1.650

−30.31 1.650

−30.31 1.650

−30.31 1.650

−30.31 1.650

−30.31 1.650

−30.31 1.650

Natural compounds

1

5

Synthetic compounds

one [ZINC08441573]

dine-2,3-dione [ZINC08440501]

Positive control

Negative control


**Table 3.** *Cont.* (2*S*)-4-hydroxy-3-(5-methylfuran-2-carbonyl)-1-[[(2*R*)-oxolan-2 Flavopiridol. [ZINC21288966] −24.76 1.875

2 2-(4-*tert*-butylphenyl)-3*H*-quinazolin-4-one [ZINC00664754] −31.38 1.575

*Biology* **2021**, *10*, x FOR PEER REVIEW 10 of 16

*3.5. Validation of Natural and Synthetic Compounds* 

tions with the active site of ERα, as shown in Figure 3a–c.

**S. # Names Structures BE (kcal/mol) RMSD (Å)** 

**Table 3.** Docking results of positive control, negative control, synthetic and natural compounds with ERα protein*.*

1 Tamoxifen −31.26 1.006

2 Arimidex −22.46 1.130

3 Letrozole −20.97 1.385

1 Progestrone −23.53 1.080

2 Testosterone −23.95 0.859

*Biology* **2021**, *10*, x FOR PEER REVIEW 11 of 16

5-(4-butoxyphenyl)-4-(2,3-dihydro-1,4-benzodioxin-6-ylcarbonyl)-1-(2-furylmethyl)-3-hydroxy-1,5-dihydro-2H-pyrrol-2-

(5*S*)-1-[3-(dimethylamino)propyl]-4-[hydroxy-(3-methyl-4 propan-2-yloxyphenyl)methylidene]-5-pyridin-4-ylpyrroli-

Average path length, N. Nodes, E. Edges, FSIN. Final strongly interacted network.3.5

12. 2.220 2.967 1.000 0.007 0.500 1.000 300.0 666.0 FSIN 2.325 2.476 1.000 0.003 0.503 1.000 923.0 2146 AD. Average degree, AWD. Average weighted degree, ND. Network diameter, GD. Graph density, Ml. Modularity, APL.

> 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 interac-

> > −32.47 1.521

−28.76 1.686

**Figure 3.** Three-dimensional interactions of Tamoxifen (**A**), Arimidex (**B**), Letrozole (**C**) with the binding site of ERα protein.

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. *Biology* **2021**, *10*, x FOR PEER REVIEW 13 of 16

**Figure 4.** Three-dimensional interactions of synthetic compounds ZINC08441573 (**A**), ZINC00664754 (**B**), ZINC00702695 (**C**), ZINC00627464 (**D**), and ZINC08440501 (**E**) with the binding site of ERα protein. **Figure 4.** Three-dimensional interactions of synthetic compounds ZINC08441573 (**A**), ZINC00664754 (**B**), ZINC00702695 (**C**), ZINC00627464 (**D**), and ZINC08440501 (**E**) with the binding site of ERα protein. *Biology* **2021**, *10*, x FOR PEER REVIEW 14 of 16

**Figure 5.** Three-dimensional interactions of natural compounds capsaicin (**A**), flavopiridol (**B**), tectorigenin (**C**), ellagic acid (**D**) with binding site of ERα protein. SATS University Islamabad, Abbottabad campus for lab facilitation. **Figure 5.** Three-dimensional interactions of natural compounds capsaicin (**A**), flavopiridol (**B**), tectorigenin (**C**), ellagic acid (**D**) with binding site of ERα protein.

opment 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 ap-

**Supplementary Materials:** The following are available online at www.mdpi.com/xxx/s1,Table S1: Results of re-docking of native inhibitors; Table S2: Cross-docking 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.

**Author Contributions:** Design, analysis, writing—B.Z. and Z.A; conceptualization, supervision, resources, visualization, proof reading, validation—A.M.A. and U.R; funding acquisition, visualization—D.A.A.F., R.M.A. (Roua M. Alkufeidy), F.S.A. and R.M.A. (Reem M. Aljowaie). All au-

**Funding:** The authors extend their appreciation to the researchers supporting project number

**Acknowledgments:** The authors extend their appreciation to the researchers supporting project number (RSP-2021/190) King Saud University, Riyadh, Saudi Arabia. We are grateful to COM-

DDI networks generated using K means clustering algorithm and Gephi tool.

thors have read and agreed to the published version of the manuscript.

(RSP-2021/190) King Saud University, Riyadh, Saudi Arabia. **Institutional Review Board Statement:** Not applicable.

**4. Conclusions** 

proaches.

#### **4. 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.

**Author Contributions:** Design, analysis, writing—B.Z. and Z.A.; conceptualization, supervision, resources, visualization, proof reading, validation—A.M.A. and U.R.; funding acquisition, visualization— D.A.A.F., R.M.A. (Roua M. Alkufeidy), F.S.A. and R.M.A. (Reem M. Aljowaie). All authors have read and agreed to the published version of the manuscript.

**Funding:** The authors extend their appreciation to the researchers supporting project number (RSP-2021/190) King Saud University, Riyadh, Saudi Arabia.

**Institutional Review Board Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available within the article and supplementary materials.

**Acknowledgments:** The authors extend their appreciation to the researchers supporting project number (RSP-2021/190) King Saud University, Riyadh, Saudi Arabia. We are grateful to COMSATS University Islamabad, Abbottabad campus for lab facilitation.

**Conflicts of Interest:** All the authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

#### **References**


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