A Comprehensive In Silico Study of New Metabolites from Heteroxenia fuscescens with SARS-CoV-2 Inhibitory Activity

Chemical investigation of the total extract of the Egyptian soft coral Heteroxenia fuscescens, led to the isolation of eight compounds, including two new metabolites, sesquiterpene fusceterpene A (1) and a sterol fuscesterol A (4), along with six known compounds. The structures of 1–8 were elucidated via intensive studies of their 1D, 2D-NMR, and HR-MS analyses, as well as a comparison of their spectral data with those mentioned in the literature. Subsequent comprehensive in-silico-based investigations against almost all viral proteins, including those of the new variants, e.g., Omicron, revealed the most probable target for these isolated compounds, which was found to be Mpro. Additionally, the dynamic modes of interaction of the putatively active compounds were highlighted, depending on 50-ns-long MDS. In conclusion, the structural information provided in the current investigation highlights the antiviral potential of H. fuscescens metabolites with 3β,5α,6β-trihydroxy steroids with different nuclei against SARS-CoV-2, including newly widespread variants.


Introduction
The global pandemic caused by coronavirus 2 (SARS-CoV-2) has taken many lives [1,2] from 2019 until the present. Several variants and outbreaks are still present in countries such as China and Korea [3].
Coronaviruses are a family of viruses that can cause a wide array of enteric, hepatic, neurological, and respiratory diseases [1]. The four subfamilies of coronavirus are α, β, γ, and δ, and are sorted based on genotype and serotype data [4]. Six species of coronaviruses cause human diseases. The most pathogenic and fatal species are Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) and Middle East Respiratory Syndrome Coronavirus (MERS-CoV). SARS-CoV-2 is the seventh type, which infects humans and is related to the β lineage of the beta-coronaviruses, which are known to cause severe disease and fatalities [4]. Based on genomic analysis, SARS-CoV-2 belongs to two bat-derived SARSlike coronaviruses with 96% similarity, whereas its similarity to SARS-CoV is 79% [5]. The lung is the primary site and the most affected organ of SARS-CoV-2 infection. The clinical manifestations range from asymptomatic to severe respiratory disease. The most common symptoms of infection are a loss of smell and taste, fever, headache, shortness of breath, cough, muscle aches, and tiredness [6].
The HMBC spectrum ( Figure 2 and Figure S6) clearly illustrated the attachment of H 3 -15 at C-4 via the presence of a correlation between the two ortho-coupling protons H-2 and H-3, and the observed correlations between H 3 -15 and C-3, C-4, and C-5. The second quaternary methyl H 3 -14 was attached to the oxygenated carbon C-10 based on the existence of cross-peaks between H 3 -14 and C-1, C-9, and C-10. The second oxygenated carbon was C-9, based on the downfield shift of H-9 δ H 4.0 (dd, J = 11.7, 3.9 Hz, H-9), while the isopropyl moiety was attached to C-7 based on the HMBC correlations between H 3 -12 and C-7, C-11, and C-13; and between H 3 -13 and C-7, C-11, and C-12, respectively. The 1 H-1 H COSY spectrum ( Figures S7 and S8) illustrated the presence of two discrete spins corresponding to H-2/H-3 in the aromatic ring and H-9/H 2 -8, H 2 -8/H-7, H-7/H-11, H-11/H 3 -12, and H-11/H 3 -13, which confirmed the overall structure of compound 1.
protons, as well as the structure of compound 1, was elucidated based on the exten analysis of 2D NMR ( 1 H-1 H COSY, HSQC, and HMBC).

Molecular Docking and Molecular Dynamic Simulation Results
To putatively determine the most probable molecular target for compounds 1-8, we virtually screened their structures against all currently available SARS-CoV-2 proteins that have been reported to be relevant to the viral life cycle or viral pathogenesis (https: //swissmodel.expasy.org/repository/species/2697049; https://www.genome.jp/keggbin/show_pathway?hsa05171+H02398 (accessed on 1 September 2022); Table S1). Both M pro and Cyclin-G-associated kinase (GAK) were the only proteins that were found to have considerable docking scores (<−8.0 kcal/mol) with compounds 4, 7, and 8 (for M pro ) and compounds 3-5 and 8 (for GAK). The subsequent MDS validation experiments (50 ns long) revealed that the structures of compounds 4-7 and 8 were not good binders to GAK, because they were significantly unstable over the course of MDS, with an average RMSD ranging from 6Å to > 10 Å. Moreover, their calculated absolute binding free energy values (∆G binding ) indicated a low affinity toward GAK's binding site, and ranged from −2.5 to −3.2 kcal/mol.
In contrast, the structures of compounds 5, 7, and 8 have achieved stable binding over the course of 50 ns MDS runs, with average RMSDs ranging from 1.8 Å to 2.2 Å, and with ∆G binding values of −8.6, −8.8, and −8.1 kcal/mol, respectively. Interestingly, the three steroids that shared the stable binding had a characteristic hydroxylation pattern at positions 3, 5, and 6 in rings A and B. In addition, we have reported that the triterpene class of compounds, which is structurally related to compounds 5, 7, and 8, is a promising scaffold to develop new M pro inhibitors [45,46].
To investigate the binding mode of each compound inside the M pro 's active site, the most populated binding poses for each compound structure was extracted from their 50-ns-long MDS trajectories.
As shown in Figure

Discussion
In the previous literature, several bioactive metabolites were isolated from soft corals, which have the ability to inhibit the SARS-CoV-2 M pro , as depresosterol, lopophytosterol, and cholest-5-ene-3β,7β-diol [47]. These compounds form stable hydrogen bonding through a polyhydroxy group in their structure with the amino acid in the active pocket of the SARS M pro [47]. In the genus Heteroxenia (family Xeniidae), several polyoxygenated steroids were isolated and showed diverse biological activity [24,25,27,28,42].

Discussion
In the previous literature, several bioactive metabolites were isolated from soft corals, which have the ability to inhibit the SARS-CoV-2 M pro , as depresosterol, lopophytosterol, and cholest-5-ene-3β,7β-diol [47]. These compounds form stable hydrogen bonding through a polyhydroxy group in their structure with the amino acid in the active pocket of the SARS M pro [47]. In the genus Heteroxenia (family Xeniidae), several polyoxygenated steroids were isolated and showed diverse biological activity [24,25,27,28,42].
From the previous data, it can be concluded that GLN-189 was the key amino acid residue interacting with the three structures (i. e., compounds 5, 7, and 8) via H-bonding, while both the LEU-27 and MET-49 amino acid residues were the only residues hydrophobically interacting with them. The active steroids (5, 7, and 8) were interacting with three of the amino acids (GLN-189, LEU-27, and MET-49), which were included in the active site pockets of the SARS-CoV-2 M pro [5], illustrating its possible activity. The dynamic behaviors of the three structures were convergent, where they were highly fluctuating over the course of the simulation, in comparison with the co-crystalized inhibitor (Figure 4). Their average RMSDs were 2.4 Å, 2.6 Å, and 1.3 Å for compounds 5, 7, and 8, respectively ( Figure 4). The calculated radius of the gyration profile of M pro in complex with each compound was also consistent, with an average of~18.4 Å ( Figure 5). With regard to the interaction energy of the three structures over the course of the simulation, they showed stable interaction energy profiles (i.e., electrostatic and van der Waals interaction energies), with an average of~-80 kcal/mol ( Figure 6). Accordingly, the calculated MM-PBSA binding energy of each compound (Table 3) was found to be convergent with or even higher than that of the co-crystalized inhibitor ML188, indicating the potential inhibitory activity of these compounds against the viral M pro . Such structural information will be of great interest during the future development of novel M pro inhibitors, depending on the scaffolds of these compounds (i.e., compounds 5, 7, and 8). active site pockets of the SARS-CoV-2 M pro [5], illustrating its possible activity. The dynamic behaviors of the three structures were convergent, where they were highly fluctuating over the course of the simulation, in comparison with the co-crystalized inhibitor ( Figure 4). Their average RMSDs were 2.4 Å, 2.6 Å, and 1.3 Å for compounds 5, 7, and 8, respectively (Figure 4). The calculated radius of the gyration profile of M pro in complex with each compound was also consistent, with an average of ⁓ 18.4 Å ( Figure 5). With regard to the interaction energy of the three structures over the course of the simulation, they showed stable interaction energy profiles (i.e., electrostatic and van der Waals interaction energies), with an average of ⁓ -80 kcal/mol ( Figure 6). Accordingly, the calculated MM-PBSA binding energy of each compound (Table 3) was found to be convergent with or even higher than that of the co-crystalized inhibitor ML188, indicating the potential inhibitory activity of these compounds against the viral M pro . Such structural information will be of great interest during the future development of novel M pro inhibitors, depending on the scaffolds of these compounds (i.e., compounds 5, 7, and 8).    With regard to the probable inhibitory activity SARS-CoV-2's M pro towards the isolated sesquiterpenes and sterols, docking, followed by MDS experiments, revealed that the polyhydroxylated sterols have good potential to bind with and to inhibit the enzyme's catalytic activity. According to the dynamic binding mode analysis, the stability of such derivatives was achieved inside the enzyme's active site through the formation of multiple H-bonds between the compounds' hydroxyl groups and a number of hydrophilic amino acids. Hence, losing such essential hydroxyl groups or masking them via acetylation might lead to unstable binding (i.e., compounds 3, 4, and 6). Previously, a number of polyhydroxylated triterpenes (e.g., ursolic and maslinic acids) were found to significantly inhibit SARS-CoV's M pro [48,49]. Hence, such steroidal or triterpenoidal scaffolds may be promising in the future development of novel M pro inhibitors.

General Experimental Procedures
Optical rotations were recorded on a Jasco DIP-370 polarimeter. The 1D ( 1 H and 13 C) and 2D (HSQC, HMBC, 1 H-1 H COSY, and NOESY) NMR experiments were recorded on a Bruker DRX 600 NMR spectrometer (Bruker, Billerica, MA, USA). HR-ESI-MS and HR-FAB-MS were measured on an LC-MS-Q-TOF (Agilent Tokyo, Japan) and a JMS-700 mass spectrometer (JEOL, Tokyo, Japan), respectively. Coupling constants are expressed in Hz, and chemical shifts in δ (ppm). Chromatographic separations were performed using column chromatography on a Merck silica gel (70-230), and Medium-Pressure Liquid Chromatography (MPLC) (Büchi Reveleris ® Prep system, Flawil, Switzerland) was performed using a silica gel cartridge (40 μM, 12 g) and a C-18 cartridge (WP, 20 μM, 4 g) with a UV-ELSD detector. Thin-layer chromatography (TLC) was performed on glass pre-coated sil- With regard to the probable inhibitory activity SARS-CoV-2's M pro towards the isolated sesquiterpenes and sterols, docking, followed by MDS experiments, revealed that the polyhydroxylated sterols have good potential to bind with and to inhibit the enzyme's catalytic activity. According to the dynamic binding mode analysis, the stability of such derivatives was achieved inside the enzyme's active site through the formation of multiple H-bonds between the compounds' hydroxyl groups and a number of hydrophilic amino acids. Hence, losing such essential hydroxyl groups or masking them via acetylation might lead to unstable binding (i.e., compounds 3, 4, and 6). Previously, a number of polyhydroxylated triterpenes (e.g., ursolic and maslinic acids) were found to significantly inhibit SARS-CoV's M pro [48,49]. Hence, such steroidal or triterpenoidal scaffolds may be promising in the future development of novel M pro inhibitors.

Animal Material
Heteroxenia fuscescens soft coral was collected and identified by Dr. Aldoushy Mahdy (Faculty of Science, Al-Azhar University, Assiut branch, Egypt) from the Red Sea in front of the National Institute of Oceanography and Fisheries, Hurghada, Egypt. A voucher sample (HF 20) has been deposited at the Pharmacognosy Department, Faculty of Pharmacy, Al-Azhar University, Assiut Branch, Egypt.

Conclusions
This study highlighted the significance of identifying antiviral secondary metabolites from the Red Sea's soft corals as naturally occurring SARS-CoV-2 inhibitors. A new steroid and a new sesquiterpene, along with six known compounds, were effectively isolated and structurally characterized, and they were essentially evaluated via in silico study against SARS-CoV-2 proteins. Through a stable hydrogen bond, the purified steroids containing a (3β,5α,6β-trihydroxy) moiety interact with the amino acid residue GLU-189, which highlights it as a perfect framework for the future creation of SARS-CoV-2 M pro inhibitory medications.