Metabolic Profile, Biotransformation, Docking Studies and Molecular Dynamics Simulations of Bioactive Compounds Secreted by CG3 Strain

Actinobacteria isolated from untapped environments and exposed to extreme conditions such as saltpans are a promising source of novel bioactive compounds. These microorganisms can provide new molecules through either the biosynthetic pathway or the biotransformation of organic molecules. In the present study, we performed a chemical metabolic screening of secondary metabolites secreted by the new strain CG3, which was isolated from a saltpan located in the Sahara of Algeria, via high-performance liquid chromatography coupled with high-resolution mass spectrometry (HPLC-ESI-HRMS). The results indicated that this strain produced five new polyene macrolactams, kenalactams A–E, along with two known compounds, mitomycin C and 6″-hydroxy-4,2′,3′,4″ tetramethoxy-p-terphenyl. Furthermore, the CG3 isolate could have excellent properties for converting the aglycone isoflavone glycitein to the compounds 6,7-dimethoxy-3-(4-methoxyphenyl)chromen-4-one (50) and 6,7-dimethoxy-3-phenylchromen-4-one (54), and the isoflavone genistein can be converted to 5,7-dimethoxy-3-(4-methoxyphenyl)chromen-4-one (52). Docking studies and molecular dynamics simulations indicated that these three isoflavones, generated via biotransformation, are potent inhibitors of the target protein aromatase (CYP19A1); consequently, they can be used to prevent breast cancer risk in postmenopausal women.


Introduction
In recent years, research on novel bioactive compounds has been more oriented towards the exploration of secondary metabolites secreted by Actinobacteria isolated from untapped environments exposed to extreme conditions [1]. Such environments offer the capacity to isolate diverse novel microorganisms, which are supposed to be potential reservoirs of novel compounds. Saltpans located in the Sahara of Algeria are one of these extreme environments, which are characterized by high temperature in the morning, severe solar radiation, low temperature at night, very low amounts of nutrients and high salt concentration [2]. Actinobacteria has adapted to such extreme conditions, acquiring complex hypha by which bioactive compounds can be produced [3]. Little is known about the The results revealed that extracts prepared from SM and ISP2 media exhibited strong antibacterial activity against all tested bacteria, except for Klebsiella pneumoniae, with MIC values ranging between 0.52-66.66 µg/mL. However, only the extract obtained from SM showed the inhibition of Candida albicans and Mucor hiemalis, with MIC values of 66.66 µg/mL and 16.66 µg/mL, respectively. The difference in activity between ISP2 and SM can be attributed to the difference in the composition of the two culture media. This composition, particularly the nature of carbon and nitrogen sources, can affect the quality and the quantity of bioactive compounds secreted by strain CG3. Therefore, SM met the Antibiotics 2022, 11, 657 3 of 18 nutritional requirements of strain CG3 for the biosynthesis of bioactive molecules with antifungal and antibacterial activities, whereas only metabolites with antibacterial activity were secreted by strain CG3 when it was grown in ISP2 medium.

Fractionation of Crude Extract
In order to localize the inhibitory activity, the crude extract from SM was fractionated via analytical HPLC and tested against Staphylococcus aureus ( Figure 1A) and Mucor hiemalis ( Figure 1B) and subsequently analyzed by HPLC-ESI-HRMS. µ g/mL and 16.66 µ g/mL, respectively. The difference in activity between ISP2 and SM be attributed to the difference in the composition of the two culture media. This comp tion, particularly the nature of carbon and nitrogen sources, can affect the quality and quantity of bioactive compounds secreted by strain CG3. Therefore, SM met the nu tional requirements of strain CG3 for the biosynthesis of bioactive molecules with a fungal and antibacterial activities, whereas only metabolites with antibacterial acti were secreted by strain CG3 when it was grown in ISP2 medium.

Fractionation of Crude Extract
In order to localize the inhibitory activity, the crude extract from SM was fractiona via analytical HPLC and tested against Staphylococcus aureus ( Figure 1A) and Mucor malis ( Figure 1B) and subsequently analyzed by HPLC-ESI-HRMS. The results in Figure 1 indicate the presence of three active areas, (1D, 1F), (3F, and (4B, 4D), in 96-well microplates. By correlation with the analytical HPLC chrom gram, the active fractions corresponding to the peaks were located at the intervals o tention times: 2-2.40 min, 10.50-11.30 min and 14.50-16.50 min, respectively. Furt more, the two active peak areas eluted at tR: 2-2.40 min and tR: 10.50-11.30 min were ac against Staphylococcus aureus, whereas the third one at tR: 14.50-16.50 min exhibited a fungal activity against Mucor hiemalis.
The comparison between the analytical HPLC chromatographic profiles of the cr extracts from SM ( Figure 1) and ISP2 media (Supplementary Figure S1) indicated that three peaks eluted between tR: 14.30-16.50 min were absent in the crude extract from I medium (Supplementary Figure S1). These peaks are responsible for the antifungal ac ity of the crude extract prepared from SM against Mucor hiemalis ( Figure 1). The results in Figure 1 indicate the presence of three active areas, (1D, 1F), (3F, 3H) and (4B, 4D), in 96-well microplates. By correlation with the analytical HPLC chromatogram, the active fractions corresponding to the peaks were located at the intervals of retention times: 2-2.40 min, 10.50-11.30 min and 14.50-16.50 min, respectively. Furthermore, the two active peak areas eluted at t R : 2-2.40 min and t R : 10.50-11.30 min were active against Staphylococcus aureus, whereas the third one at t R : 14.50-16.50 min exhibited antifungal activity against Mucor hiemalis.
The comparison between the analytical HPLC chromatographic profiles of the crude extracts from SM ( Figure 1) and ISP2 media (Supplementary Figure S1) indicated that the three peaks eluted between t R : 14.30-16.50 min were absent in the crude extract from ISP2 medium (Supplementary Figure S1). These peaks are responsible for the antifungal activity of the crude extract prepared from SM against Mucor hiemalis ( Figure 1).

HPLC-UV-HRESIMS Analysis of Crude Extracts
In order to identify the interesting peaks, the crude extract prepared from SM was analyzed by HPLC-UV-HRESIMS ( Figure 2). R PEER REVIEW 4 of 18

HPLC-UV-HRESIMS Analysis of Crude Extracts
In order to identify the interesting peaks, the crude extract prepared from SM was analyzed by HPLC-UV-HRESIMS ( Figure 2). In order to correlate the peaks from Figure 2 with those in Figure 1, we follow two steps: (i): The first step consists of comparing the chromatographic profiles in both Figures  1 and 2, which are shown to be similar (almost the same morphology). Therefore, each peak in Figure 2 can be correlated to its homologue in Figure 1 according to its position in the chromatogram. (ii): To confirm the correlations, in the second step, comparison of the UV-Vis spectrum of each peak in Figure 1 (measured by analytical HPLC) to the UV-Vis spectrum of the corresponding peak in Figure 2 (measured by HPLC-UV-HRESIMS) is carried out. The UV profile of the peaks in Figure 1 should be exactly identical/similar to that of the corresponding peaks in Figure 2.
By comparing the HPLC chromatographic profile in Figure 1 with the LC-UV-MS chromatographic profile in Figure 2, the first zone (1D, 1F) of activity in Figure 1 (tR = 2-2.40 min) was assigned to peak numbers 12 (tR = 3.77 min) and 13 (tR = 3.95 min) in Figure  2. Peaks 12 and 13 exhibited the same UV-Vis absorption maxima at 216, 245 and 362 nm and the same molecular ion cluster [M+H] + at m/z 368.2390, which provided the same molecular formula of C23H29NO3. This indicates that both metabolites (12 and 13) are isomers.
The search for molecules with the same character in natural chemical product databases, such as Antibase and Dictionary of Natural Products, revealed that compounds 12 and 13 are correlated to mitomycin C and its stereoisomers ( Figure 3). In order to correlate the peaks from Figure 2 with those in Figure 1, we follow two steps: (i): The first step consists of comparing the chromatographic profiles in both Figures 1 and 2, which are shown to be similar (almost the same morphology). Therefore, each peak in Figure 2 can be correlated to its homologue in Figure 1 according to its position in the chromatogram. (ii): To confirm the correlations, in the second step, comparison of the UV-Vis spectrum of each peak in Figure 1 (measured by analytical HPLC) to the UV-Vis spectrum of the corresponding peak in Figure 2 (measured by HPLC-UV-HRESIMS) is carried out. The UV profile of the peaks in Figure 1 should be exactly identical/similar to that of the corresponding peaks in Figure 2.
By comparing the HPLC chromatographic profile in Figure 1 with the LC-UV-MS chromatographic profile in Figure 2, the first zone (1D, 1F) of activity in Figure 1 (t R = 2-2.40 min) was assigned to peak numbers 12 (t R = 3.77 min) and 13 (t R = 3.95 min) in Figure 2. Peaks 12 and 13 exhibited the same UV-Vis absorption maxima at 216, 245 and 362 nm and the same molecular ion cluster [M+H] + at m/z 368.2390, which provided the same molecular formula of C 23 H 29 NO 3 . This indicates that both metabolites (12 and 13) are isomers.
The search for molecules with the same character in natural chemical product databases, such as Antibase and Dictionary of Natural Products, revealed that compounds 12 and 13 are correlated to mitomycin C and its stereoisomers ( Figure 3).
Mitomycin C is a methylazirinopyrroloindoledione purified from Streptomyces caespitosus and presents potent antineoplastic activity against a variety of cancers due to its ability to crosslink DNA with high efficiency [13]. Furthermore, this compound has a wide spectrum of antibacterial activities [14]; therefore, the antimicrobial activity against Staphylococcus aureus observed in Figure 1 at t R : 2.00-2.40 min can be correlated to mitomycin C. Mitomycin C has been previously isolated from several strains belonging to Streptomyces. However, it should be noted that this compound was identified for the first time in the crude extract prepared from the culture fermentation of a strain closely related to the genus Nocardiopsis.
Antibiotics 2022, 11, 657 5 of 18 2. Peaks 12 and 13 exhibited the same UV-Vis absorption maxima at 216, 245 and 362 and the same molecular ion cluster [M+H] + at m/z 368.2390, which provided the same lecular formula of C23H29NO3. This indicates that both metabolites (12 and 13) are isom The search for molecules with the same character in natural chemical product d bases, such as Antibase and Dictionary of Natural Products, revealed that compound and 13 are correlated to mitomycin C and its stereoisomers ( Figure 3). The structures of 39 and 43, assigned from 1D and 2D NMR spectra ( 1 H 1 H COSY, 1 H 13 C HMBC and HSQC), revealed that both molecules form 22-membered macrocyclic amide rings; therefore, they belong to the class of polyene macrolactams ( Figure 3) [15]. The names kenalactams A and B are attributed to 43 and 39, respectively ( Figure 3). Due to the low amount and instability of compound 39, only compound 43 was tested for antimicrobial and cytotoxic activities at concentrations ≥ 66.66 µg/mL, and the results indicate that 43 did not show any antimicrobial activity against Staphylococcus aureus [15]. Consequently, the antibacterial activity in Figure 1 at t R : 10.50-11.30 min can be linked to kenalactam B (39).
The third active area (4B, 4D) eluted between t R : 14.50-16.50 min and exhibiting antifungal activity against Mucor hiemalis ( Figure 1) corresponds to peaks 50, 52 and 54 in Figure 2. These three peaks showed the same UV-Vis absorption maxima at 220, 254 and 310 nm.
The molecular formula of 54 was determined by HPLC-UV-HRESIMS to be C 17 H 14 O 4 with a molecular ion cluster [M+H] + at m/z 283.0967. The 1 H NMR and 13 C NMR data of compound 54 (Supplementary Table S2) contained all structure elements of 50, and the only difference was the absence of methoxy carbons (δ H/C 3.86/55.9) linked to the C-4 position; therefore, compound 54 was identified as 6,7-dimethoxy-3-phenylchromen-4-one.
However, the molecular formula of C 18 H 16 O 5 of compound 52 with a molecular ion cluster [2M+Na] + at m/z 647.1900 was determined by HPLC-UV-HRESIMS. A survey of chemical databases (Antibase and Dictionary of Natural Products) of compounds with similar properties identified compound 52 as 5,7-dimethoxy-3-(4-methoxyphenyl)chromen-4-one.
To the best of our knowledge, compounds 50, 52 and 54 have never been tested to evaluate their antifungal activity, and the antifungal activity seen in Figure 1 at t R : 14.50-16.50 min against Mucor hiemalis, as well as the antifungal activity of the crude extract prepared from the culture of strain CG3 in SM (Table 1), can be attributed to one of these three compounds or to the synergistic action between them.
Compounds 50, 52 and 54, belonging to the isoflavone subclass, are isomers of flavones. Both subclasses, isoflavones and flavones, pertain to the class of flavonoids, and they share an almost identical structure. The only difference in the structure compared to flavones is the position of the phenyl group, which is linked to C-3 instead of C-2 for flavones [17,18]. Flavonoids are polyphenolic plant-derived secondary metabolites, generally found in various plant species [18].
Isoflavones 50, 52 and 54 were detected only in the crude extract prepared from the culture of strain CG3 in SM (Supplementary Figure S2). On the other hand, the three compounds were completely absent when the crude extract from ISP2 medium was analyzed by HPLC-UV-HRESIMS (Supplementary Figure S3). SM contains soybeans (20.0 g/L); however, this ingredient is absent in ISP2 medium. The soybeans in SM are the major natural source of isoflavones [19]; therefore, compounds 50, 52 and 54, detected in the crude extract of SM, likely originate from the culture medium and are not biosynthesized by strain CG3. To exclude this hypothesis, SM was incubated without strain CG3 at 37 • C for 14 days. The HPLC-UV-HRESIMS analysis of the crude extract prepared from this last culture indicated the absence of compounds similar to metabolites 50, 52 and 54 in SM (Supplementary Figure S4).
Compounds 50, 52 and 54 are aglycone isoflavones. Glycitein is the structure most closely related to 50 and 54; in fact, the three compounds have a methoxy group linked to the C-6 position of the isoflavone core. However, the two hydroxy groups of glycitein attached to C-7 and C-4 are methylated in 50. Compound 54 is methylated at C-7, in addition to C-6, whereas the methoxy group of 50 linked to C-4 is absent in 54 ( Figure 3).
Compound 52 is structurally close to genistein; the only difference is that the hydroxyl groups at C-5, C7 and C-4 in genistein are replaced by methyl groups in 52 ( Figure 4).
We can expect that strain CG3 generates compounds 50, 52 and 54 via biotransformation of the aglycone isoflavone, genistein and glycitein (Figure 4), which were detected in the crude extract prepared from SM without the CG3 strain (t R = 4.40-7.50 min) (Supplementary Figure S4). In fact, we suppose that strain CG3 generates compound 50 in the first step via the O-methylation of both hydroxyl groups of glycitein, attached at positions 7 and 4 ( Figure 4). These reactions would be catalyzed by methyl transferase enzymes such as 7-O-methyltransferase (7-OMT) and 4 -O-methyltransferase (4 -OMT), respectively. The source of methyl groups linked to the 4 -O and 7-O positions is S-Adenosyl-L-methionine (SAM), which is one of the major methyl donors in all living organisms [20] ( Figure 5).
Compound 54 was possibly generated by strain CG3 through the direct demethoxylation of 50 at the 4 -O position ( Figure 4). In fact, after a long period of incubation (up to 21 days), the amount of 50 decreased gradually, whereas the production of metabolite 54 increased ( Supplementary Figures S5 and S6).
Setchell et al. [21] explained that the isoflavone daidzein can be generated via demethoxylation of glycitein at the 6-position. This reaction (demethoxylation) is a minor biotransformation pathway. attached to C-7 and C-4' are methylated in 50. Compound 54 is methylated at C-7, in ad dition to C-6, whereas the methoxy group of 50 linked to C-4' is absent in 54 (Figure 3).
Compound 52 is structurally close to genistein; the only difference is that the hy droxyl groups at C-5, C7 and C-4̍ in genistein are replaced by methyl groups in 52 (Figur 4). We can expect that strain CG3 generates compounds 50, 52 and 54 via biotransfo mation of the aglycone isoflavone, genistein and glycitein (Figure 4), which were detecte in the crude extract prepared from SM without the CG3 strain (tR = 4.40-7.50 min) (Sup plementary Figure S4). In fact, we suppose that strain CG3 generates compound 50 in th first step via the O-methylation of both hydroxyl groups of glycitein, attached at position  Compound 54 was possibly generated by strain CG3 through the direct demethoxylation of 50 at the 4'-O position (Figure 4). In fact, after a long period of incubation (up to 21 days), the amount of 50 decreased gradually, whereas the production of metabolite 54 increased ( Supplementary Figures S5 and S6).
Setchell et al. [21] explained that the isoflavone daidzein can be generated via demethoxylation of glycitein at the 6-position. This reaction (demethoxylation) is a minor biotransformation pathway.
Furthermore, methylation of genistein at C-6, C-7 and C-4' led to the formation of compound 52 (Figure 4).
Compounds 52 and 54 were obtained via organic synthesis using eleven phenol derivatives and six phenylacetic acids [22]. However, compound 50 was generated from the chemical transformation of the isoflavone 6-methoxyisoformononetin, which was isolated from the roots of the medicinal plant Amphimas pterocarpoides [16].
Isoflavones have been considered chemoprotective compounds; consequently, they can be used to reduce the risk of a wide range of chronic diseases, such as diabetes, osteoporosis and cardiovascular diseases. Additionally, they may protect the body from hormone-related cancers, such as breast cancer and prostate cancer. Seo et al. [23] indicated that compound 50 acted synergistically with glycitin to promote wound healing and reduce scarring. Therefore, it could potentially be developed in conjunction with glycitin as Furthermore, methylation of genistein at C-6, C-7 and C-4 led to the formation of compound 52 (Figure 4).
Compounds 52 and 54 were obtained via organic synthesis using eleven phenol derivatives and six phenylacetic acids [22]. However, compound 50 was generated from the chemical transformation of the isoflavone 6-methoxyisoformononetin, which was isolated from the roots of the medicinal plant Amphimas pterocarpoides [16].
Isoflavones have been considered chemoprotective compounds; consequently, they can be used to reduce the risk of a wide range of chronic diseases, such as diabetes, osteoporosis and cardiovascular diseases. Additionally, they may protect the body from hormone-related cancers, such as breast cancer and prostate cancer. Seo et al. [23] indicated that compound 50 acted synergistically with glycitin to promote wound healing and reduce scarring. Therefore, it could potentially be developed in conjunction with glycitin as a bioactive therapeutic agent for wound treatment.
It should be noted that compounds 50, 52 and 54, which have previously been reported as synthetic compounds, are described for the first time as natural products from the fermentation of CG3.
The p-terphenyl derivative 65 was isolated for the first time from the halophilic actinomycete Nocardiopsis gilva YIM 90087, which is closely related to strain CG3 [24].
Compound , respectively. Structure elucidation based on the NMR and HPLC-UV-HRESIMS data of these three metabolites isolated from the CG3 strain was described previously by Messaoudi et al. [15], and these unprecedented compounds, named kenalactams C-E (69, 75 and 78), were identified as the first family of polyenic macrolactams from the genus of Norcadiopsis.
Compounds 69, 75 and 78 exhibited weak to moderate antimicrobial and cytotoxic activities against a panel of human pathogenic microorganisms and human cancer cell lines.
Estrogenic hormones in females are synthesized primarily by the ovaries from androgens, such as testosterone and androstenedione. The reactions are catalyzed by the cytochrome P450 19A1 (CYP19A1; EC 1.14.14.1), commonly known as aromatase. Estrogens are also produced in smaller amounts by other tissues, such as breasts [27].
The quantity of estrogens produced in female organisms is related to the emergence of some kinds of cancers, particularly estrogen-dependent breast cancer. In this case, the attachment of estrogen to specific estrogen receptors releases a signal, which stimulates the proliferation of breast cancer tumor cells. Approximately 80% of breast cancer is estrogen receptor (ER)-positive [28].
Blocking the enzyme aromatase through the use of aromatase inhibitors is able to stop the production of estrogen, which contributes to decreased breast cancer risk [29]. One group of potent inhibitor compounds is isoflavones, which can be used to decrease cancer risk by inhibiting aromatase enzyme activity and CYP19 gene expression in human tissues [30].
It should be noted that aromatase inhibitors cannot stop estrogen production in the ovaries, but they block their synthesis in secondary sources of estrogens, particularly in the breast; consequently, aromatase inhibitors are only effective for postmenopausal women [31].
Molecular docking using AutoDock Tools 1.5.4 was performed in order to evaluate the inhibitory potential of 50, 52 and 54 against the target protein aromatase (CYP19A1). Obtained binding modes and docking energies of 50, 52 and 54 were compared with those of the reference androgen, androstenedione. The results of the free binding energies (∆Gb), calculated by AutoDock, are summarized in Table 2.
A careful analysis of the enzyme binding pocket indicated that it is highly hydrophobic, and it was assembled by the condensation of non-polar aliphatic amino acid residues. Therefore, only inhibitors bearing alkyl or aromatic groups are expected to bind with high affinity [35].
Compounds 50, 52 and 54 are small hydrophobic molecules consisting of two benzene rings linked by a heterocyclic pyran ring. These interact with the protein CYP19A1, mainly through hydrophobic and some hydrogen bonds ( Figure 6).
The most potent ligand, 52, was found to exhibit hydrophobic alkyl interactions with the key residues Leu477, Met374, Val370, Ala306 and Ile133 at the active site, whereas a H-bond interaction was observed between the NH group of Arg115 and the oxygenbearing ketone group of 52, which tend to form a stable binding interaction ( Figure 6A). Compounds 50 and 54 interact with important amino acids in the active site of the target CYP19A1, specifically via electrostatic and hydrophobic interactions. H-bonding to the target CYP19A1 was not observed for either 50 or 54 ( Figure 6B,C). In addition, the reference hormone, androstenedione, which fits within the active site cavity, displayed two hydrogen bonds with Arg115 and Met374, one unfavorable donor-donor bond with Asp309 and one carbon-hydrogen bond with Ala306. However, the two residues Trp224 and Val370 were involved in the alkyl interaction ( Figure 6D). Antibiotics 2022, 11, x FOR PEER REVIEW 10 of 18

Molecular Dynamics Simulation
Molecular dynamics (MD) simulations were performed for a 100 ns time interval to understand the dynamic behavior and evaluate the stability of the docked complexes 3EQM-50, 3EQM-52 and 3EQM-54. Following the completion of MD simulations, a root mean square deviation (RMSD) evaluation was carried out and used to measure the natural change in particle removal for a given frame as compared to a standard constant frame. Protein-ligand RMSD values are depicted in Figure 7.  Intramolecular interactions of 50, 52 and 54 with the human placental aromatase cytochrome P450 target are observed in Figure 9.
Hydrophobic interactions were identified with higher numbers during the MD simulation event for the three inhibitors ( Figure 9). Furthermore, the interactions of the residues of the target protein, 3EQM, with the three inhibitors 50, 52 and 54 showed that Phe134 and Met374 interacted with 52 through a hydrophobic moiety bond and hydrogen bond through a water molecule for 58% and 45% of the simulation time, respectively (Figure 9B). However, the catalytic residue Phe134 of 3EQM formed a hydrophobic interaction with 54 for 46% of the simulation time ( Figure 9C). The RMSD plots in Figure 7 reveal that the complex 3EQM-50 ( Figure 7A) stabilized after 35 ns, while the complex 3EQM-54 ( Figure 7C) remained stable over the trajectory of the 100 ns simulation, with a slight perturbance near 78 ns. However, the complex 3EQM-52 ( Figure 7B) showed stability from initiation to the end of the MD simulation event. Furthermore, the RMSD values of the three complexes remained under the 3 Å range, which is within the acceptable region and indicates that these complexes were stable throughout the simulation. Figure 8 illustrates the ligand properties, including RMSD, rGyr (radius of gyration), intraHB (intramolecular hydrogen bonds), MolSA (molecular surface area), SASA (solventaccessible surface area) and PSA (polar surface area), of all MD complexes.
The RMSD values for the three complexes 3EQM-50 ( Figure 8A), 3EQM-52 ( Figure 8B) and 3EQM-54 ( Figure 8C     Hydrophobic interactions were identified with higher numbers during the MD simulation event for the three inhibitors ( Figure 9). Furthermore, the interactions of the residues of the target protein, 3EQM, with the three inhibitors 50, 52 and 54 showed that Phe134 and Met374 interacted with 52 through a hydrophobic moiety bond and hydrogen bond through a water molecule for 58% and 45% of the simulation time, respectively ( Figure 9B). However, the catalytic residue Phe134 of 3EQM formed a hydrophobic interaction with 54 for 46% of the simulation time ( Figure 9C).
Molecular identification of strain CG3, based on the sequencing of 16s rDNA, indicated that this isolate is closely related to Nocardiopsis rosea YIM 90094 T (99.2%). Strain CG3 was distinguished from its closest species based on the results of phylogenetical, morphological, physiological and biochemical characterization. Therefore, strain CG3 was identified as a novel species within the genus Nocardiopsis [36].

Preparation of Suspension
Antimicrobial activity was evaluated against ten different microorganisms, including eight bacteria and two fungi, which displayed a wide array of differences in their taxonomic positions, morphological and physiological characteristics, and their virulence.
Among bacteria, four are Gram-positive and belong to two different phyla, among which three are Firmicutes (Micorococcus luteus DSM1790, Staphylococcus aureus Newman and Bacillus subtilis DSM10), and one belongs to the Actinobacteria phylum (Mycobacterium smegmatis ATCC 700084). This last one is a fast grower and non-pathogenic model for research on a new anti-tuberculosis drug. In addition, four Gram-negative bacteria were used, which all belong to the phylum of Proteobacteria (Chromobacterium violaceum DSM 30191, Pseudomonas aeruginosa PA14, Klebsiella pneumoniae ATCC and Escherichia coli TolC).
Three of the bacteria used belong to the ESKAPE group, namely, Pseudomonas aeruginosa PA14, Klebsiella pneumoniae ATCC and Staphylococcus aureus Newman, which includes highly virulent and antibiotic-resistant pathogens. Furthermore, two fungi were used, including one yeast (Candida albicans DSM1665) and one mold (Mucor hiemalis DSM 2656).
The selected microorganisms were obtained from DSMZ (German Collection of Microorganisms and Cell Cultures), Braunschweig, Germany, and ATCC (American Type Culture Collection), Manassas, VA, USA.
After two weeks of incubation in a rotary shaker at 37 • C and 160 rpm, 20 mL of each culture was taken and mixed with 20 mL of ethyl acetate in two Falcon tubes (50 mL). The two tubes were shaken for 20 min, followed by a centrifugation step at 9000 rpm for 10 min. The ethyl acetate was evaporated at 40 • C using a rotary evaporator, and the residue was dissolved in 1 mL of methanol and then centrifuged at 14,000 rpm for 10 min [39].

Serial Dilution Method for Antimicrobial Activity
Minimum inhibitory concentrations, corresponding to the lowest concentration of the tested extract that prevents visible growth of tested microorganisms, were determined by serial dilution in 96-well microplate. Twenty-microliter aliquots with a concentration of 1 mg/mL (the final concentration in the first well was 67 µg/mL) of crude extracts prepared from the culture of strain CG3 in SM and ISP2 media were tested against different tested microorganisms. Oxytetracycline and nystatin were used as positive controls for antibacterial and antifungal activities, respectively, while methanol was used as a negative control [40].

Analytical HPLC and Fractionation of Crude Extract
Analytical RP-HPLC and fractionation were conducted with an Agilent 1260 HPLC system equipped with a fraction collector. Detection of peaks was performed using a diodearray UV detector (DAD-UV, Santa Clara, CA, USA) or a Corona Ultra detector (Dionex, Germering, Germany). Analytical HPLC conditions: column 100 × 2.1 mm XBridge C 18  In order to localize the biological activity, the crude extract prepared from the culture of strain CG3 in SM was fractionated. Each fraction was collected in a 96-well microplate every 30 s. The solvent was removed with heated nitrogen in MiniVap (Porvair Sciences, Wrexham, UK) for 45-60 min at 40 • C. Afterward, each well of the 96-well microplate was inoculated with 150 µL of the suspension prepared from the former inhibited test microorganism. After incubation at 30 • C for 24 h, the inhibited wells can be correlated to the retention time (t R ) and the corresponding peak.

Metabolic Profile
Metabolomics is an approach used to study metabolites secreted by microorganisms as well as plants; it can be defined as the "systematic study of the unique chemical fingerprints that specific cellular processes leave behind". The metabolic profile is determined using different techniques; however, high-performance liquid chromatography coupled with high-resolution mass spectrometry (HPLC-ESI-HRMS) is a powerful analytical tool for metabolic profiling that can detect a wide range of chemical compounds at the same time without purification [41,42].
The crude extract prepared from the culture of strain CG3 in SM was analyzed by LC-HRESIMS, and results were recorded on a MaXis ESI-TOF mass spectrometer (Bruker) equipped with an Agilent 1260 series RP-HPLC system: column 50 × 2.1 mm Acquity UPLC BEH C 18 (Waters); solvent A: 0.1% formic acid in H 2 O, B: 0.1% formic acid in acetonitrile; gradient system: 5% B for 0.5 min, increased to 100% B in 19.5 min and maintained at 100% B for 5 min; flow rate: 0.6 mL/min; 40 • C; DAD-UV detection at 200−600 nm. Molecular formulas were calculated, including the isotopic pattern, with the SmartFormula algorithm (Bruker, Billerica, MA, USA). Detected compounds were identified by comparison of molecular weight, molecular formula and UV-Visible spectrum with already-known compounds registered in chemical databases, such as Dictionary of Natural Products, which is the most comprehensive resource of natural chemical products.

Protein and Ligand Preparation
The crystal structure of the target protein, aromatase, was retrieved from Research Collaboratory for Structural Bioinformatics (RCSB) in PDB format (PDB code: 3EQM). The aromatase protein structure was prepared for docking using BIOVIA Discovery Studio Visualizer 2020 after removal of water molecules and the original ligand attached to the target protein. Polar hydrogen atoms and Kollman charges were added using AutoDock Tools 1.5.6. Finally, the target protein was saved as a PDBQT file, whereas the three ligands, 50, 52 and 54, were prepared for docking by energy minimization using the Gasteiger algorithm, detecting root and a set of torsions [43].

Molecular Docking Analysis
Molecular docking calculations were performed with the program AutoDock Tools 1.5.4 using the Lamarckian Genetic Algorithm. AutoGrid was used to generate a grid box size of 50 × 64 × 78 Å points with a grid spacing of 0.375 Å, centered at x, y and z coordinates of 83.35, 49.60 and 50.60, around the hotspot residues in the active site of the target 3EQM [34].
The employed docking parameters for each docked compound were derived from 100 independent docking runs that were set to terminate after a maximum of 2.5 × 10 6 energy evaluations with mutation rate of 0.02 and crossover rate of 0.8. The population size was set to 250 randomly placed individuals. The Lamarckian genetic algorithm was used, and the output was saved in docking parameter file (DPF) format. The predicted binding poses for each compound were processed by 0 clustering analysis (1.0 Å RMSD tolerance), and the lowest energy conformation from the largest cluster was selected as representative. Discovery Studio and PyMOL were implemented to visualize and scrutinize the interactions between the ligand fragments and aromatase protein [34].

Molecular Dynamics Simulation
Molecular dynamics simulation was used to assess the physical motions of atoms and molecules in a protein-ligand docked complex. Desmond [44] (Schrödinger Release March 2019) was used to run molecular dynamics simulations with the human placental aromatase cytochrome P450 and three compounds. A simulation length of up to 100 ns for each run was conducted, and different parameters were computed to determine if the systems were stable. To allow complex relaxation, these complexes were prepared using a protein preparation wizard. Certain predefined parameters were considered for the simulation of cell preparation, which includes adding hydrogens, removing water, assigning bond orders, and filling in missing side chains and loops with optimization of hydrogen-bond assignment (sampling of water orientations and use of pH 7.0). The simulation periodic box was created with the System Builder module and transferable intermolecular potential with 3 points (TIP3P) water model and an all-atom force field from optimized potentials for liquid simulations (OPLS). An orthorhombic box shape with dimensions of 10 * 10 * 10 was used to define boundaries for the Na and Clneutralization process. The NPT ensemble (number of atoms, pressure and temperature were constant) contains 300 K temperature and 1.01325 bar pressure to equilibrate the unrestrained processing process for 100 ns time interval. The isotropic Martyna-Tobias-Klein barostat (relaxation time = 2 ps) and the Nosé-Hoover thermostat (relaxation time = 1 ps) were used. The smooth particle mesh Ewald (PME) method (PME) was used to calculate short-(cutoff = 9 Å) and long-range Coulombic interactions using RESPA integrator. The conformations captured within the simulation trajectories were exported every 5 ps. The system stability was assessed through root mean square fluctuations (RMSF), hydrogen bond analysis, radius of gyration (Rg) and a histogram for torsional bonds after the completion of the MD simulation.