In Silico Studies of Some Isoflavonoids as Potential Candidates against COVID-19 Targeting Human ACE2 (hACE2) and Viral Main Protease (Mpro)

The Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused the “COVID-19” disease that has been declared by WHO as a global emergency. The pandemic, which emerged in China and widespread all over the world, has no specific treatment till now. The reported antiviral activities of isoflavonoids encouraged us to find out its in silico anti-SARS-CoV-2 activity. In this work, molecular docking studies were carried out to investigate the interaction of fifty-nine isoflavonoids against hACE2 and viral Mpro. Several other in silico studies including physicochemical properties, ADMET and toxicity have been preceded. The results revealed that the examined isoflavonoids bound perfectly the hACE-2 with free binding energies ranging from −24.02 to −39.33 kcal mol−1, compared to the co-crystallized ligand (−21.39 kcal mol–1). Furthermore, such compounds bound the Mpro with unique binding modes showing free binding energies ranging from −32.19 to −50.79 kcal mol–1, comparing to the co-crystallized ligand (binding energy = −62.84 kcal mol–1). Compounds 33 and 56 showed the most acceptable affinities against hACE2. Compounds 30 and 53 showed the best docking results against Mpro. In silico ADMET studies suggest that most compounds possess drug-likeness properties.


Introduction
In December 2019, an outbreak of severe pneumonia caused by the novel severe SARS-CoV-2 originated in Wuhan, China. The infection spread all over the world causing coronavirus disease   [1,2]. By October 2020, COVID-19 caused more than 33 million infections and more than 1 million deaths according to the WHO [3]. Unfortunately, till now there is no specific antiviral drug available for the treatment of COVID-19-infected people. However, some drugs such as remdesivir showed modest activity through decreasing the mortality rate and treatment time [4].
Angiotensin-converting enzyme (ACE-2) is a crucial enzyme in the renin-angiotensin system. It is a significant target for antihypertensive drugs [7]. It is primarily expressed in renal tubular epithelium and vascular endothelium cells [8]. It was also reported to be

Experimental
Drug-likeness properties were calculated using Lipinski's rule of five, which suggested that the absorption of an orally administered compound is more likely to be better if the molecule satisfies at least three of the following rules: (i) H bond donors (OH, NH, and SH) ≤5; (ii) H bond acceptors (N, O, and S atoms) ≤10; (iii) molecular weight <500; (iv) logP <5. Compounds violating more than one of these rules could not have good oral bi-

Experimental
Drug-likeness properties were calculated using Lipinski's rule of five, which suggested that the absorption of an orally administered compound is more likely to be better if the molecule satisfies at least three of the following rules: (i) H bond donors (OH, NH, and SH) ≤5; (ii) H bond acceptors (N, O, and S atoms) ≤10; (iii) molecular weight <500; (iv) logP <5. Compounds violating more than one of these rules could not have good oral bioavailability [38]. The pharmacokinetic properties (ADMET) of isoflavonoids and adherence with Lipinski's rule of five were calculated using Discovery studio 4.0 software(Accelrys software Inc., San Diego, CA, USA) [39].
The title molecules were investigated with the aid of docking studies using Discovery At first, water molecules were removed from the complex. Using the valence monitor method, the incorrect valence atoms were corrected. The energy minimization was then accomplished through the application of force fields CHARMM and MMFF94 [42][43][44][45]. The binding sites were defined and prepared for docking processes. Structures of the tested isoflavonoids and the co-crystallized ligands were sketched using ChemBioDraw Ultra 14.0 (PerkinElmer, Waltham, MA, USA) [46] and saved as MDL-SD files. Next, the MDL-SD files were opened, 3D structures were protonated, and energy was minimized by implementing force fields CHARMM and MMFF94, then adjusted for docking. CDOCKER protocol was used for docking studies using CHARMM-based molecular dynamics (MD) to dock the co-crystallized ligands into a receptor binding site [47,48]. In the docking studies, a total of 10 conformers were considered for each molecule. Finally, according to the minimum free energy of binding interaction, the most ideal pose was chosen.
The toxicity parameters for the examined compounds were calculated using Discovery studio 4.0 software (Accelrys software Inc., San Diego, CA, USA). Simeprevir was used as a reference drug. At first, the CHARMM force field was applied then the compounds were prepared and minimized according to the preparation of small molecule protocol. Then different parameters were calculated from the toxicity prediction (extensible) protocol. In the present study, an in silico computational study of compounds (1-59) was performed to determine their physicochemical properties according to the directions of Lipinski's rule of five [38] (Table 1). Moreover, all the tested isoflavonoids were predicted to have good absorption behavior better than that of remdesivir. Also, the solubility levels of most compounds were expected to be in the good range (Table 2 and Figure 2). a BBB level, blood brain barrier level, 0 = very high, 1 = high, 2 = medium, 3 = low, 4 = very low. b Absorption level, 0 = good, 1 = moderate, 2 = poor, 3 = very poor. c PBB, plasma protein binding, 0 means less than 90%, 1 means more than 90%, 2 means more than 95%. d solubility level, 0 = extremely low, 1 = very low, 2 = low, 3 = good, 4 = optimal.

Validation Process
Validation of the docking procedures was achieved via re-docking of the co-crystallized ligands against the active pocket of hACE2 and M pro . The calculated RMSD values

HACE2
Coronavirus spike receptor-binding domain complexed with its receptor hACE-2 (PDB: 6LZG) used as a target for the docking studies of selected isoflavonoids. The results demonstrated that all isoflavonoids bound strongly to hACE-2 with binding energies bitter than that of the co-crystallized ligand (NAG). This indicated that the affinity of the tested isoflavonoids toward hACE-2 is higher than that of the co-crystallized ligand ( Table  3). Moreover, almost all the tested isoflavonoids exhibited binding modes similar to that of NAG.

HACE2
Coronavirus spike receptor-binding domain complexed with its receptor hACE-2 (PDB: 6LZG) used as a target for the docking studies of selected isoflavonoids. The results demonstrated that all isoflavonoids bound strongly to hACE-2 with binding energies bitter than that of the co-crystallized ligand (NAG). This indicated that the affinity of the tested isoflavonoids toward hACE-2 is higher than that of the co-crystallized ligand (Table 3). Moreover, almost all the tested isoflavonoids exhibited binding modes similar to that of NAG. Table 3. Free binding energies of the selected isoflavonoids and the co-crystallized ligand (NAG) against hACE-2 and amino acid residues involved in H. bonds and hydrophobic interaction.

Comp.
Binding Energy (kcal mol −1 )  The binding pattern of co-crystallized ligand (NAG) demonstrated single hydrogen bonding interaction with Ser371 residue (Figure 4). NAG showed binding energies of −21.39 kcal mol −1 . It was found that most of the tested isoflavonoids exhibited binding modes similar to the reference molecule. Compounds 1 (−30.90 kcal mol -1 ) and 8 (−27.41 kcal mol −1) demonstrated an additional hydrogen bond with Asn343 residue (Figures 5 and 6). This extra hydrogen bond may account for the relatively high binding affinity of both compounds. Furthermore, compounds 33 ( Figure 7) and 56 ( Figure 8) were found to have good binding energy values of −36.35 and −34.90 kcal mol −1 , respectively. Compound 33 formed a binding mode like that of the reference ligand as it formed one hydrogen bond with Ser371 and seven hydrophobic interactions with Phe374, Phe342, Ser371, Asn343, Cys336, Glu340, and Ser373. Interestingly, compound 56 formed two hydrogen bonds with Ser371 and Cys336 in addition to seven hydrophobic interactions with Phe374, Phe338, Ser371, Val367, Cys336, Leu368, and Ser373.
Such results indicate the significance of the tested isoflavonoids as potential inhibitors for hACE-2. Consequently, such compounds may inhibit the entrance of coronavirus into human cells.

Main Protease (M pro )
The docking results of isoflavonoids into the active site of coronavirus M pro (PDB: 6LU7) were listed in Table 4. The results showed that all tested isoflavonoids can bind to M pro with one or more hydrogen bonds. At the same time, the tested compounds bound to the receptor with free binding energies ranging from −32.19 to −50.79 kcal mol −1 , compared to the co-crystallized (binding energy = −62.84 kcal mol −1 ).
These results revealed that the affinities of the presented isoflavonoids against M pro are lower than that of N3. Despite that, the binding energies are still considerable, and their binding modes are great which making these isoflavonoids seem to be biologically active ligands to some extent. Compound 30 formed a binding mode like that of the reference ligand as it formed three hydrogen bonds with Glu166, Tyr54, and Asp187. Furthermore, it formed eight hydrophobic interactions with His41, Gln189, His163, Met165, Tyr54, Asp187, Leu167, and Glu166. For compound 53, it formed two hydrogen bonds with Glu166, Phe140. Besides, it formed six hydrophobic interactions with Glu166, Gln189, Leu141, Met165, His172, and Phe140.

Main Protease (M pro )
The docking results of isoflavonoids into the active site of coronavirus M pro (PDB: 6LU7) were listed in Table 4. The results showed that all tested isoflavonoids can bind to M pro with one or more hydrogen bonds. At the same time, the tested compounds bound to the receptor with free binding energies ranging from −32.19 to −50.79 kcal mol −1 , compared to the co-crystallized (binding energy = −62.84 kcal mol −1 ). Glu166. For compound 53, it formed two hydrogen bonds with Glu166, Phe140. Besides, it formed six hydrophobic interactions with Glu166, Gln189, Leu141, Met165, His172, and Phe140. Figure 9. the co-crystallized ligand (N3) docked into M pro , forming four H. bonds with Gln189, Tyr 54, Asp 142, Asp187.
Observing binding affinities of isoflavane derivatives. It was found that compound 4 incorporating an additional methoxy group at 6 position of phenyl ring showed better binding affinity against hACE-2 than the unsubstituted derivative (compound 5). Such a result may be attributed to the electron donating effect of the methoxy group.
Concerning the activity of different pterocarpan derivatives, it was noted that compound 11, which contained an additional tetrahydrofuran ring attached to the chromene ring, showed better binding affinity inside the hACE-2 than compounds 9 and 10, which contained free OH groups at the chromene ring.  For isoflavone derivatives (compounds 34, 33, 35 and 37), it was found that compound 34 incorporating 3-hydroxy-3-methylbutyl moiety at 6-position was more active that compound 33 incorporating 3-methylbut-2-en-1-yl moiety at the same position. The latter was more active than compound 35 incorporating 2-hydroxy-3-methylbut-3-en-1-yl moiety at the same position. Compound 37 incorporating 3-methylbut-2-en-1-yl moiety at 8-position was less active than the corresponding members.
Then, we investigated the effect of substitutions at isoflavone derivatives on the binding affinity. It was found that the substitutions at 5-position with hydroxyl (compound 1) and methoxy (compound 3) group, increase the binding of isoflavones against hACE-2, with an increased affinity of hydroxyl derivative.
Regarding the effect of substitutions at isoflava-3-ene, it was found that the derivative with additional pyran ring (compound 6) was more active than the corresponding member with free OH group at position-1 of phenyl ring (compound 8) which was more potent than compound 7 incorporating a dioxolan ring .
Observing binding affinities of isoflavane derivatives. It was found that compound 4 incorporating an additional methoxy group at 6 position of phenyl ring showed better binding affinity against hACE-2 than the unsubstituted derivative (compound 5). Such a result may be attributed to the electron donating effect of the methoxy group.
Concerning the activity of different pterocarpan derivatives, it was noted that compound 11, which contained an additional tetrahydrofuran ring attached to the chromene ring, showed better binding affinity inside the hACE-2 than compounds 9 and 10, which contained free OH groups at the chromene ring.

Toxicity Studies
Toxicity prediction was carried out based on the validated and constructed models in Discovery studio 4.0 software [49,50] as follows. (i) FDA rodent carcinogenicity test which computes the probability of a compound to be a carcinogen. (ii) Carcinogenic potency TD 50 which predicts the tumorigenic dose rate 50 (TD 50 ) of a drug in a rodent chronic exposure toxicity test of carcinogenic potency [51]. (iii) Rat maximum tolerated dose (MTD) [52,53]. (iv) Rat oral LD 50 which predicts the rat oral acute median lethal dose (LD 50 ) of a chemical [54]. (v) Rat chronic LOAEL which predicts the rat chronic lowest observed adverse effect level (LOAEL) value [55,56]. (vi) Ocular irritancy predicts whether a particular compound is likely to be an ocular irritant and how severe the irritation is in the Draize test [57]. (vii) Skin irritancy predicts whether a particular compound is likely to be a skin irritant and how severe it is in a rabbit skin irritancy test [57].
As shown in Table 5, most compounds showed in silico low toxicity against the tested models. FDA rodent carcinogenicity model indicated that most of the tested compounds are non-carcinogens. Only compounds 6, 9, and 10 were predicted to be carcinogens so that, these compounds do not have the likeness to be used as drugs.