Isolation, Crystal Structure, and In Silico Aromatase Inhibition Activity of Ergosta-5, 22-dien-3 β -ol from the Fungus Gyromitra esculenta

exhibited copious promising biological activities. The fungus Gyromitra esculenta is widely distributed in Europe and North America. In order to examine the chemical properties of Gyromitra esculenta , a phytochemical study has been preceded and resulted in the isolation of the steroid, ergosta-5, 22-dien-3 β -ol (brassicasterol), from its methanol extract. The complete identiﬁcation and absolute conﬁguration of the isolated compound have been established by X-ray structural analysis to be (22E, 24R)-24-methylcholesta-5, 22-dien-3beta-ol. The reported cytotoxicity and the great structural similarity of the isolated compound with the cocrystallized ligand of the aromatase enzyme inspired us to run molecular docking studies against that protein. Ergosta-5, 22-dien-3 β -ol occupied the target protein with a binding mode almost the same as the cocrystallized ligand and a binding aﬃnity of − 33.55kcal/mol, which was better than that of the cocrystallized ligand ( − 22.61kcal/mol). This promising result encouraged us to conduct in silico ADMETand toxicity studies of ergosta-5, 22-dien-3 β -ol against 6 models, and the results expected the likeness of the isolated compound to be a drug. In conclusion, ergosta-5, 22-dien-3 β -ol has been isolated from Gyromitra esculenta , identiﬁed by X-ray structural analysis, and exhibited promising in silico activities against aromatase enzyme.

Gyromitra esculenta is a fungus that belongs to the genus Gyromitra which is widely distributed across Europe and North America. It normally fruits in spring and early summer in sandy soils under the coniferous trees. e fruiting body (mushroom) is an irregular brainshaped dark brown cap that can reach 10 cm in height and 15 cm in width, perched on a stout white stipe up to 6 cm in hight [27,28]. Despite the reported toxicity of G. esculenta, it is still consumed and used in some countries in North America and Europe due to its high nutritive value [29,30].
Ergosterol and its derivatives were reported to have various cytotoxic effects. As an example, ergosterol could inhibit in vitro and in vivo cancer growth through upregulation of multiple tumor suppressors [31,32]. Furthermore, ergosterol peroxide exerted promising antitumor activities in colorectal cancer [33] and several other tumor types [34]. Additionally, dehydroergosterol derivatives induced apoptosis in human malignant melanoma cells [35] and inhibited the growth of human breast adenocarcinoma MCF-7 cells [36]. Interestingly, a dehydroergosterol derivative isolated from Ganoderma lucidum inhibited the proliferation of human cervical carcinoma cells with an IC 50 value of 8.58 μM through induction of apoptosis [37]. Some ergosterol derivatives displayed anticancer activities through the inhibition of aromatase protein [38,39].
Compound 1 (brassicasterol) has been isolated before from several plant sources such as the steam distillate of rapeseed oil [40] and Brassica juncea seeds [41]. Moreover, it was found in algae [42] and marine organisms [43]. In addition to the anti-inflammatory effect [44], brassicasterol could inhibit bladder carcinogenesis in an in vivo study [45].
X-ray crystallography is the technique that uses the ability of X-rays to be diffracted by a crystalline structure into different specific directions. e angles and intensities of the diffracted X-ray beams could be used to determine the three-dimensional picture of the electron density of the diffracting crystal. Consequently, the atoms' positions, chemical bonds, absolute configuration, and several other information can be determined [46].

X-Ray Analysis.
In order to confirm the absolute configuration of 1, the crystal structure of its crystalline hydrate was investigated by X-ray diffraction analysis. Compound 1 crystallized in the Sohnke space group P2 1 , with the asymmetric unit consisting of two dehydroergosterol molecules and two hydrate water molecules ( Figure 2).
It follows from the data obtained that the bond lengths and bond angles in compound 1 are close to the usual ones [48]. Ring A takes a somewhat distorted chair conformation in the first (1a) and second (1b) crystallographically independent molecules (the minimum parameters of cycle asymmetry [49] and intracyclic torsion angles are given in Table 2). e conformation of cycle B in 1a and 1b, containing the C5 = C6 double bond, is close to the slightly distorted 9α, 8-half-chair. e third carbocycle C in molecules 1a and 1b deviates more significantly from the ideal chair. e 5-membered cycle D in molecule 1a takes the conformation of a 13β-envelope, strongly distorted towards the 14α, 13β-half-chair. In the second molecule 1b, this cycle takes the conformation of the 14α, 13β-half-chair, strongly distorted towards the 13β-envelope. In general, distortions of the conformation of A-D cycles are close to those observed in the crystal structures of 3β-hydroxy-Δ5-sterols, for example, in 24 (R), 25-epoxycholesterol [50], stigmast-5-en-3-ol [51], (24E)-26-hydroxydesmosterol [52], and (24E)-26hydroxydesmosterol monohydrate [53].  In the crystal, molecules 1a and 1b and molecules of hydration water are linked by intermolecular hydrogen bonds (Table 3), forming infinite ribbons in the (−1 0 2) plane along the b-axis (Figure 3).

Crystallographically independent molecule 1a
Crystallographically independent molecule 1b Torsion angles τ ΔC Torsion angles molecule. We depended on the binding free energy (∆G) and the correct binding mode between the docked molecules and the active site of aromatase. e binding free energies are summarized in Table 4.
At first, 3D-flexible alignment of ergosta-5, 22-dien-3βol with the cocrystallized ligand (EXM) was carried out (Figure 4). From 3D-flexible alignment, it was observed that the structure of ergosta-5, 22-dien-3β-ol has a good overlap with the cocrystallized ligand (EXM). en, validation of the docking process was checked through the running of the docking procedure for only the cocrystallized ligand (EXM) against the active pocket of aromatase. It was found that the produced RMSD value between the generated pose of the docked molecule and the original one equals 0.90. is indicates the validity of the docking process ( Figure 5). e binding mode of the cocrystallized ligand (EXM) as a reference molecule showed a binding free energy of −22.61 kcal/mol (Table 3). e binding interaction showed that the steroidal nucleus overlapped with the hydrophobic environment of the binding pocket of the aromatase receptor.
e methyl group at position-13 formed two hydrophobic interactions with Leu477 and Val370. Besides, the C ring of steroidal moiety was involved in hydrophobic interactions with Ile133. e B ring was engaged in two hydrophobic interactions with Val370 and Cys437. e methyl group at position-10 formed two hydrophobic interactions with Cys437 and Val370. Finally, the methylene group at position-6 was involved in two hydrophobic interactions with Cys437and Val373 (Figure 6).
Brassicasterol interacted with the active site of aromatase showing a binding mode almost the same as that of the cocrystallized ligand with extra hydrogen bonding and hydrophobic interactions. Ergosta-5, 22-dien-3β-ol exerted a binding affinity of −33.55 kcal/mol, which was higher than that of the cocrystallized ligand. e binding mode revealed the orientation of the steroidal nucleus of the docked molecule toward the hydrophobic pocket of the aromatase receptor. e D ring of the steroidal moiety was involved in hydrophobic interaction with Val370, Val373, and Cys437.
e methyl group at position-13 formed two hydrophobic interactions with Trp224 and Ile133. Also, the C ring of the steroidal moiety was involved in hydrophobic interaction with Ile133. e B ring was engaged in two hydrophobic interactions with Ile133 and Cys437. e A ring formed three hydrophobic interactions with Ile133, Ala306, and Cys437. e methyl group at position-10 formed two hydrophobic interactions with Cys437 and Ile133. e hydroxyl group at position-3 was involved in hydrogen bonding interaction with Gly349. Finally, the side chain (5, 6-dimethylhept-3-ene) moiety formed extra hydrophobic interactions with Val369, Val370, Leu477, Phe221, and Trp224 ( Figure 7).

In Silico ADMET Analysis.
ADMET studies were carried out for ergosta-5, 22-dien-3β-ol and the cocrystallized ligand. Discovery studio 4.0 was used to predict ADMET descriptors for all compounds. e predicted descriptors are listed in Table 5.

Isolation of Brassicasterol (1).
e raw material of G. esculenta was collected in summer in the vicinity of the city of Karkaraly, Karaganda Region, Kazakhstan. G. esculenta was extracted via adding 300 mL of MeOH to 207.5 g of a semidried powder and sonicated at 40-50°C for 3 hrs. e procedure was repeated 3 times per day. e obtained extracts were combined and evaporated under reduced pressure. Total weight of the obtained extract was -62.9 g. e total extract was subjected to a SiO 2 column (400 g) using hexane-EtOAc and CH 2 Cl 2 -MeOH as mobile phases in a manner of increasing polarity. Pure white-colored crystal of 1 was obtained from fraction 54 (hexane-EtOAc 1 : 10).

X-Ray
Analysis. X-ray intensity data for the compound C 28 H 46 O·H 2 O were collected at 100 K, on a Rigaku Oxford Diffraction Supernova Dual Source (Cu at zero) diffractometer equipped with an Atlas CCD detector using ω scans and CuKα (λ = 1.54184Å) radiation. e images were interpreted and integrated with the program CrysAlisPro [60]. Using Olex2 [61], the structure was solved by direct methods using the ShelXT structure solution program and refined by full-matrix least squares on F [2] using the ShelXL program package [62,63]. Nonhydrogen atoms were anisotropically refined, and the hydrogen atoms in the riding mode were with isotropic temperature factors fixed at 1.2 times U (eq) of the parent atoms (1.5 times for methyl and hydroxyl groups). e absolute configuration was established showing a refined Flack parameter of 0.0 (2).
CCDC-2060747 contains the supplementary crystallographic data for this paper. ese data can be obtained free of charge from e Cambridge Crystallographic Data Centre via http://www.ccdc.cam.ac.uk/structures. Details of the X-ray crystal structure data collection and refinement are given in Table 7.

Conclusions
Ergosta-5, 22-dien-3β-ol (1) was isolated from a methanol extract of the fungus Gyromitra esculenta. e absolute configuration of 1 was determined by X-ray structural crystallography. Ergosta-5, 22-dien-3β-ol occupied the binding site of the aromatase enzyme with a binding mode very similar to that of the cocrystallized ligand and a binding affinity of −33.55 kcal/mol, which was higher than that of the cocrystallized ligand (−22.61 kcal/ mol). In silico ADMET and toxicity studies against 6 models have been conducted, and the results expected the safety of 1 with a disadvantage of poor water solubility and absorption.

Data Availability
Details of the in silico experimental part and NMR data of compound 1 are available in the supplementary data. Also, CCDC-2060747 contains the supplementary crystallographic data for this paper. ese data can be obtained free of charge via https://www.ccdc.cam.ac.uk/structures.

Conflicts of Interest
e authors declare no conflicts of interest.

Authors' Contributions
Yerlan Melsuly Suleimen was responsible for collection of raw material, extraction of raw material, isolation of compounds, and identification of spectra. Ahmed M. Metwaly contributed to identification of spectra and doking studies of the compound. Ahmad E. Mostafa and Eslam B. Elkaeed contributed to doking studies of the compound. Hong-wei Liu and Buddha Bahadur Basnet took part in isolation of compound. Raigul Nurbekkyzy Suleimen was responsible for identification of spectra. Margarita Yulayevna Ishmuratova contributed to collection of raw material and identification of mushroom. Koblandy Muboryakovich Turdybekov took part in interpretation of X-ray analysis. Kristof Van Hecke was responsible for X-ray analysis and interpretation of results of it.