In Vitro and in Silico Study of New Biscoumarin Glycosides from Paramignya trimera against Angiotensin-Converting Enzyme 2 (ACE-2) for Preventing SARS-CoV-2 Infection

Abstract

In Vietnam, the stems and roots of the Rutaceous plant Paramignya trimera (Oliv.) Burkill (known locally as “Xáo tam phân”) are widely used to treat liver diseases such as viral hepatitis and acute and chronic cirrhosis. In an effort to search for Vietnamese natural compounds capable of inhibiting coronavirus based on molecular docking screening, two new dimeric coumarin glycosides, namely cis-paratrimerin B (1) and cis-paratrimerin A (2), and two previously identified coumarins, the trans-isomers paratrimerin B (3) and paratrimerin A (4), were isolated from the roots of P. trimera and tested for their anti-angiotensin-converting enzyme 2 (ACE-2) inhibitory properties in vitro. It was discovered that ACE-2 enzyme was inhibited by cis-paratrimerin B (1), cis-paratrimerin A (2), and trans-paratrimerin B (3), with IC₅₀ values of 28.9, 68, and 77 µM, respectively. Docking simulations revealed that four biscoumarin glycosides had good binding energies (∆G values ranging from −10.6 to −14.7 kcal/mol) and mostly bound to the S1′ subsite of the ACE-2 protein. The key interactions of these natural ligands include metal chelation with zinc ions and multiple H-bonds with Ser128, Glu145, His345, Lys363, Thr371, Glu406, and Tyr803. Our findings demonstrated that biscoumarin glycosides from P. trimera roots occur naturally in both cis- and trans-diastereomeric forms. The biscoumarin glycosides Lys363, Thr371, Glu406, and Tyr803. Our findings demonstrated that biscoumarin glycosides from P. trimera roots hold potential for further studies as natural ACE-2 inhibitors for preventing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection.

Introduction

The world is facing a serious, often fatal flu-like infection pandemic caused by a coronavirus, namely severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Since 2020, it has spread to more than 200 countries, infected over 775 million people, and caused the deaths of about 1% of those afflicted.¹⁾ Numerous scientific studies were carried out to help stop the spread of the virus, and many preventative health policies were promulgated to find solutions to protect people’s health.

The respiratory tract is the principal route by which SARS-CoV-2 enters the human body. To enable it to invade the lung cells of humans, it produces a set of enzymes or proteins, including the nonstructural proteins (nsp) 1–16 and the main structural proteins, which constitute the membrane (M), envelope (E), nucleocapsid phosphoprotein (N), and spike (S).²⁾ Via the S1 subunit of the receptor-binding domain (RBD), the S1 and S2 domains of this spike S can interact with the human protein enzyme angiotensin-converting enzyme-2 (ACE-2), which is frequently present on the surface of lung cells. Coronaviruses, including HoV-NL63, SARS-CoV, and SARS-CoV-2, fuse their envelope with the host cell membrane after adhering to the ACE-2 protein enzyme, which serves as a receptor for virus entry. They then release their genetic material, a positive RNA strand of about 20–32 kb, into the cytoplasm for replication and begin to cause respiratory illness.³⁾ Therefore, the human ACE-2 enzyme became a prominent drug target for coronavirus disease 2019 (COVID-19) inhibitors, as inhibiting its activity can block viral entry, proliferation, and invasion.^4,5)

Recently, molecular docking techniques with computer-based support have been widely utilized to identify novel therapeutic drugs in general and ACE-2 inhibitors for SARS-CoV-2 treatment in particular. For example, a protein-peptide composite from buckwheat (Fagopyrum sp.) and quinoa (Chenopodium quinoa, Willd) established a new vista for food-derived peptides having ACE-2 inhibitory potential as a tentative strategy to develop SARS-CoV-2 therapeutics.⁶⁾ Through the use of molecular docking, a series of bioactive compounds like flavonoids (rutin, astragalin, quercitin, isoquercitrin (from the Himalayan stinging nettle version of Urtica dioica⁷⁾), glyasperin A, broussoflavonol F, isorhamnetin (from Sulawesi propolis),⁸⁾ chalcone (butein),⁹⁾ triterpenoids (cucurbitacin E, cucurbitacin B, and isocucurbitacin B¹⁰⁾) were discovered to be powerful S-protein-ACE-2 inhibitors. In addition, docking simulation is very useful for structural optimization of novel inhibitors through the analysis of key binding interactions within the enzyme active site and then offers more insights regarding the action of residues involved in catalysis and substrate specificity. So far, the search for ACE-2 inhibitors from medicinal plants and their natural constituents based on molecular docking simulation is one of the most effective approaches to the discovery of potential drug candidates for the treatment of coronavirus infections.

Paramignya trimera (Oliv.) Burkill is a valuable medicinal plant belonging to the family Rutaceae. It is a small tree, a climbing or crawling bush, with a trunk up to 4–5 m long and typical light-yellow roots. The tree has its fruiting season around May–August. In Ninh Van commune, Khanh Hoa province, in the south of Vietnam, the stems and roots of this herb are commonly used by people to treat liver diseases such as hepatitis and chronic cirrhosis.¹¹⁾ However, to the best of our knowledge, there are no reports on the use of Paramignya trimera in traditional medicines in other Asian countries, such as Thailand, Malaysia, or Singapore. The plant contains natural compounds such as alkaloids,¹²⁾ chromene derivatives,¹³⁾ coumarins, and especially dimeric monoterpene-linked coumarin glycosides.^14,15)

The water extract from the roots of P. trimera is reported to possess hepatoprotective activity in a mouse model of liver injury induced by paracetamol.¹⁶⁾ The methanol extract from its roots showed an anti-cancer effect on a human breast cancer cell line three dimensional (3D) model (MCF-7)¹⁷⁾; in cytotoxicity assays¹⁸⁾; and antibacterial activity against some multiple-drug-resistant bacterial strains.¹⁹⁾ The coumarins isolated from P. trimera are reported to be compounds with remarkable antioxidant,²⁰⁾ and anti-inflammatory activity,²¹⁾ and in accordance with these findings, they can act as free radical scavengers.²²⁾ The acridone alkaloids, like paratrimerin I are cytotoxic.¹²⁾ The coumarin ostruthin has shown anti-proliferative activity against five cancer cell lines, i.e., hepatocarcinoma Hep-G2, colon cancer HTC116, breast cancer MDA MB231, ovarian cancer OVCAR-8, and cervical cancer Hela,²¹⁾ and shows a strong inhibition of α-glucosidase.²³⁾

We screened more than five hundred natural compounds from diverse Vietnamese medicinal plants using molecular docking screening for numerous protein targets associated with SARS-CoV-2 infection, such as ACE-2, M^pro, and 3Cl^pro, among others (data not shown). Several biscoumarin glycosides from P. trimera were found to be potential ACE-2 enzyme inhibitors by docking screening. In vitro, it was discovered that both water and EtOH 96% extracts of P. trimera root inhibited the ACE-2 enzyme. We herein report the isolation and structural elucidation of two new dimeric coumarin glycosides, namely cis-paratrimerin B (1) and cis-paratrimerin A (2), along with the known coumarins, paratrimerins A (3) and B (4), from the roots of P. trimera and their anti-ACE-2 inhibitory properties in vitro and in silico. This report demonstrates that the typical biscoumarin glycosides of P. trimera exhibited their activity against ACE-2 in both in vitro and in silico experiments, with a high binding affinity and distinctive interactions in the S1′ subsite of the ACE-2 protein.

Results and Discussion

Isolation and Structural Elucidation

From the water-soluble fraction, some biscoumarin glycosides were isolated. The chemical structures of the new and known compounds were identified by comparison with the physicochemical parameters and spectroscopic data published in the literature.

Compound 1 was obtained as a white amorphous powder with a negative optical rotation ([α]_D²⁵ −25.0°, c = 0.02, MeOH). Its molecular formula was determined to be C₄₅H₅₂O₂₀ (M = 912.3 g/mol) based on the quasi-molecular ion peak observed at m/z 947.2756 [M + Cl]⁻ (Calcd for C₄₅H₅₂O₂₀Cl⁻ 947.2740) (Supplementary Fig. S1), similar to a series of monoterpene-linked biscoumarin glycosides containing three sugar moieties, such as paratrimerin B, and K–V, found previously in the roots of Paramignya trimera collected in Khanh Hoa province, Vietnam.^14,24) On acid hydrolysis, compound 1 gave D-glucose and D-apiose units on the basis of co-TLC with an authentic sample. The fragment at m/z 781.2707 also indicated the presence of an apiosyl fragment ([M − api + H]⁺) (Supplementary Figs. S1, S10).

The ¹H- and ¹³C-NMR and DEPT spectra of 1 (in CD₃OD) with 45 carbon signals (13 × C, 23 × CH, 7 × CH₂, 2 × CH₃), displayed patterns similar to those of paratrimerin B,¹⁴⁾ with characteristic proton signals of two coumarin nuclei, a monoterpene bridge, and three sugar moieties (Table 1, Supplementary Figs. S2–S4). The binding positions of each structural unit were determined based on the analysis of heteronuclear multiple bond connectivity (HMBC), correlation spectroscopy (COSY), nuclear Overhauser effect spectroscopy (NOESY), and rotating frame nuclear Overhauser enhancement spectroscopy (ROESY) spectra (Table 1, Fig. 1, Supplementary Figs. S5–S9).

Table 1. ¹H-NMR and ¹³C-NMR Data of cis-Paratrimerin B (1) and cis-Paratrimerin A (2)

Position	cis-Paratrimerin B (1)*		cis-Paratrimerin A (2)*
	13C-NMR	1H-NMR	13C-NMR	1H-NMR
2	163.6 (s)	—	163.3 (s)	—
3	114.3 (d)	6.28, d, 9.6 Hz	114.3 (d)	6.22, d, 9.0 Hz
4	146.2 (d)	7.94, d, 9.6 Hz	146.0 (d)	7.96, d, 9.0 Hz
4a	115.3 (s)	—	115.2 (s)	—
5	126.6 (d)	7.45, s	126.6 (d)	7.48, s
6	127.6 (s)	—	127.7 (s)	—
7	158.5 (s)	—	158.4 (s)	—
8	104.4 (d)	7.09, s	104.0 (d)	7.04, s
8a	155.2 (s)	—	155.3 (s)	—
2′	163.5 (s)	—	163.5 (s)	—
3′	114.1 (d)	6.21, d, 9.6 Hz	114.1 (d)	6.28, d, 9.6 Hz
4′	145.9 (d)	7.80, d, 9.6 Hz	145.9 (d)	7.89, d, 9.6 Hz
4′a	114.4 (s)	—	114.4 (s)	—
5′	131.0 (d)	7.37, s	131.0 (d)	7.39, s
6′	131.7 (s)	—	131.7 (s)	—
7′	160.1 (s)	—	160.1 (s)	—
8′	103.7 (d)	7.08, s	103.7 (d)	7.10, s
8′a	155.0 (s)	—	155.1 (s)	—
9	121.3 (d)	6.38, s	121.3 (d)	6.40, d, 8.4 Hz
10	140.9 (d)	6.38, s	141.0 (d)	6.40, d, 8.4 Hz
11	40.2 (s)	—	40.2 (s)	—
12	32.2 (t)	1.85 ma) 1.66, dt like, 6 and 18 Hzb)	31.9 (t)	1.66, dt-like, 13.3, 5.9 Hz, & 1.88, m, part. overlapped
11-CH3	25.8 (q)	1.29, s	25.8 (q)	1.32, s
9′	44.4 (d)	4.12, br s	44.4 (d)	4.14, s
10′	124.6 (d)	5.36, br s	124.6 (d)	5.36, s
11′	135.6 (s)	—	135.6 (s)	—
12′	28.5 (t)	2.28, dt-like, 4.5 and 18 Hza) 2.14, dt-like, 6.6 and 14.5 Hzd)	28.6 (t)	2.29, dt-like, 5.04 and 17.8 Hz 2.18, m
11′-CH3	23.7 (q)	1.83, s	23.7 (q)	1.86, s
1″	102.6 (d)	4.91, d, 7.8 Hz	101.7 (d)	4.99, d, 7.8 Hz
2″	74.8 (d)	3.42, m	74.8 (d)	approx. 3.50, ma,d)
3″	78.5 (d)	approx. 3.5, ma,d)	78.2 (d)	approx. 3.47, ma,d)
4″	71.9 (d)	3.03, m	71.7 (d)	3.23, t, 9.0 Hz
5″	78.3 (d)	3.48,d) m	78.4 (d)	approx. 3.56, ma,d)
6″	62.7 (t)	3.83, dd, 11.4; 1.8 Hz 3.46, m	62.7 (t)	3.37, ma,d)
1‴	101.9 (d)	4.88, d, 7.8 Hzc)	102.5 (d)	4.93, d, 7.8 Hz
2‴	75.0 (d)	3.58,a) m	75.0 (d)	3.57, dd, 9.2, 7.7 Hz
3‴	78.4 (d)	3.48, md)	78.4 (d)	approx. 3.56, ma,d)
4‴	71.7 (d)	3.25, t, 9 Hz	71.2 (d)	approx. 3.36, ma,d)
5‴	77.2 (d)	3.62, md)	78.4 (d)	approx. 3.56, ma,d)
6‴	69.1 (t)	4.02, d, 9 Hz,d) 3.61, md)	62.5 (t)	3.89, dd, 12.2, 2.3 Hz 3.71, ma,d)
1″″	111.2 (d)	4.91, d, 5.5 Hz	—	—
2″″	78.3 (d)	3.97, d, 2.4 Hz	—	—
3″″	80.5 (s)	—	—	—
4″″	75.0 (t)	4.04, d, 9.6 Hz,d) 3.77, d, 10.2 Hzd)	—	—
5″″	65.7 (t)	3.62, md)	—	—

* All compounds were measured in the CD₃OD solvent, 600 MHz for ¹H- and 150 MHz for ¹³C-NMR spectra. a) Strongly overlapped signals. b) Chemical shift of HSQC correlation peak. c) Overlapped by solvent signal. d) May be interchanged.

Fig. 1. Correlations (COSY, ROESY and HMBC) of Biscoumarin Glycosides 1 and 2

Similar to paratrimerin B, both two coumarin nuclei in compound 1 were substituted at C-6 and C-7 positions, as indicated by the presence of two pairs of ortho-located olefinic protons at δ_H 6.28 (H-3) and 7.94 (H-4), and δ_H 6.21 (H-3′) and 7.80 (H-4′) with J = 9.6 Hz, as well as two pairs of para-singlet aromatic protons at δ_H 7.45 (H-5) and 7.09 (H-8), and δ_H 7.37 (H-5′) and 7.08 (H-8′). Their oxygenated carbons C-7/C-7′ were connected to the two glucopyranosyl moieties determined by the HMBC correlations between the anomeric protons H-1″ (δ_H 4.91, d, J = 7.8 Hz) and H-1‴ (δ_H 4.88, d, J = 7.8 Hz) to carbons C-7 (δ_C 158.5) and C-7′ (δ_C 160.1), respectively, and by the ROESY cross-peaks from the sugar protons H-2″ (δ_H 3.42) and H-2‴ (δ_H 3.58) to the singlet aromatic protons H-8 (δ_H 7.09) and H-8′ (δ_H 7.08), respectively (Table 1, Fig. 2). The position of the monoterpene bridge attached to two glucopyranosyl coumarin units at the non-protonated carbons C-6 (δ_C 127.6) and C-6′ (δ_C 131.7) was determined based on the HMBC cross-peaks (³J-correlations) from the coumarinic protons H-5 (δ_H 7.45) and H-5′ (δ_H 7.37) to the tertiary carbons of the vinylcyclohexene moiety C-9 (δ_C 121.3) and C-9′ (δ_C 44.4), respectively, and the ROESY interactions from H-5 and H-5′ to the vinylic proton H-9 (δ_H 6.38) and methine proton H-9′ (δ_H 4.12), respectively (Table 1, Fig. 2). The spectroscopic data supported the connection at C-6/C-9 and C-6′/C-9′, and the monoterpene (C10) (1,4-dimethyl-4-vinylcyclohexene) served as a bridge between the two glucosidic coumarins, comparable to several monoterpene-linked biscoumarins isolated from P. trimera.¹⁴⁾ But different from paratrimerin B, two olefinic protons H-9 (δ_H 6.38)/H-10 (δ_H 6.38) appeared as two single signals, indicating that they were in cis-position (Fig. 1). In paratrimerin B, these two vicinal protons, H-9 and H-10, were trans-located with a J = 16.5 Hz.^14,24) However, a significant difference in the ¹³C-NMR values of C-9 and C-10 carbons compared to their trans-isomers was not observed.¹³⁾

Fig. 2. The Structures of Biscoumarin Glycosides 1–4

The presence of two glucosyl moieties in the β-configuration was confirmed by two anomeric proton signals observed at δ_H 4.91 (d, H-1″) and δ_H 4.88 (d, H-1‴) (each J = 7.8 Hz), and fully assigned by 2D-NMR data (HMBC, COSY, ROESY) (Table 1). For example, the anomeric proton H-1‴ (4.88, d, J = 7.8 Hz) had the ROESY cross-peak to H-5‴ (δ_H 3.62), which further had COSY interaction with the -CH₂-O-C methylene protons H-6″ (δ_H 4.02, H-6‴a) and (δ_H 3.61, H-6‴b) of the substituted glucopyranosyl unit. The C-6‴ had the following HMBC correlation with the anomeric proton H-1″″ of a sugar: an apiose or a xylose moiety.

This sugar was then determined as an apiose by the presence of two characteristic methylene carbons bearing oxygen (-CH₂-O-) at δ_C 75.0 (C-4″″) and at δ_H 3.62 (C-5″″). This apiose moiety was attached to the glucosyl unit at its anomeric proton at δ_H 4.91 (H-1″″) and has a HMBC correlation with the glucosyl methylene carbon at δ_C 69.1 (C-6‴) of the glucosyl moiety (Supplementary Fig. S6). The anomeric apiosyl carbon C-1″″ appeared at δ_C 111.2, indicative of the β-D-configuration, as the ¹³C-NMR data of α-D-apiofuranoside appeared at δ_C 104.5 and the β-D-apiofuranoside at δ_C 111.5.

Additionally, the structure of 1 was confirmed by analysis of high resolution (HR)-MS/MS fragments, as shown by the presence of main fragments including m/z 781.2707 [M − api + H]⁺, 619.2180 [M − api − glc + H]⁺_, and 352.3405 amu (Supplementary Figs. S1, S10).

A circular dichroism (CD) spectrum was also measured and revealed that compound 1 had isomeric configurations of 9′S,11S, similar to those of other paratrimerins (A, B, L, N, S)^14,24) (Fig. 3). Based on these spectroscopic results, compound 1 was identified as a new dimeric monoterpene-linked coumarin glycoside; a geometric isomer of paratrimerin B, which was accordingly named cis-paratrimerin B. Another new monoterpene-linked biscoumarin, cis-paratrimerin A (2), was also isolated and structurally evaluated by NMR spectroscopic data and the CD spectrum (Table 1, Supplementary Figs. S12–S18). The above evidence indicates that these two biscoumarins are not artifacts, as previously suggested for one cis-derivative of paratrimerin J.²⁴⁾ A number of natural products were discovered to exist as diastereomeric mixtures, each of which can exhibit a different level of bioactivity. For example, dl-1-benzoyloxy-2-dimethylamino-1,2,3,4-tetrahydronaphthalene exists in both trans- and cis-forms, and both configurations have local anesthetic activities. The cis-compound was 2.9 to 6 times more effective than its trans-isomer in its analgesic, intracutaneous, and sciatic nerve blocking effects, surpassing those of procaine, lidocaine, and cocaine in guinea pigs.²⁵⁾ Captopril is a drug widely used in hypertension treatment and has two isomeric forms (cis and trans), both of which have an effect on the cardiovascular system.²⁶⁾ A possible biosynthetic pathway for the presence of both cis- and trans-forms of monoterpene-linked biscoumarins from P. trimera roots is suggested in Supplementary Fig. S19.^27,28)

Fig. 3. CD Spectra of Biscoumarin Glycosides 1 and 2

In Vitro Evaluation of Anti-ACE-2 Activity

A commercial ACE-2 inhibitor screening kit (Merck KGaA, Darmstadt, Germany) was used to measure how well the test substances block the enzyme. The kit used glycoprotein- and biotin-labeled (≥90%) recombinant human ACE-2 expressed in human HEK 293 cells. The ACE2-biotin complex that is formed was visualized with a streptavidin-HRP conjugate probe, and the specific activity of the recombinant ACE-2 was measured by its ability to cleave a fluorogenic peptide substrate, 4-methoxycoumarin-Mca-YVADAPK(Dnp)-OH, to release a free fluorophore. In the presence of an inhibitor, the enzyme loses its peptidase activity, which results in a decrease in fluorescence intensity. One unit is defined as the amount of enzyme required to cleave 1 pmol of the fluorogenic peptide substrate, Mca-YVADAPK(Dnp)-OH, in one minute at 37 °C, pH 7.5.²⁹⁾ MLN-4760, an ACE-related carboxypeptidase inhibitor, was used as a positive control.

The results in Table 2 showed that the hot water and ethanol-96% extracts of P. trimera had a weak inhibitory effect on the ACE-2 activity. Cis-paratrimerin B (1), cis-paratrimerin A (2), and trans-Paratrimerin B (3) inhibited ACE-2 with IC₅₀ values of 28.9, 68, and 77 µM, respectively. Compound (1) (cis-paratrimerin B) showed the highest ACE-2 inhibitory effect (103.11 ± 0.4%) with an IC₅₀ value of 26.4 (µg/mL) or 28.9 (µM). Under the same condition, the IC₅₀ value of the positive control was 33 (ng/mL) (Table 2). These results indicate that biscoumarin glycosides of P. trimera have some potential as anti-ACE-2 inhibitors in particular and thus anti-SARS-CoV-2 in general.

Table 2. In Vitro ACE-2 Inhibitory Activity Assay

Sample name	% ACE-2 inhibition	IC50 (µg/mL)	IC50 (µM)
Water P. trimera extract	19.1 ± 0.51	>100
EtOH-96% P. trimera extract	12.1 ± 0.12	>100
cis-Paratrimerin B (1)	103.1 ± 0.42	26.3	28.9
cis-Paratrimerin A (2)	84.0 ± 0.33	53.1	68.1
trans-Paratrimerin B (3)	71.2 ± 0.16	70.3	77.1
Enzyme control (−)	0	0
Inhibitor control (+) (MLN-4760)	99.7 ± 0.22	0.033

It is well reported that the interaction of SARS-CoV-2 with ACE-2 is crucial for viral entry in the early stages of COVID-19 infection. Moreover, the macrophage phagocytosis-associated virus may also move through the blood from the lungs to other organs that have high levels of ACE-2. It has been discovered that ACE-2 plays two roles: first, it facilitates the entry of viruses into the host; and second, it protects against serious lung damage caused by angiotensin II damage.^5,30) Therefore, plants containing ACE-2-inhibiting compounds imply mitigation of viral invasion and protection against lung injury. By preventing the down-regulation of the angiotensin-converting enzyme 2, osthole, a prenylated monocoumarin, has been shown by Shi et al. to reduce lipopolysaccharide (LPS)-induced acute lung damage in mice.³¹⁾ To assess their ability to prevent COVID-related lung injury, the biscoumarins found in Paramignya trimera require further study employing in vivo assays. Some other Rutaceous plant biscoumarins, like toddalosin from Toddalia asiatica,³²⁾ hassmarin from Citrus hassaku,³³⁾ and bisparasin from the root mixtures of Citrus plants,³⁴⁾ might be useful in discovering their biological activities in general as well as their anti-ACE-2 effects specifically. Biscoumarins of Hypericum sp. have also shown cytotoxic and antiviral properties.^35,36)

Molecular Docking

In order to gain more insight into the interactions of paratrimerins against ACE-2, molecular docking simulations were carried out, using the human ACE-2 structure (hACE-2, PDB ID: 1R4L) reported by Towler et al.³⁷⁾

Validation of Docking Protocol Using hACE-2 Binding Site

The accuracy of docking protocol was validated through the re-docking process. The high overlap between the co-crystallized ligand MLN-4760 (PDB ID: 1R4L) and the re-docked ligand is depicted in Fig. 4.³⁶⁾ The root means square deviation (RMSD) between the coordinates of the MLN-4760 redocked and the original co-crystallized ligand was 0.816 Å. As revealed by MLN-4760 interactions, the hACE-2 binding site can be divided into three subsites, i.e., S1, S1′, and the zinc binding site,³⁷⁾ with zinc binding being part of the catalytic motif of this enzyme. The S1 subsite is composed of Thr347, Phe504, Arg514, and Tyr510, which define the selectivity of binders mainly through Van der Waals interactions. Meanwhile, the S1′ subsite is much larger than S1 and can adopt a wide range of substrates that can form H-bonding interactions with Arg273, His345, Thr371, Pro346, and His505, as well as stacking interactions with residues like Phe274, Cys344, Pro346, Met360, Cys361, and Thr371. According to an experimental report,³⁷⁾ MLN-4760 is able to chelate the Zn²⁺ ion and interact with multiple residues in the S1 and S1′ cavities of ACE-2, as shown in Fig. 4. These key interactions of MLN-4760 were conserved during our re-docking, indicating the high reliability of the docking protocol.

Fig. 4. 2D and 3D Redocked Results of MLN-4760 Showing the Binding Cavity Shape of ACE-2 (PDB ID: 1R4L)

Docking of P. trimera Biscoumarin Glycosides against the hACE-2

The validated docking protocol was used to examine the structural interactions of the target protein ACE-2 with the four compounds 1–4 from the roots of P. trimera and to calculate the binding energies of the complexes. The flexible docking results (Table 3) showed that all compounds 1–4 and the positive control MLN-4760 can bind to the ACE-2 enzyme with ΔG values ranging from −10.69 to −14.70 kcal/mol. The ΔG values of biscoumarin glycosides were better than those of several natural polyphenolic compounds, such as quercetin (−9.0 kcal/mol), kaempferol (−8.7 kcal/mol), catechin (−8.3 kcal/mol), gallic acid (−6.0 kcal/mol), the alkaloid indirubin (−8.9 kcal/mol),³⁸⁾ or the stilbene glucoside rhaponticin (−9.3 kcal/mol).³⁹⁾ It is also worth noting that the ranking order of 1–3 based on ΔG values is in line with experimental results (Table 2). In addition, one can observe the difference between the docking scores of the tested compounds and the control, MLN-4760. This fact is owing to the structural difference between carbohydrates 1–4, which are notoriously flexible and hydrophilic molecules compared to rigid, small-molecule ligands with few rotatable bonds like MLN-4760. It is recognized that docking scores may not completely reflect the binding ability of carbohydrates.³⁹⁾ Notwithstanding, understanding how glycoside ligands 1–4 can interact with ACE-2 protein targets at the atomic level is of considerable scientific value for further optimization in drug development. In this regard, molecular docking simulation, which is a popular computational tool can, in principle, fill the gap. The main interactions between these compounds and ACE-2 were subsequently analyzed.

Table 3. Binding Affinities, Hydrogen Bonding and Other Interactions of the Biscoumarin Glycosides (1–4) in the Active Site of the ACE-2 Enzyme

Cpd. No.	Name	Docking scores (kcal/mol)	Hydrogen bonds*	Other interactions*
1	cis-Paratrimerin B	−14.70	Ser128, Glu145, His345, Lys363, Thr371, Glu406, Tyr803	Ala153, Phe274, Arg273, Pro346, Glu375
2	cis-Paratrimerin A	−12.95	Tyr127, His345, Asp367, Thr371, Glu406, Thr445, Arg518	Glu145, Glu150, Ala153, Arg273, Cys344, Cys361 (unfavorable), Lys363
3	trans-Paratrimerin B	−11.94	Asn154, Asn277, Lys363, Thr365, Glu406, Tyr515, Arg518	Arg273, Cys344, Pro346, Glu375
4	trans-Paratrimerin A	−10.69	Glu145, Asn149, Glu406, Thr445, Tyr515	Glu150, Ala153, Arg273, Phe274, His345, Pro346, Lys363, Glu375
5	MLN-4760	−9.12	Arg273, His345, Pro346, Thr371, Tyr515	Tyr510, Phe504, Arg273, His345, Pro346, Arg514, His374, Met360, Arg518, Phe274

* Types of interactions are marked as different colors as annotated in Fig. 5.

Computational Analysis of the Interactions between Biscoumarin Glycosides (1–4) and the Amino Acid Residues within the ACE-2 Complex

Table 3 and Fig. 5 provide a summary of the interactions between biscoumarin glycosides (1–4) and the amino acid residues in the active site of the ACE-2 enzyme protein. After the energy-minimization, all compounds (1–4) could accommodate well within the active site of ACE-2. As demonstrated in Fig. 5 and Supplementary Fig. S20, they can bind to the Zn ion through a coumarin carbonyl group (lactone group) at a close distance of 2.3–2.6 Å. This metal chelation interaction has been described as the main catalytic mechanism of ACE-2 inhibition.³⁴⁾ Unlike MLN-4760, all biscoumarin glycosides generally interacted primarily with residues in the S1′ subsite.³⁴⁾ Given the polyphenol structures, biscoumarin glycosides 1–4 thereby exhibited a higher number of hydrogen bonds than the inhibitor MLN-4760. This compound only forms five H-bonds, while most of the docked biscoumarin glycosides (1–3) exhibit seven H-bonds. Because biscoumarin glycosides have substantially greater surface areas than MLN-4760, they have the ability to interact with more residues throughout the lengthwise channel of the S1′ cavity and have demonstrated higher binding energies than MLN-4760.

Fig. 5. 2D and 3D Interactions of (A) cis- and (B) trans-Paratrimerin A (2 and 4, Respectively); (C) cis- and (D) trans-Paratrimerin B (1 and 3, Respectively) with Residues in the ACE-2 Active Site

cis-Paratrimerin B (1), which has the highest docking score, showed seven H-bonds (Table 3) with Ser128, Glu145, His345, Lys363, Thr371, Glu406, and Tyr803. As can be seen in Fig. 5 and Supplementary Fig. S20, π-stacking interactions between the side chains of Ala153, Arg273, Pro346, and Glu375 against coumarin rings played an essential role in the binding of this compound. Similar to 1, biscoumarin 2, forms seven hydrogen bonds with Tyr127, His345, Asp367, Thr371, Glu406, Thr445, and Arg518. Particularly, cis-paratrimerin A (2) has a hydrogen bond interaction with residue Thr371, similar to the inhibitor MLN-4760 and other natural compounds like the flavonoids apigenin, isovitexin, quercetin, and isorhamnetin.^8,40) It also engages in further interactions with Glu145, Glu150, Ala153, Arg273, Cys344, Cys361, and Lys363. However, the interaction from Cys361 to a glycoside moiety of 2 is composed of two acceptor molecules that can hinder the formation of stable protein-ligand complexes. trans-Paratrimerin B (3) formed the same number of H-bonds as 1 and 2, but had lower interactions with hydrophobic residues in the pocket. As can be observed in Fig. 5 and Supplementary Fig. S20, a disaccharide structure of 3 was accommodated in the far end of the S1′ subsite, which weakened the interactions with the key residues in the active site, such as His345 and Thr371. Among the four docked compounds, trans-paratrimerin A (4) showed the lowest binding energy with a ∆G value of −10.69 kcal/mol. This compound formed five H-bonds with Glu145, Asn149, Glu406, Thr445, and Tyr515, which is quite different from MLN-4760. Despite having a lower number of H-bonds, compound 4 showed higher stacking interactions with hydrophobic residues in the S1′ subsite than the other three biscoumarins. Interestingly, the flavonoids quercetin 3-glucuronide-7-glucoside and isosakuranetin 7-O-neohesperidoside also show hydrophobic interactions similar to 4.⁴¹⁾ As the predominant interaction in the S1′ subsite is H-bonding, the binding of 4 may be weaker in comparison to 1–3.

Conclusion

Two new biscoumarin glycosides, cis-paratrimerin B (1) and cis-paratrimerin A (2), were isolated from the roots of Paramignya trimera. The chemical structure of these compounds was elucidated using modern spectroscopic techniques, including 1D, 2D-NMR, HR-MS, UV, and CD spectra. The biscoumarin glycosides (1–3) were found to have ACE-2 inhibitory effects in vitro with IC₅₀ values of 28.9, 68.1, and 77.1 µM, respectively. Using a computer-based molecular docking method, these four biscoumarin glycosides (1–4) had the highest binding affinity to ACE-2 protein, with calculated ΔG values of −14.70, −12.95, −11.94, and −10.69 kcal/mol, respectively, which were even lower than that of the positive control MLN-4760 (−9.12 kcal/mol). As a result, biscoumarin glycosides from P. trimera roots merit additional research for treating SARS-CoV-2 entrance and maybe producing a plant-based medication.

Experimental

General

¹H-NMR (600 MHz), ¹³C-NMR (150 MHz) spectra were measured on a Bruker AVANCE 600 spectrometer. The CD spectrum was measured on a Chirascan spectrometer (Applied Photophysics, U.K.) using a 1 mm cuvette. Column chromatography (CC) was carried out on silica gel (Si 60 F₂₅₄, 230–400 mesh, Merck KGaA). All solvents were distilled before use. Precoated plates of silica gel 60 F₂₅₄ were used for analytical purposes. Diaion HP-20 (Mitsubishi Chemical, Tokyo, Japan) was used for absorption column chromatography. Compounds were visualized under UV radiation (254, 365 nm) and by spraying plates with 10% H₂SO₄ followed by heating with a heat gun. HPLC was carried out using an Agilent 1260 HPLC system with a DAD detector and an Optima Pak C18 column (10 × 250 mm I.D., 10 µm particle size) and all purified solvents with analytical grade were from Merck KGaA.

The ACE-2 enzyme inhibition of the test substances was measured under the manufacturer’s instructions for the ACE-2 inhibitor screening kit (MAK378, Merck KGaA), which utilizes the ability of an active ACE-2 to cleave a synthetic 4-methoxycoumarin-7-acetic acid (MCA) based peptide substrate to release a free fluorophore. Dulbecco’s modified eagle medium and its supplements, including fetal bovine serum (FBS), were from GIBCO, Invitrogen.

Materials

The roots of Paramignya trimera (Oliv.) Burkill were collected in Ninh Van commune, Khanh Hoa province (May, 2022), and identified by botanist Dr. Nguyen Quoc Binh, Vietnam National Museum of Nature, VAST, Hanoi, Vietnam. A voucher specimen (C-765) was deposited in the herbarium of the Institute of Natural Products Chemistry, VAST, Hanoi, Vietnam. This material was used to prepare the ethanol and water extracts for the ACE-2 inhibitory test.

Preparation of EtOH 96% and Water Extracts of P. trimera Roots for ACE-2 Inhibitory Assay

The dried and ground roots of P. trimera (50 g) were extracted with ethanol-96% twice (using 250 mL each) at 25 °C in an ultrasonic bath (Elma, 220 V, and 50 Hz). Whatman paper was used to filter the extracts, and the combined filtrates were then vacuum-dried to produce the EtOH-96% extract (1.5 g, yield 3% of dry weight). Another 50 g of P. trimera roots were boiled twice with 250 mL each of distilled water. The boiling was kept at 100 °C for ten minutes. The water filtrates were combined and evaporated to dryness to give the water extract (2.35 g, yielded 4.7% of dry weight).

Separation of Biscoumarins 1 and 2

Following the procedures described in an article published by Cuong et al. discussing the isolation and structural determination of paratrimerins A and B,¹⁴⁾ the sub-fraction PW-5D3 of the water extract of P. trimera roots containing biscoumarins 1 and 2 was obtained. Sub-fraction PW-5D3 (300 mg) was chromatographed on a Sephadex LH-20 column eluting with methanol-water (1 : 1, v/v) and further purified on a YMC column eluting with acetone-water (1 : 2, v/v) to yield CPT3B (100 mg) and CPT3C (50 mg).

Fraction CPT3B was purified by Agilent 1260 HPLC system using an Optima PAK C18 column (10 × 250 mm, 10 µm particle size), eluted with an isocratic solvent system of ACN–H₂O + 0.1% formic acid (flow rate 2.0 mL/min) over 50 min, UV detections at 200, 260, and 330 nm, resulted in the isolation of compounds 1 (26.3 mg; t_R = 39.9 min) and 2 (37.8 mg; t_R = 43.0 min), respectively (Supplementary Fig. S18).

Spectroscopic Data

• Compound 1: cis-Paratrimerin B, ivory amorphous powder; C₄₀H₄₄O₁₆
  • ¹H-NMR (600 MHz, CD₃OD) and ¹³C-NMR (150 MHz, CD₃OD): see Table 1. HR-ESI-QTOFMS (m/z): 947.2756 [M + Cl]⁻ (Calcd for C₄₅H₅₂O₂₀Cl⁻: 947.2740, ∆m/m = 1.69), 781.2707 [M − api + H]⁺, 619.2180 [M − api − glc + H]⁺, 413.2182, 352.3405, 310.2007, 223.2047. CD (MeOH, c 0.2 mg/mL) λ_max (θ: mdeg): 209 (39.59), 230 (14.73), 263 (−8.84), 290 (−0.36), 324 (−3.22).

• Compound 2: cis-Paratrimerin A, white amorphous powder; C₄₀H₄₄O₁₆
  • ¹H-NMR (600 MHz, CD₃OD) and ¹³C-NMR (150 MHz, CD₃OD): see Table 1. HR-ESI-QTOFMS (m/z): 781.2673 [M + H]⁺, 619.2132 [M − Glc + H]⁺, 617.1963 [M − Glc − H]⁻, 457.1642 [M − 2Glc + H]⁺. CD (MeOH, c 0.2 mg/mL) λ_max (θ: mdeg): 209 (+16.13), 229 (7.02), 263 (−4.88), 295 (−0.32), 322 (−1.45).

In Vitro ACE-2 Inhibitory Assay

The enzyme inhibition of the test substances was measured in accordance with the manufacturer’s instructions for the ACE-2 inhibitor screening kit (MAK378, Merck KGaA).

Sample preparation: Samples consisting of plant extracts (water and EtOH extracts) as well as isolated compounds were prepared in DMSO in concentration ranges from 100→6.25 µg/mL, with three replicates each. Testing samples (S) along with the negative control (enzyme control, EC), blank (solvent control, SC), positive control MLN-4760 (inhibitor control, IC), and standard background control samples (background control, BC) were prepared in ACE-2 dilution buffer. Fifty microlitters of ACE-2 enzyme solution was added to each well in a 96-well microtiter plate. Then 10 µL of test sample was added to well S, and 10 µL of buffer to wells EC and BC, respectively. Subsequently, 10 µL of ACE-2 inhibitor [DC(+)] and sample diluent were added to IC- and SC wells, respectively. After incubation for 15 min at room temperature, 40 µL of enzyme substrate (MCA-ACE-2) was pipetted into each well. The enzyme inhibition of the samples was measured with λ_Ex/_Em = 320/420 nm (Spark multimode reader, Tecan, Switzerland) in kinetic mode for one hour at room temperature. The enzyme inhibition of the test substance was calculated by comparing with standards provided in the kit, which reduces peptidase activity of ACE-2, resulting in a decrease in fluorescence intensity (relative fluorescence units, ΔRFU). The percentage of ACE-2 inhibitory activity was determined by the formula⁴²⁾:

  

where RFU (S) is the fluorescence signal obtained from the sample well at the selected time of T₁ and T₂; RFU (EC) is the fluorescence signal obtained from the EC well at the time of T1 and T2 selection; σ is the standard deviation calculated by Duncan’s formula. Samples exhibiting ACE-2 inhibitory activity were determined as IC₅₀ (µg/mL or µM) as the sample concentration that inhibits 50% of ACE-2 activity, using TableCurve 2D program (SPSS Statistical Software, Chicago, IL, U.S.A.).

Molecular Docking

In this study, a two-step docking procedure (rigid and flexible) was performed using AutoDock Vina v.1.2.3.^43,44) The structures of paratrimerins (1–4) and MLN-4760 were modeled using Marvin JS and optimized using MMFF94s. The lowest energy conformations of the ligands were determined and used as the starting structures for molecular docking. The target protein (PDB ID: 1R4L) was prepared by removing ligands and water molecules using UCSF Chimera.³⁷⁾ All docking studies were performed in the active site based on a grid box centered on the MLN-4760 co-crystallized ligand with dimension X  =  41.4, Y  =  5.85, and Z  =  28.08, setting a maximum exhaustiveness of 400 and all other parameters as their default values. After obtaining the best conformer, each ligand was flexibly docked into the active site of ACE-2, allowing 11 residues to freely move within the grid box, including Arg273, Thr371, Cys344, His345, Met360, Lys363, Asp368, Tyr510, Arg514, Tyr515, and Arg518. They have been identified as important residues that make close contact with MLN-4760 (radius <4.5 Å). The binding energy (kcal/mol) was computed based on the affinity scoring function implemented into AutoDock Vina v.1.2.3. The visualization of the docking results was mainly carried out using Discovery Studio Visualizer v2021.

Acknowledgments

This research is funded by Grants from the Vietnam Academy of Science and Technology (codes: NCVCC07.05/22-23 and KHCBSS.01/21-23).

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

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