Chemical studies on Ophiocoma dentata have resulted in discovering a new steroid, 5α-cholesta-4(27), 24-dien-3β, 23 β-diol. The structure of the isolated compound was determined with the help of spectroscopic studies. The isolated compound’s NMR spectra matched those of 5 α-cholesta-9(11),24- dien-3 β,6 α,20β-triol-23-one 3-sulphate, reported by Yang et al.16, except for an absence of hydroxyl group at C-6, C-20 position and the double bond between C-9 and C-11 as well as additional double bond at C-4 and this was confirmed by the HMBC experiment. Inspection of its NMR data indicated a considerable similarity to those of ophidianoside F17 except for an absent hydroxyl group at C-6, C-20 and the double bond between C-9 and C-11, and the presence of a double bond between C-24 and C-25 position.
Docking studies were carried out using MOE14.0 software, yielding free energy (ΔG) values that indicate the examined molecule’s binding interaction with the selected protein.
The isolated compound demonstrated good binding affinities with COVID-19 main protease (ΔG = − 24.68 kcal/mol), nsp10 (ΔG = − 23.47 kcal/mol), and RNA-dependent RNA polymerase (ΔG = − 29.86 kcal/mol), compared to the co-crystallized ligands PRD_002214 (ΔG = − 27.72 kcal/mol), SAM (ΔG = − 17.86 kcal/mol), and F86 (ΔG = − 23.56 kcal/mol), respectively (Table 3).
The docking mode of the co-crystallized ligand (PRD_002214) and COVID-19 main protease formed four hydrogen bonds and three hydrophobic interactions. The first pocket of Mpro was occupied by 2-oxopyrrolidin-3-yl moiety, forming two hydrogen bonds with Thr190 and Gln189. Additionally, the isopropyl moiety occupied the second pocket of Mpro forming three hydrophobic interactions with His41 and Met165. Furthermore, the third pocket was occupied by the benzyl acetate moiety, whereas the 5-methylisoxazole-3-carboxamide moiety was buried in the fourth pocket, forming a hydrogen bond with Thr26. Finally, one hydrogen bond was formed between an amide group and Met165 (supplementary Fig. S10).
Moreover, the co-crystallized ligand (SAM) formed three hydrogen bonds and seven hydrophobic interactions against the COVID-19 nsp10 protein. In detail, the tetrahydrofuran-3,4-diol moiety formed three hydrogen bonds with Asn6899, Tyr6930, and Asp6928. Besides, the 9H-purin-6-amine moiety formed three hydrophobic interactions with Phe6947 and Leu6898. Also, the (S)-(3-amino-3-carboxypropyl) dimethylsulfonium moiety formed many hydrophobic, electrostatic, and hydrogen bonding interactions with Asp6897, Lys6968, Lys6844, and Asp6928 (supplementary Fig. S11).
With respect to the binding mode of the co-crystallized ligand (F86) against COVID-19 RNA-dependent RNA polymerase, it formed three hydrogen bonds, six hydrophobic interactions, and two electrostatic interactions. Furthermore, the pyrrolo[2,1-f] [1,2,4] triazin-4-amine moiety formed six hydrophobic interactions with Urd20, Ade11, Arg555, and Val557. As well, the sugar moiety formed a hydrogen bond with Asp623. Additionally, the phosphate derivative moiety formed two electrostatic interactions and a hydrogen bond with Asp760, Asp623, and Cys622 (supplementary Fig. S12).
Considering the isolated compound’s binding mode against the COVID-19 main protease protein, it occupied three pockets of the protein with a similar orientation to that of the co-crystallized ligand inside the active pocket of Mpro. Exhaustively, the (S)-2-methylenecyclohexan-1-ol moiety occupied the first pocket of Mpro, forming one hydrophobic interaction with Met49. Moreover, the (3aR,7aS)-3a-methyloctahydro-1H-indene moiety was buried in the second pocket, forming a hydrogen bond with His41. In addition, the (R)-3-methylhex-1-en-3-ol moiety was incorporated in hydrophobic interaction with Met165 in the third pocket of Mpro (Fig. 4).
(a) 3D of the isolated compound docked into the active site of COVID-19 main protease. (b) 2D of the compound docked into the active site of COVID-19 main protease and superimposed with the co-crystallized ligand. (c) Mapping surface showing the compound occupying the active pocket of COVID-19 main protease.
The compound’s interaction with the active pocket of NSP10 formed three hydrophobic interactions and a hydrogen bond. Closely, the hydroxyl group of (S)-2-methylenecyclohexan-1-ol moiety formed a hydrogen bond Asp6912. Such moiety formed two hydrophobic interactions with Leu6898. In addition, the (3aR,7aS)-3a-methyloctahydro-1H-indene moiety formed one hydrophobic interaction with Pro6932. The orientation of the compound inside the active pocket is similar to that of the co-crystallized ligand to some extent (Fig. 5).
(a) 3D of the compound docked into the active site of COVID-19 NSP10. (b) 2D of the compound docked into the active site of COVID-19 NSP10 and superimposed with the co-crystallized ligand. (c) Mapping surface showing the compound occupying the active pocket of COVID-19 NSP10.
The binding mode of the compound against RNA-dependent RNA polymerase formed twelve hydrophobic interactions and two hydrogen bonds with the active pocket. Comprehensively, the hydroxyl group of (S)-2-methylenecyclohexan-1-ol moiety formed two hydrogen bonds with Thr680 and Cys622. Such moiety formed a hydrophobic interaction with Urd20. Additionally, the (3aR,7aS)-3a-methyloctahydro-1H-indene moiety formed five hydrophobic interactions with Urd20 and Ade11, whereas the (R)-3-methylhex-1-en-3-ol moiety formed five hydrophobic interactions with Lys545, Urd10, Val557, and Ala547 (Fig. 6).
(a) 3D of the isolated compound docked into the active site of COVID-19 RNA-dependent RNA polymerase. (b) 2D of the compound docked into the active site of COVID-19 RNA-dependent RNA polymerase and superimposed with the co-crystallized ligand. (c) Mapping surface demonstrating the compound occupying the active pocket of COVID-19 RNA-dependent RNA polymerase.
ADMET studies were carried out for the isolated compound using lopinavir as a reference compound. Discovery studio 4.0 was used to predict the following ADMET descriptors; blood–brain barrier (BBB) penetration, aqueous solubility, intestinal absorption, hepatotoxicity, cytochrome P450 inhibition, and plasma protein binding. The predicted descriptors are listed in (Table 4).
ADMET -BBB penetration studies predicted that the isolated compound possesses a high level compared to lopinavir, which is very low level. Although the compound showed a very low level of ADMET aqueous solubility, it was predicted to have a good level of intestinal absorption. The isolated compound was predicted to be a non-hepatotoxic and non-inhibitor of CYP2D6. Consequently, the liver dysfunction side effect is not expected upon administration of this compound. The plasma protein binding model predicts that the isolated compound can bind plasma protein over 90% (Fig. 7).
The expected ADMET study.
The isolated compound’s toxicity was predicted using the validated and constructed models in the Discovery studio 4.0 software18,19 as follows: (i) FDA rodent carcinogenicity which expects the probability of a molecule to be a carcinogen. (ii) The tumorigenic dose rate 50 (TD50) of a chemical in a rodent chronic exposure toxicity test is predicted by the carcinogenic potency TD5020. (iii) The rat maximum tolerated dose (MTD) of a chemical is estimated using Rat maximum tolerated dose21,22. (iv) In the toxicity test of a chemical, the rat oral LD50 predicts the rat oral acute median lethal dose (LD50)23. (v) The rat chronic LOAEL predicts a compound’s rat chronic lowest observed adverse effect level (LOAEL) value24,25. (vi) Ocular irritancy predicts if a certain compound is probable to be an ocular irritant and how severe the irritation will be in the Draize test26. (vii) skin irritancy predicts if a compound would irritate the skin and how severe it will be in a rabbit skin irritancy test26.
The isolated compound exhibited in silico low adverse effects and toxicity towards the tested models as shown in (Table 5). About FDA rodent carcinogenicity, the tested compound was predicted to be non-carcinogenic. For carcinogenic potency TD50 rat model, the tested compound showed TD50 value of 0.621 mg/kg body weight/day which is less than lopinavir (TD50 = 3.553 mg/kg body weight/day). Regarding the rat maximum tolerated dose model, the tested compound showed a maximum tolerated dose value of 0.040 g/kg body weight which is less than lopinavir (0.117 g/kg body weight). The examined compound demonstrated an oral LD50 value of 1.715 mg/kg body weight/day which is higher than lopinavir (1.154 mg/kg body weight/day). For the rat chronic LOAEL model, the isolated compound showed a LOAEL value of 0.002 g/kg body weight which is less than lopinavir (0.049 g/kg body weight). Moreover, the compound was predicted to be an irritant in the ocular irritancy model. Finally, it was expected to have moderate irritancy against the skin irritancy model.
According to Mostafa et al.27, Azithromycin, which is an FDA-approved antimicrobial drug with promising antiviral activity for repurposing against COVID-19, its mode of action was during the viral replication, which demonstrated up to 70% inhibition at concentration 10.4 µM and exhibited moderate virucidal effect with 37% viral inhibitory effect. Additionally, the Niclosamide drug exhibited a high virucidal effect with a 78% viral inhibitory effect at concentration 10.4 µM, suggesting that our isolated compound is a promising viral inhibitor against SARS-CoV2 and its mode of action could be during the viral replication or had a virucidal effect.
The results revealed that the isolated compound has low toxicity to normal cells and could be used as an antiviral agent after performing in vivo assays. Interestingly, the effective dose which inhibited 95% of the viral count was 5 ng/µl (5 μg/ml) and that below the IC50 value (11.35 ± 1.5 μg/ml) of the normal fibroblast cells. The obtained IC50 for the isolated compound was lowered than the reference drug (Diclofenac), indicating that the compound was more potent than diclofenac and could be used as an anti-inflammatory drug.
Echinoderms are rich bioactive components that provide tremendous pharmaceutical and clinical medicine values. Similarly, like many marine invertebrates, survival requirements have led to the evolution of these complex substances. Interestingly, several secondary metabolites were proven to have a potent antiviral activity as well as a relatively non-cytotoxicity. As a result, these promising echinoderm natural compounds could be used to develop new antiviral drugs. In this study, 5α-cholesta-4(27), 24-dien-3β, 23 β-diol was isolated from the brittle stars, Ophiocoma dentata showing 95% inhibition of COVID-19 virus at concentration 5 ng/µl, which is safe to the normal cells. The current study’s findings are promising regarding finding an effective cure for the COVID-19 pandemic. Further in-vivo studies regarding the clinical and structure–activity relationship are needed to confirm the isolated compound’s potential against the infectious virus SARS-CoV-2.
Marine natural products remain a promising source for discovering high structural diversity and various bioactivities that can be directly developed or used as starting points for the development of novel medications. In the future, we must consider the final dosage form for human use (e.g., tablets, capsules, and injections) to have the potential to be translated into a clinically applicable therapeutic. Biological challenges, large-scale manufacturing, biocompatibility, intellectual property, stable storage, government regulations, and overall cost-effectiveness should all be considered during clinical development. Future research could focus on determining the biodistribution, Pharmacodynamics, and pharmacokinetics in humans, as well as screening for safety and demonstrating the preliminary efficacy of the isolated compound. It is critical to comprehend how the in-vivo environment influences its long-term structural organization, retention, and clearance.
