key: cord-0975694-z3gdv2tp
authors: Elgoud Said, Asmaa Abo; Afifi, Ahmed H.; Ali, Taha F. S.; Samy, Mamdouh Nabil; Abdelmohsen, Usama Ramadan; Fouad, Mostafa A.; Attia, Eman Zekry
title: NS3/4A helicase inhibitory alkaloids from Aptenia cordifolia as HCV target
date: 2021-10-06
journal: RSC advances
DOI: 10.1039/d1ra06139a
sha: 2173991f3c3d16da652bd6f036aa66f1f70968cc
doc_id: 975694
cord_uid: z3gdv2tp

Chemical investigation of Aptenia cordifolia roots extract, using chromatographic and spectroscopic techniques, resulted in isolation and identification of eight known compounds. The basic ethyl acetate fraction (alkaloidal fraction) afforded O-methylsceletenone, epinine, 4-methoxy phenethylamine, and N-methyl tyramine while, the acidic ethyl acetate fraction (non-alkaloidal fraction) afforded only cis-N-coumaroyl tyramine. Moreover, the petroleum ether fraction afforded capric acid, tricosanol, and a mixture of β-sitosterol & stigma sterol. Upon screening of anti HCV activity of these three fractions, only the basic ethyl acetate fraction had high activity against HCV with an IC(50) value equal to 2.4 μg mL(−1) which provoked us to carry out structure based in silico virtual screening on the drug targets of HCV of isolated alkaloidal compounds as well as the previously dereplicated alkaloids through metabolomics from the antiviral active fraction. The tortuosamine compound exhibited the strongest binding to the active site of NS3/4A helicase with a binding affinity (−7.1 kcal mol(−1)) which is very close to the native ligand (−7.7 kcal mol(−1)).

Hepatitis C virus (HCV) was rst identied in 1989. 1 It is an enveloped, positive stranded RNA virus belonging to the family Flaviviridae and considered to be a public health concern which infects more than 185 million people worldwide. 2,3 HCV is the major etiological agent of non-A non-B hepatitis as the infection occurs principally through blood or blood-derived products. 4 This viral infection is the leading cause of cirrhosis and, as a result, liver transplantation around the world. 5, 6 The conventional HCV treatment has advanced quickly, as several protease inhibitors, such as boceprevir and telaprevir have recently been recommended as hepatitis C therapy. These novel inhibitors, however, have been linked to drug toxicity, the creation of resistance mutants, high cost and efficacy limitation. 7 Indeed, the challenge is to develop a potent, inexpensive and widely available antiviral drug.

Medicinal plants contain a variety of compounds that have the potential to treat ailments, particularly infectious disorders. Different studies declared benecial effects of secondary metabolites derived from medicinal plants against viral diseases. A broad variety of these secondary metabolites, such as alkaloids, coumarins, lignans and polyphenolic compounds affect cellular functions and replication of various infectious viruses. Medicinal plants' history dates back to the origin of human civilization on this planet. Inhibitory activity of medicinal plants extracts against replication of several viruses was reported latterly, such as, herpes simplex virus type2 (HSV-2), 8 HIV, 9, 10 hepatitis B virus (HBV), 11, 12 emerging viral infections associated with poxvirus, and severe acute respiratory syndrome (SARS) virus. 13 Recently, various studies have been conducted to investigate the antiviral activity of a variety of plants. As the methanolic extract of both Terminalia bellerica seeds and Enicostemma axillare showed antiviral activity against hepatitis B. 14 On the other hand, Stixis scandens Lour leafe extract showed powerful antiviral activity against porcine epidemic diarrhea virus. 15 Moreover, silymarin, epigallocatechin gallate, naringenin, 16 phenolic compounds of grape seed and aqueous extract of Eclibta alba leaves exhibited anti HCV activity. 14 The alcoholic and/or water soluble extracts of medicinal plants were utilized during furthermost of these antiviral studies. Genus Aptenia is endemic to South Africa and includes four species, which are currently recognized as Aptenia cordifolia L.F. Schwantes, Aptenia geniculiora L. Bittrich ex Gerbaulet, Aptenia haeckeliana A. Berger Bittrich ex Gerbaulet, and Aptenia lancifolia L. Bolus. 17 Aptenia cordifolia, a well-known groundcover, was named heart-leaved ice plant and has only one synonym "Mesembryanthemum cordifolium". 18 So as to provide a complementary therapy for previously existing remedies, the antiviral activity of different fractions (petroleum ether, acidic ethyl acetate and basic ethyl acetate) derived from the total ethanol extract of A. cordifolia roots were examined against HCV by in vitro cells culture using luciferase assay and the cytotoxic effect was accessed by MTT assay. Additionally, we aimed to identify which compound/s in the basic ethyl acetate fraction responsible for the anti HCV activity as well as the mechanism of action by undergoing docking studies on the drug targets of HCV. Although there are many HCV targets, our docking study will focus on the most attractive viral proteins required for HCV replication. These targets are NS5B HCV RNAdependent RNA polymerase, NS3/4A protease, NS3/4A helicase, and a new allosteric pocket in the HCV NS3-NS4A protein located at the interface between the protease and helicase domains. The compounds used for docking studies were the isolated alkaloids from the active fraction (basic ethyl acetate fraction), as well as, the previously nine dereplicated alkaloids from the basic ethyl acetate fraction through metabolomic analysis. 19 

The petroleum ether fraction afforded four compounds. Mixture of b-sitosterol & stigma sterol (8 mg) was isolated as white amorphous powder, and was simply identied by comparison with authentic sample. This mixture was previously isolated from the aerial parts of the plant. 20 Tricosanol alcohol (5 mg) was isolated as white powder and the complete assignment was conrmed by investigation of 1 H-NMR, DEPT-Q and ESI-MS analyses. The molecular formula was determined to be C 23 H 48 O by ESI-MS that showed a pseudo molecular ion peak at 339 [M-H] À . The compound was identi-ed by comparison of the ESI-MS and NMR data, with the reported data 21 and this is the rst time for its isolation from family Aizoaceae.

Capric acid was isolated as white powder (4 mg), the molecular formula was conrmed to be C 10 H 20 O 2 by ESI-MS that showed a molecular ion peak at 172. 8 [M] + . The compound was identied by comparison of the ESI-MS and 1 H-NMR data with the reported data, 22 and it was previously iden-tied by GC-MS in Attalea dubia family Arecaceae, but this is the rst time for its isolation from family Aizoaceae.

Cis-N-coumaroyl tyramine (3),which was isolated as white powder (5 mg data with the literature, 23 and this is the rst report for its isolation from family Aizoaceae. 4-Methoxyphenethylamine (1) was isolated as white powder (3 mg, purity 97%) HR-ESI-MS of compound 1 showed a pseudo molecular ion peak at m/z 152.1066 [M + H] + (calculated mas 152.1075), corresponding to the molecular formula C 9 H 14 NO. From the HR-ESI-MS, 1 H-NMR data and from the reported data in literature 24 compound 1 was identied as 4-methoxyphenethylamine which was previously isolated from Coryphantha pectinata 25 and this is the rst report for its isolation from family Aizoaceae.

N-Methyltyramine (2) was isolated as white powder (4 mg). HR-ESI-MS of compound 2 showed a pseudo molecular ion peak at m/z 152.0697 [M + H] + (calculated mas 152.1031), corresponding to the molecular formula C 9 H 14 NO. From ESI-MS, 1 H-NMR data and from the data reported in literature 26 compound 2 was identied as N-methyltyramine which was previously isolated from Sawa millet seeds 26 and this is the rst report for its isolation from family Aizoaceae.

O-methylsceletenone (4) was isolated as reddish brown powder (20 mg, purity 98%), the complete assignment of compound 4 was conrmed by investigation of 1 H-NMR, DEPT and HR-ESI-MS. Positive HR-ESI-MS of compound 4 showed a quassi molecular ion peak at m/z 258.1462 [M + H] + , (calculated mass at m/z 258.1494) corresponding to the molecular formula C 16 H 20 NO 2 . From the HR-ESI-MS, NMR data and in comparison with the literature, 27 compound 4 was identied as O-methyl sceletenone. It was previously isolated from different organs of the investigated plant. 28 Epinine (5) was isolated as yellow powder (4 mg, purity 97%) HR-ESI-MS of compound 5 showed a pseudo molecular ion peak at m/z 168.9802 [M + H] + , (calculated mass at m/z 167.1024) corresponding to the molecular formula C 9 H 14 NO 2 . Comparison of the HR-ESI-MS and 1 H-NMR data of compound 5, with the data reported in literature 29, 30 revealed that compound 5 is epinine alkaloid which was previously isolated from Cytisus scoparius Family Leguminosae 31 and this is the rst report for its isolation from family Aizoaceae. 

Upon screening the HCV antiviral activity of different fractions of A. cordifolia roots extract, only the basic ethyl acetate fraction (alkaloidal fraction) displayed high potential against HCV with IC 50 value equal to 2.4 mg mL À1 . According to the HCV genome, there are four structural (core, E1, E2, and p7) and six nonstructural (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) proteins. 32, 33 The non-structural proteins that accounted for viral production and replication are the primary targets for the currently FDA-approved drugs designed against HCV. 34 In addition, there is a newly discovered binding pocket between the protease and helicase domains, also considered a druggable target. Of the six non-structural viral proteins, NS2/3 autoprotease, which mediates a single cleavage in the polyprotein, has been less well explored. Moreover, the NS5A protein, involved in both RNA replication and infectious virus assembly, is not a promising target because it has no known enzymatic activity. 35 Therefore, our main target is in silico prediction of the binding affinities of the four alkaloids isolated from the alkaloidal fraction of the A. cordifolia roots named, 4-methoxyphenethylamine (1), N-methyl tyramine (2), O-methylsceletenone (4) and epinine (5) (Fig. 1 ) as well as previously reported nine alkaloids dereplicated from alkaloidal fraction through metabolomic analysis (3 and 6-12) (Fig. 2) 19 against the four non-structural HCV antiviral targets: namely NS5B HCV RNA-dependent RNA polymerase, NS3/4A protease, NS3/4A helicase, and a new allosteric pocket in the HCV NS3-NS4A protein located at the interface between the protease and helicase domains.

Before docking of the screened compounds, we assessed our MOE induced docking protocol by re-docking the co-crystallized ligand within the active site of NS5B HCV RNA-dependent RNA polymerase (PDB code: 3H2L), NS3/4A protease (PDB code: 6NZT), NS3/4A helicase (PDB code: 4OKS), and a new allosteric pocket in the HCV NS3-NS4A protein located at the interface between the protease and helicase domains (PDB code: 4B73).

As seen in Fig. 3-6 , MOE successfully re-docked the native ligand in its original pose with acceptable RMSD values of 0.28, 0.62, 0.31, and 0.16 A respectively. The binding free energies of the re-docked ligand, as well as the screened compounds, were summarized in Table 1 .

Among the screened compounds, compound 6 showed the highest binding affinity (7.4 kcal mol À1 ) to the active site of NS5B HCV RNA-dependent RNA polymerase. Fig. 3C demonstrated that compound 6 binds within the palm I region, where the phenyl ring makes three H-p interactions with three important amino acid residues (Tyr 415, Met 414 and Cys 366) in addition to two important hydrogen bonds made by NH atom to two key amino acid residues (Tyr 415 and Ser 367). These binding interactions explain the strong binding affinity of compound 6 to the active site of NS5B HCV RNA-dependent RNA polymerase ( Table 2) .

Compounds 6, 8 and 12 demonstrated the best binding affinities (À6.7 and À6.9 and À6.9 kcal mol À1 , respectively), among the screened compounds, to the active site of NS3/4A protease. Fig. 4C revealed that the phenyl ring of compound 8 binds within protease active site via a single H-p interaction with amino acid residue (Lys 1136). This binding interaction elucidates the weak binding affinity of compound 8 to the active site of NS3/4A protease (Table  3) . Therefore, NS3/4A HCV protease protein is less likely to be a considerable target for our screened compounds.

As depicted in Fig. 5C , compound 6 binds strongly to the active site of NS3/4A helicase with binding affinity (À7.1 kcal mol À1 ) very close to the native ligand (À7.7 kcal mol À1 ). This strong binding affinity could be explained by two reasons. Firstly, compound 6 binds mainly through two strong hydrogen bonds to the key amino acid residues (Asp 496 and Glu 493) as well as a strong ionic bond to Glu 493 (Table 4 ). Secondly, the alkylamine side chain of compound 6 extended deeply in the lipophilic pocket of helicase active site which also increases the binding affinity of this compound to the target helicase protein. Consequently, NS3/4A helicase protein is more likely to be a potential target for compound 6, among our screened compounds. Compound 6 displayed the highest binding affinity (À7.9 kcal mol À1 ) to the allosteric site of the HCV NS3-NS4A protein. As summarized in Table 5 , compound 6 binds to the curial amino acid residue Asp 81 forms two bonds, one hydrogen bond and one ionic bond. Fig. 6C and D showed the 3D and 2D depiction of compound 6 within the allosteric site of the HCV NS3-NS4A protein displaying hydrogen and ionic bonds with the key amino acid residue (Asp 81). Subsequently, the new allosteric site of the HCV NS3-NS4A protein is considered as a plausible target for compound 6.

According to our molecular docking simulation study, compound 6 was the most promising anti-HCV candidate among the twelve members of the alkaloidal fraction of A. cordifolia roots as it gives an excellent binding affinity; specically against NS3/4A helicase and a new allosteric pocket in the HCV NS3-NS4a protein located at the interface between the protease and helicase domains with a relative good binding energy and distance values from the amino acids of the active binding site compared with the native ligands in these proteins.

The ADME analysis has revealed that all the studied compounds show no violations toward Lipinski's rule and hence prove acceptable drug-likeness and pharmacokeintic properties with no potential toxicity (Tables 6 and 7 ).

All chemicals and solvents were acquired from Merck (Germany) and Sigma Chemical Co. (USA), respectively and were of analytical grades.

The roots of A. cordifolia (250 g) were extracted using 95% ethyl alcohol, the dried ethanol extract (21.5 g) was then suspended in the least amount of distilled water, transferred to separating funnel, then defatted with light petroleum ether to afford pet. ether fraction (3 g). The remaining aqueous solution was then acidied and extracted with ethyl acetate to give acidic ethyl acetate fraction (2.8 g) (non-alkaloidal fraction). The mother liquor was then completely basied and extracted again with ethyl acetate to give basic ethyl acetate fraction (1.8 g) (alkaloidal fraction). The petroleum ether soluble fraction was subjected to silica gel column chromatography and eluted with pet. ether : EtOAc gradient elution starting with 100% pet. ether till 70 : 30 pet. ether : EtOAc to afford four compounds named, capric acid, tricosanol and mixture of b-sitosterol & stigma sterol. Then the acidic EtOAc fraction was subjected to silica gel column chromatography eluted with DCM : MeOH gradient elution to afford ve subfractions. Subfraction no. three was further subjected to sephadex LH-20 (Merck, Germany) column chromatography using isocratic elution with DCM : MeOH (50 : 50) to yield cis-N-coumaroyl tyramine compound. Finally the alkaloidal fraction was subjected to silica gel column chromatography eluted with DCM : MeOH gradient elution to afford seven subfractions. Subfraction no. 3 was further eluted on sephadex LH-20 (Merck, Germany) column chromatography using isocratic elution with MeOH (100%) to yield compound Omethyl sceletenone (4), while 4-methoxyphenethylamine (1), Nmethyl tyramine (2) and epinine (5) compounds were afforded by further purication of subfraction no. 4 on semi preparative HPLC (Knauer, Smartline, Germany) using H 2 O/methanol (95 : 5) initially for 5 min, followed by a linear gradient to 100% methanol within 40 min and maintained isocratically for 5 min. Separation was achieved on a preparative C18 column (5 mm, 10 Â 250 mm, Waters XBridge, Eschborn, Germany), with a ow rate of 2.0 mL min À1 and solvent mixture complemented by 0.05% triuoroacetic acid. protocol was applied, where the co-crystallized ligand was used as a center for the docking simulation grid. All water molecules as well as other non-essential ligands were removed before docking. Other parameters were le in their default value.

In silico drug-likeness, ADME proling, and toxicity risk assessment

The drug-likeness and pharmacokinetic properties of all iden-tied compounds were predicted using Swiss ADME database. 36 

The investigation of A. cordifolia roots revealed isolation and structure elucidation of eight compounds. Additionally, the alkaloidal fraction showed potent anti HCV activity with IC 50 value of 2.4 mg mL À1 so, we undergo docking studies of compounds isolated from alkaloidal fraction as well as, alkaloidal compounds dereplicated from the same fraction through metabolomics. The structure-based screening of the compounds was elucidated against four non-structural HCV antiviral targets and revealed that tortuosamine compound could be a promising anti-HCV candidate as it showed the strongest binding to the active site of NS3/4A helicase among the others. Accordingly, our in silico study highlights the importance of the alkaloidal fraction of the A. cordifolia roots as a potential complementary remedy for HCV infections aer further validation.

There are no conicts to declare.

Plants of southern Africa: an annotated checklist

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This publication was funded by Minia University.