Comprehensive In Silico Screening of the Antiviral Potentialities of a New Humulene Glucoside from Asteriscus hierochunticus against SARS-CoV-2

Chromatographic fractionation of the methanolic extract of Asteriscus hierochunticus whole plant led to the identiﬁcation of a new humulene glucoside ( 1 ). The chemical structure of the isolated compound was elucidated by IR, 1D, 2D NMR, and HRESIMS data analysis to be (-)-(2 Z ,6 E ,9 E )8 α -hydroxy-2,6,9-humulatrien-1(12)-olide. In this study, we report the in silico binding aﬃnities of 1 against four diﬀerent SARS-CoV-2 proteins (COVID-19 main protease (PDB ID: 6lu7), nonstructural protein (PDB ID: 6W4H), RNA-dependent RNA polymerase (PDB ID: 7BV2), and SARS-CoV-2 helicase (PDB ID: 5RMM)). The isolated compound showed excellent binding aﬃnity values ( Δ G) of − 21.65, − 20.05, − 28.93, and − 21.73kcal/mol, respectively, against the target proteins compared to the cocrystallized ligands that exhibited Δ G values of − 23.75, − 17.65, − 23.57, and − 15.30kcal/mol, respectively. Further in silico investigations of the isolated compound ( 1 ) for its ADMETand toxicity proﬁles revealed excellent drug likeliness. On the other hand, the results obtained from in vitro antitrypanosomal, antileishmanial, and antimalarial activities of ( 1 ) were not promising.


Introduction
e world witnessed the emergence of SARS-CoV-2 in the later part of 2019 from the Wuhan district of China, leading to millions of deaths worldwide and a total lockdown of most economies with attendants' socioeconomic impact [1]. By November 2020, more than 33 million humans have been infected and more than other 1.3 million have died all over the world according to the WHO [2]. Unfortunately, there is no available treatment for COVID- 19. e symptomatic treatment based on anti-inflammatory agents such as dexamethasone, some research drugs, and ventilators (oxygen perfusion) in severe cases is currently used for treatment of affected persons [3]. Coronaviruses generally have been linked with debilitating diseases that have previously affected a wide range of the population, Middle East Respiratory Syndrome (MERS-CoV) and Severe Acute Syndrome (SARS-CoV) that appeared in 2003 [4,5].
Humans always relied on the nature as the major curative of diseases [6,7]. Increased use of natural products in the last few decades has been witnessed for various purposes, especially as a source for primary healthcare to most of the world population [8]. Natural products are not only used as a major source of new bioactive molecules for managing of several diseases but also as a starting material in the synthesis of new potent pharmacological compounds [8]. Plants and microbes could be a source for the effective natural products [9][10][11][12][13]. e efficacy of natural products as curatives is enhanced due to the presence of varied types of compounds as saponins [14,15], alkaloids [16], pyrones [17], isochromenes [18], flavonoids [19,20], diterpenes [21], and sesquiterpenes lactones [22,23].
Asteriscus hierochunticus (Michon) A. Wiklund is a widely distributed shrub that belongs to family Asteraceae and found in tropical regions, including Asia, the Americas, Africa, the Middle East and the Mediterranean region, Europe, and some islands in the Pacific and Atlantic oceans [24]. e genus Asteriscus is used in folk medicine for treatment and managing of different disease conditions, such as inflammations, fevers, cancers, stomach aches, bronchitis, hemorrhoids, and other disorders [25]. Phytochemical and pharmacological investigations on other members of the genus have been reported and resulted in the isolation of bioactive secondary metabolites such as sesquiterpenes, diterpenes, and phenolics [26], but for A. hierochunticus, there was no enough phytochemical investigation. e isolation and characterization of bioactive humulene sesquiterpenes lactones, such as asteriscunolides A-D [27,28], steriscanolide, and aquatolide [29], from some species of genus Asteriscus had been reported. ese isolated compounds have been screened for their antitrypanocidal, cytotoxic, antidiabetic, antiprotozoal, and antiparasitic activities [30][31][32][33]. e current study investigates the phytochemistry of A. hierochunticus whole plant through chromatographic isolation and in silico anti-COVID-19 screening for the isolated compounds.

Materials and Methods
2.1. General. It is detailed in the supporting information.

Plant Material.
It is detailed in the supporting information.

Extraction and Isolation.
ey are detailed in the supporting information.
2.5. In Silico Studies 2.5.1. Conformational Search for Humulene-Glucoside 1. Conformational search for humulene-glucoside (1) was carried out using the LowModeMD Search protocol using MOE2014 [34]. See the supporting information for more details.
2.5.2. Molecular Docking. Molecular Operating Environment (MOE) was used for the docking analysis, see the supporting information.
2.5.4. In Silico Toxicity. Discovery studio 4.0 was used [36], see the supporting information.

In Vitro Macrophage Amastigote Assay.
is was performed according to the parasite-rescue and transformation assay described in [37,38], see the supporting information.

Antitrypanosomal Assay.
is assay was carried out according to a method previously described [39], see the supporting information.

Results
3.1. Isolation of Humulene-Glucoside (1) and Structure Elucidation. e phytochemical investigation of A. hierochunticus extract resulted in the purification of a new humulene-glucoside (1) (Figure 1).  , and one oxygenated methylene (δ H 3.40 (dt, J � 11.8, 5.7 Hz, H-6′a) and 3.63 (ddd, J � 11.8, 6.1, 2.1 Hz, H-6′b)). e 13 C NMR and DEPT-135 spectra showed the presence of c-lactone, together with six olefinic carbons, three methyls, two methylenes, two methines, and one quaternary carbon, as well as one sugar moiety. e 13 C and 1 H data of compound (1) are shown in Table 1. e analysis of 1 H, 13 C, and DEPT-135 NMR spectra of (1) revealed the presence of sesquiterpene lactone of humulene skeleton in the glycoside form. e protons' connectivities were confirmed by both HMQC and HMBC spectrum, and the humulanolide structure was established ( Figure 2). By considering the coupling constant between (C9�C10) (J 9-10 � 16.1 Hz), its stereochemistry was determined to be E, as well as the geometry of double bond between (C6�C7) [41,42]. By comparing the NMR data, with the reported one, it revealed the structure of asteriscunolide D [28], except for the absence of a carbonyl signal at C-8 which was replaced by an oxygenated methine at δ C 83.9, attached for a sugar moiety. e α-orientation of the hydroxy group at C-8 was deduced from the chemical shift and coupling constant value of H-8 [43]. Based on the NMR data and comparison with an authentic sample after hydrolysis, the sugar moiety was concluded to be β-D-glucose [44]. e correlations of H-1′ (δ H 3.86) with C-8 (δ C 83.9) and H-8 (δ H 4.79) with the anomeric carbon (δ C 99.9) in the HMBC spectrum revealed the sugar connection at C-8 ( Figure 2). e structure of 1 was established to be (-)-(2Z,6E,9E)8α-hydroxy-2,6,9humulatrien-1(12)-olide.

Conformational Search for Humulene-Glucoside (1).
In this work, conformational search for humulene-glucoside (1) was carried out using the LowModeMD protocol. e best conformer was with a potential energy (E) of 75.48 kcal/ mol. e strain energy of this conformer relative to the lowest energy conformation with the same stereochemistry configuration (dE) was 0.00 kcal/mol. Besides, the integer encoding stereochemistry configuration of such conformer (CHI) was equal 1. e radius of gyration of this conformer (RGYR) was 4.09. It was found that the globularity of the conformation (Glob) was 0.24 and the eccentricity of the conformation (Ecc) was 0.89 (Table 2 and Figure 3).

Molecular Docking.
e main protease (M pro ) is a vital chymotrypsin-like cysteine protease that belongs to the nonstructural type and plays a crucial role in the replication process of coronavirus. It releases the 16 nonstructural proteins (NSPs 1-16) via cleavage of the C-terminal end of the two polyproteins (PP1a and PP1ab) [45,46]. e nonstructural protein (NSP10) is a vital cofactor for activation of the SARS-CoV replicative enzyme (replicase) [47].    RNA-dependent RNA polymerase or RNA replicase is the enzyme that is responsible for the replication process of RNA through catalyzing the synthesis of the RNA template complementary from the RNA strand [48]. Helicases are a group of enzymes that have the potentiality to separate double-stranded DNA or RNA. Helicase enzyme is very essential for the process of RNA replication and repair [49].
Redocking of the cocrystallized ligands (PRD_002214, SAM, F86, and VXG) against the active pockets of COVID-19 main protease, NSP10, RNA-dependent RNA polymerase, and SARS-CoV-2 helicase, respectively, has been preceded to validate the docking procedure. e calculated RMSD values between the redocked poses and the cocrystallized were 3.10, 1.07, 1.34, and 1.17 indicating the efficiency and validity of the docking processes ( Figure 4).
Comprehensive docking studies were carried out using MOE14.0 software and showed generally low binding energies for compound (1) ( Table 3).
Regarding the binding mode of the cocrystallized ligand (VXG) against SARS-CoV-2 helicase, VXG occupied the protein forming four hydrogen bonds besides two hydrophobic interactions. e (S)-1-acetylpyrrolidine-3-carboxylic acid moiety made four hydrogen bonds with Asn177, Asn516, and Ser486. Also, it was incorporated in two hydrophobic interactions with Tyr515 and His554 ( Figure 8).

ADMET.
Following the exciting results obtained from the docking studies and the fact that ADMET studies are a fundamental factor in drug discovery, we decided to carry   out in silico ADMET studies. e studies were carried out on humulene-glucoside (1) using simeprevir as a reference drug. e parameters investigated with the ADMET studies included the following descriptors: (i) blood-brain barrier penetration, (ii) intestinal absorption, (iii) aqueous solubility, (iv) CYP2D6 binding, (v) hepatotoxicity prediction, and (vi) plasma protein binding. e predicted descriptors are listed in Table 4. e results revealed that humulene-glucoside (1) has a very low BBB penetration level. Accordingly, the compound may be assumed to have a high degree of CNS safety.
Compound (1) showed an adequate level of ADMET aqueous solubility and intestinal absorption levels compared to simeprevir. As such, intestinal absorption of humuleneglucoside (1) could be significant.
Compound (1) was also predicted to be a noninhibitor of CYP2D6 and nonhepatotoxic. Consequently, the liver dysfunction effect is not expected to happen upon administration of humulene-glucoside (1). Regarding the plasma protein binding study, it was revealed that humulene-glucoside (1) could bind to plasma protein ˂90% (Figure 13).
As shown in Table 5, compound (1) exhibited in silico low adverse effect and toxicity against the tested models. Humulene-glucoside (1) did not show any carcinogenic tendencies in the FDA rodent carcinogenicity model and showed a TD 50 value of 1.968 mg/kg body weight/day, higher than that of the reference drug simeprevir (0.280 mg/ kg body weight/day).
Humulene-glucoside (1) showed a maximum tolerated dose of 0.114761 g/kg body weight higher than simeprevir (0.002967 g/kg body weight) and showed an oral LD 50 value of 0.621649 mg/kg body weight/day. is value was higher than that of simeprevir (0.208835 mg/kg body weight/day).
Humulene-glucoside (1) showed an LOAEL value of 0.00643343 g/kg body weight, which was higher than that of simeprevir (00.00210575 g/kg body weight).

Biological Activities.
e humulene-glucoside (1) was tested against transformed human monocytic (THP1) cells and showed no cytotoxic effect against THP1. is result agreed with the experimental in silico toxicity properties. On

Data Availability
Details of the experimental part and NMR charts of compound 1 are available on request.

Conflicts of Interest
e authors declare no conflicts of interest.
Acknowledgments is work was supported partially by USAID/HED grant 153 -6200BF A15 -01. e authors also acknowledge the University of Benin as a shared cost partner in this grant. Finally, the authors would like to thank the National Center for Natural Product Research (NCNPR), School of Pharmacy, University of Mississippi, for the use of their laboratory.