Antcins have less cytotoxicity than GC376. Prior to delving into the SARS-CoV-2 3CLPro inhibitory effects of antcins, it is vital to assess the cytotoxic effects of antcins on A549 cells. To evaluate cytotoxicity, we treated A549 cells with various antcins (antcin-A, antcin-B, antcin-C, antcin-H, antcin-I, and antcin-M), non-antcin compounds (citronellol and limonene), and GC376 at increasing concentrations (5–80 µM) for 24, 48, and 72 h. GC376 was developed as a pharmacological inhibitor of 3CLPro14. Based on the MTT assay, we found that treatment with GC376, a positive drug control, caused significant cytotoxicity over a concentration of 40 µM at 48 and 72 h, and 20 µM GC376 decreased cell viability to 30% after 72 h of incubation (Fig. 2a). Except antcin-A, other antcins did not exhibit significant cytotoxicity towards A549 cells even at a concentration of 80 µM for 24 h (Fig. 2b-g). Whereas antcin-A and antcin-M exhibited significant cytotoxicity at a dose of 80 µM at 48 and 72 h. Likewise, citronellol did not induce cytotoxicity up to a dose of 20 µM, whereas 40 µM and 80 µM caused cytotoxicity at 72 h (Fig. 2h). Indeed, limonene did not exhibit significant cytotoxicity neither in a concentration- nor time-dependent manner (Fig. 2i). These findings collectively suggest that antcins have lower cytotoxicity towards A549 cells compared to GC376. Furthermore, antcins appear to have no potential adverse effects on cells, making them suitable for drug screening and further investigation. Consequently, based on the MTT assay results, we selected all the aforementioned compounds for screening in the docking study and other experiments.
Drug-likeness, physicochemical properties, toxicity risk assessment, and ADMET prediction of antcins. Drug discovery and development pose significant challenges and costs when targeting specific diseases involving multiple stages such as selection, target identification, validation, lead discovery, and optimization, as well as preclinical and clinical trials24. Therefore, crucial factors such as chemical absorption, distribution, metabolism, excretion, and toxicity (ADMET) play a vital role in the drug discovery and development process25. In-silico models can accurately predict ADMET properties based on specific parameters, aiding in the identification of viable drug candidates for particular diseases. To evaluate the drug-likeness of the ligand's physicochemical properties and assess its toxicity risk, we employ the OSIRIS Property Explorer. This tool allows us to assess parameters such as solubility (ranging from 0 to − 6), TPSA (topological polar surface area ≤ 130 Å2), drug-likeness (scores with a positive value, are likely to be an oral drug), drug score (0–1), and molecular weight (MW) lower than 500 grams/mole. Based on our analysis, all the tested compounds exhibit moderate soluble, TPSA values of 130 Å2, and drug scores within a range of 0–1 (Table S1), which indicate favorable drug-likeness and low toxicity risks. Notably, this prediction aligns with the cytotoxicity of the tested compounds (Fig. 2). Next, we screened the ADMET properties of test compounds using AdmetSAR server2 http://lmmd.ecust.edu.cn/admetsar226. In-silico toxicity testing of our hit compounds reveals insights into their pharmacokinetics and pharmacodynamics compared to the selected control drugs. Regarding absorption, Caco-2 permeability testing predicted that all compounds, including GC376, to be absorbed by the human intestine. The combinations were also expected to exhibit human oral availability, p-glycoprotein inhibition, and act as substrates. None of the compounds were deemed AMES mutagenic or carcinogenic, and they were anticipated to be non-toxic to the liver cells. The predicated acute oral toxicity (kg/mol) is presented in Table S2. Two properties, plasma protein binding (PPB) and blood-brain barrier (BBB) permeability, were utilized to assess the compound’s distribution. All the compounds exhibited the potential to reach their target site at high to moderate doses, as their ability to bind to plasma proteins was predicted to be over 95% in most cases. However, GC376 (58.38%) and limonene (86.38%) showed a lower likelihood of binding to plasma proteins, as indicated in Table S2. Moreover, all the compounds were predicted to function as substrates or inhibitors of various cytochrome enzymes, including CYP1A2, CYP2C19, CYP2C19, CYP2C9, CYP2C9, CYP2D6, CYP2D6, and CYP3A4 as shown in Table S2. Additionally, the predicted acute oral toxicity (kg/mol) for the compounds is provided in Table S2.
Antcin-B has the lowest binding affinity energy determined by molecular docking. Molecular docking is a method to determine the interaction between a small molecule and a protein at the atomic level27. Therefore, the interactions between antcins and non-antcin compounds with SARS-CoV-2-3CLPro were determined by molecular docking. 3D crystal structure of 3CLPro (7LME) was retrieved from RCSB. The detailed structure of 3CLPro is depicted in Figure S1a. Molecular docking for each compound with 3CLPro was determined one by one using CB-docking228, which predicted a more than 85% success rate compared to FitDock, MTiAutoDock, SwissDock, and COACH-Dn. After docking, the conformation with the least binding energy (BE, kcal/mol) was considered the most suitable docking pose of the compounds, finding the active site of the 3CLPro, as presented in Table 1. Docking with 3CLPro, all the antcins, including antcin-A, antcin-B, antcin-C, antcin-H, antcin-I, and antcin-M were produced the least BE of − 7.2, − 8.2, − 7.2, − 7.6, − 8, and − 8.1, kcal/mol, respectively, whereas citronellol and limonene showed much higher BE of − 4.2 and − 4.1 kcal/mol, respectively. Interestingly, GC376 produced less BE of − 7.1 kcal/mol, which is comparable with antcins. The binding site of 3CLPro is contacted by various residues, including Glu166, Leu141, Phe140, His,172, His163, Asn142, Thr25, Thr24, Met165, Met49, Asp187, His41, Thr54, Leu167, Phe185, Gln192, Gln189, Met165 168, Ala191, and Thr190, located in substrate subsites of 3CLPro such as S1, S1' S2, S4, and S5, as shown in Fig. 3a-c30. All these compounds bound at active sites with residues His41 and Cys14529, as shown in Table 1 and Fig. 3d-l.These residues might explain the inhibitory activity of these compounds. For example, antcin-B exhibits the lowest binding affinity energy, contacting 19 amino acids within the subsite binding cleft, including S1'-Thr25 and Th24, S1-Glu166, Leu141, Phe140, Asn142, and His163, S2-Met165, Met49, His41, S4-Gln189 whereas Cys44, Thr45, Ser46, Ser144 Cys145, Arg188 are not present in subsites cleft, which differentiates it more other ligands with 3CLPro. Details of the interactions with other compounds are proved in Table 1. Moreover, citronellol does contact with Cys145 and His41, as shown in Table 1. Molecular docking relies on specific parameters, where the docked complexes must reproduce the original poses of the native ligands with precision. Therefore, the complex was redocked using the Auto-Dock Vina module; UCSF-Chimera© (version 1.16) revealed that antcin-B exhibited the lowest binding energy of -9.3 kcal/mol, followed by antcin-H, antcin-M, antcin-I, antcin-A, antcin-C, GC376, citronellol, and limonene with BE of − 7.6, − 7.4, − 7.2, − 7.2, − 6.7, − 7.4, − 4, and − 4.1 kcal/mol, respectively. These results align with the CB-dock2 binding presented in Table 1. All these compounds, except limonene, bound to the active sites with residues His41 and Cys145. These results suggest that the antcins, especially antcin-B, have the potential inhibitory capacity against 3CLPro, while citronellol and limonene lack inhibitory ability.
Table 1
Results of binding affinity (Kcal/mol), cavity and docking size, and contacts residues from non-antcins and GC376, which were docked against the SARS-CoV-2 3CLPro protein using CB-dock2 and Auto-Dock Vina module; UCSF-Chimera.
PubChem CID | Compounds name | Kcal/mol (1) | Kcal/mol (2) | Cavity Volme (Å3) | Center (x, y,z) | Docking size (x,y,z) | Contact residues |
44424392 | Antcin-A | −7.2 | −7.2 | 397 | 27, 0, 14 | 25, 25, 25 | Thr25 His41 Val42 Cys44 Thr45 Ser46 Met49 Phe140 Leu141 Asn142 Cys145 His163 His164 Met165 Glu166 Arg188 Gln189 |
387397 | Antcin-B | −8.7 | −9.3 | 397 | 27, 0, 14 | 24, 24, 24 | Thr24 Thr25 His41 Cys44 Thr45 Ser46 Met49 Phe140 Leu141 Asn142 Gly143 Ser144 Cys145 His163 His164 Met165 Glu166 Arg188 Gln189 |
10050412 | Antcin-C | −7.2 | −6.7 | 397 | 27, 0, 14 | 25, 25, 25 | Thr25 Leu27 His41 Val42 Cys44 Thr45 Ser46met49 Phe140 Leu141 Asn142 Gly143 Ser144 Cys145 His163 His164 Met165 Glu166 Arg188 Gln189 |
68150384 | Antcin-H | −7.6 | −7.6 | 397 | 27, 0, 14 | 25, 25, 25 | Thr24 Thr25 Thr26 Leu27 His41 Ser46 Met49 Asn142 Gly143 Cys145 His164 Met165 Glu166 Leu167 Arg188 Gln189 |
Not Yet | Antcin-M | −8 | −7.4 | 397 | 27, 0, 14 | 23, 23, 23 | Thr25 Thr26 His41 Cys44 Thr45 Ser46 Met49 Phe140 Leu141 Asn142 Ser144 Cys145 His163 His164 Met165 Glu166 His172 Gln189 |
Not Yet | Antcin-I | −8.1 | −7.2 | 397 | 27, 0, 14 | 23, 23, 23 | Thr24 Thr25 Thr26 His41 Cys44 Thr45 Ser46 Met49 Phe140 Leu141 Asn142 Ser144 Cys145 His163 His164 Met165 Glu166 His172 Gln189 |
8842 | Citronellol | −4.1 | −4 | 397 | 27, 0, 14 | 19, 19, 19 | His41 Met49 Phe140 Leu141 Asn142 Gly143Ser144 Cys145 His163 His164 Met165 Glu166 Asp187 Arg188 Gln189 |
22311 | Limonene | −4.2 | −4.1 | 397 | 27, 0, 14 | 17, 17, 17 | His41 Met49 Asn142 His164 Met165 Glu166 Val186 Asp187 Arg188 Gln189 |
71481119 | GC376 | −7.1 | −7.4 | 397 | 27, 0, 14 | 24, 24, 24 | Thr25 Thr26 Leu27 His41 Met49 Phe140 Leu141 Asn142 Gly143 Ser144 Cys145 His163 His164 Met165 Glu166 Leu167 His172 Arg188 Gln189 |
Antcin-B inhibits 3CL Pro activity via hydrophobic interaction, hydrogen bonding, and salt bridging. We employed PLIP31 and ProteinPlus32 to determine and visualize the binding pose and interaction patterns obtained from the molecular docking. These ligands were found to bind to specific sites of the 3CLPro (Table 1 and Fig. 3a-l). Despite the fact that the ligands fit similar cavities, their interaction with amino acids varied when analyzed with PLIP (Table 2). GC376 displayed interactions with 10 amino acids. These interactions included Thr25, Met165, Glu166, and Gln189 via hydrophobic interaction at 3.67, 3.78, 3.62, and 3.92 Å, respectively. Additionally, Phe140, Leu141, Asn142, Gly143, and Glu166 formed hydrogen bonds with GC376 at bound lengths of 3.42, 2.94, 2.91, 3.56, and 2.97 Å, respectively. Furthermore, a salt bridge was formed with His163 (Fig. 4a and Table 2). Indeed, antcin-B exhibited the lowest BE and interaction with 6 amino acids. It formed hydrophobic interaction with Glu169 and Gln189, while the other four amino acids (His41, Leu141, Asn142, and Glu166) engaged in hydrogen bonding. Moreover, a salt bridge was formed with His165. All bond length is < 4 Å (Fig. 4b and Table 2). These patterns closely resembled GC376 (Fig. 4a,b, and Table 2), which explains why antcin-B displayed the highest inhibitory activity. Antcin-A engaged hydrophobic interaction with Leu 141, Met165, and Glu166, which formed a hydrogen bond with Thr25 and a salt bridge with His41 (Fig. 4c and Table 2), Despite these differences, antcin-A shared the same binding cavity with antcin-B. Antcin-C only contacted Met165 and Glu166 via hydrophobic interaction, interacted with Thr25 and Thr45 via hydrogen bonding, and formed a salt bridge with His41 (Fig. 4d and Table 2). Although both antcin-H and antcin-M have to BE, neither of these compounds interacted with His41 nor Cys145, (Fig. 4e,g and Table 2). Conversely, antcin-I interacts with His41 at a bound length of 2.49 Å through hydrogen bonding (Fig. 4f). Citronellol and limonene displayed lower BE. Citronellol only interacted with His41 through hydrogen bonding at a bond length of 2.23 Å. Limonene engaged in hydrophobic interactions with three amino acids, Met49, Met165, and Gln89, at bond lengths of 3.87, 3.78, and 3.87 Å, respectively (Fig. 4h, i). The docking results and ligand-protein interactions were further validated using ProteinPlus, which offers detailed 2D protein-ligand interaction. ProteinPlus performs various pre-processing tasks, including structure quality assessment (EDIA), hydrogen placement (Protoss), and the identification of alternative conformations (SIENA). Additionally, it addresses common challenges such as generating 2D-interaction diagrams (PoseView) and http://proteins.plus33. The detailed interaction is provided in Figure S2a-h. Furthermore, the docking results demonstrated that GC376 and antcin-B exhibited similar interactions with Thr25 residue with their benzene functional group as a backbone, rather than the active group (Fig. S2a, b). Interestingly, both antcins and non-antcins displayed similar interaction with 3CLPro, suggesting inhibitory activity (Fig. S2c,d). These results indicate that the compact structures of different antcins generate similar functions and interactions with 3CLPro, potentially inhibiting its activity. Notably, the interaction pattern of antcin-B with amino acid residues in 3CLPro resembles that of GC376. Both compounds engage in hydrophobic interaction with Met165 and Gln189, as well as hydrogen bonding with Leu141, Asn142, and Glu166. However, salt bridges with His163, which were absent in the antcins (A, C, H, I, and M), citronellol, and limonene might not contribute to the inhibition of 3CLPro activity, which is crucial for anti-COVID-19. Nonetheless, antcin-B showed the highest potential as a candidate for anti-COVID-19 treatment.
Table 2
Details of binding interactions of the antcins (A, B, C, H, I, and M), non-antcins (citronellol and limonene), and DC376 docked into the active site of the SARS-CoV2-3CLPro and formed ligand-protein complex.
Ligand-protein complex | Interaction | Residues (AA) | Bond length (Å) |
Antcin-A-SARS-CoV2-3CLPro | Hydrophobic | Leu141, Me165, Glu166 | 3.89,3.93, 3.7 |
| Hydrogen bonds | Thr25 | 3.76 |
| Salt Bridges | His41 | 4.8 |
Antcin-B-SARS-CoV2-3CLPro | Hydrophobic | Met165, Gln189A | 3.94, 3.95 |
| Hydrogen bonds | His 41, Leu141, Asn142 Glu166 | 2.71, 2.77, 2.67 3.1 |
| Salt Bridges | His163 | 4.07 |
Antcin-C-SARS-CoV2-3CLPro | Hydrophobic | Met165, Glu166 | 3.8, 3.76 |
| Hydrogen bonds | Thr25, Thr45 | 3.78, 3.51 |
| Salt Bridges | His41 | 4.69 |
Antcin-H-SARS-CoV2-3CLPro | Hydrophobic | Met49, Gln189 | 3.65, 3.79 |
| Hydrogen bonds | Asn142, Glu166 | 3.15, 2.47 |
| Salt Bridges | × | × |
Antcin-I-SARS-CoV2-3CLPro | Hydrophobic | Thr25, Met49, Glu166 Gln189 | 3.49, 3.82, 3.56 3.91 |
| Hydrogen bonds | His41 | 2.71 |
| Salt Bridges | His163, His172 | 4.46, 5.45 |
Antcin-M-SARS-CoV2-3CLPro | Hydrophobic | THr25, Met49, Glu166 Gln189 | 3.83, 3.79, 3.59 3.6 |
| Hydrogen bonds | Phe140, Gln189 | 2.47, 3.275 |
| Salt Bridges | His163, His172 | 4.42, 5.45 |
Citronellol-SARS-CoV2- 3CLPro | Hydrophobic | Met165 | 3.85 |
| Hydrogen bonds | Leu141 | 2.62 |
| Salt Bridges | × | × |
| Interaction | Residues (AA) | Bond length (Å) |
Limonene-SARS-CoV2- 3CLPro | Hydrophobic | Met49, Met165, Gln189 | 3.87, 3.78, 3.87 |
| Hydrogen bonds | × | × |
| Salt Bridges | × | × |
GC376-SARS-CoV2-3CLPro | Hydrophobic | Thr25, Met165, Glu166 Gln189 | 3.67, 3.78, 3.62 3.92 |
| Hydrogen bonds | Phe140, Leu141, Asn142 Gly143, Glu166 | 3.42, 2.94, 2.915, 3.56, 2.975 |
| Salt Bridges | His163 | 4.76 |
Note: Interaction details the ligand-protein complex was performed by using the PLIP. Three types of interaction were observed: hydrophobic interaction, hydrogen bonds, and salt bridges. Residues (AA), Amino acid, and Bond length are measured in the (Å). X represents the absence of the salt bridge bond formation between the ligand and target protein. |
100 nanoseconds (ns) molecular dynamics (MD) simulation exhibiting antcin-B has the highest stable interaction with 3CL Pro . To validate the predicted molecular interactions, 100 ns molecular dynamics (MD) simulations were conducted for all ligand-protein complexes obtained from the molecular docking outcomes. The interaction between GC376 with Glu166 involved both a hydrogen bond and a water bridge and persisted for approximately 70% of the simulation time. Additionally, GC376 demonstrated sustained interaction with Gln189, and to a lesser extent, with Thr190 and Gln192. In the cases of Gln189 and Thr190, both hydrogen bonds and water bridges were formed, while Gln192 only exhibited a water bridge (Fig. 5a and Fig. S3a, and Fig. S4a-i). Noteworthy interactions were observed with antcin-B. These interactions included hydrogen bonds with Asn142 and Gly143, as well as a consistent water bridge with Glu166, which occurred for approximately 40%, 20%, and 70% of the simulation duration, respectively (Fig. 5b and Fig. S3b). However, other antcins displayed weaker interactions. Antcin-A formed only two hydrogen bonds with Arg4, each persisting for approximately 35% of the total simulation time. Additionally, a hydrophobic interaction with Phe3 was observed for slightly over 20% of the simulation time (Fig. 5c and Fig. S3c). Antcin-C, which exhibited higher potency compared with antcin-A, formed a hydrogen bond with Thr26 for approximately 45% of the simulation time. Short-term water bridges were also observed with Asn119, Asn142, and Gln189, each present for less than 20% of the simulation time. Among these, a hydrogen bond with Gln189 was formed for a similar duration (Fig. 5d and Fig. S3d).
Moreover, antcin-H, antcin-I, and antcin-M demonstrated a moderate interaction compared to antcin-A and antcin-C. During the simulation, antcin-H established hydrogen bonds with Thr26 (approximately 90% of the time) and formed a water bridge with Gln189 for around half of the simulation time (Fig. 5e and Fig. S3e). Antcin-I exhibited significant interactions, including hydrogen bonds with Thr26 (~ 90%), Asn119 (~ 50%), and Gln189 (~ 60%). Additionally, water bridges were formed with Met49 (~ 70%), Asn142 (~ 50%), Arg188 (~ 40%), and Gln189 (~ 70%) (Fig. 5f and Fig. S3f). Antcin-M displayed weaker interaction, the most notable being a hydrogen bond with Gln189, present for 60% of the simulation time. Water bridges were also observed with Thr26 and Gln189, respectively, for 30% and 20% of the time (Fig. 5g and Fig. S3g). Furthermore, citronellol formed a hydrogen bond with His41 for approximately 45% of the simulation time, while hydrophobic interactions with Met49 and Met165 were observed for around 30% and 20% of the simulation time, respectively (Fig. 5h and Fig. S3h). Whereas limonene did not exhibit significant interactions throughout the simulation (Fig. 5i and Fig. S3i). The observed patterns of protein-ligand interactions in the MD simulations corresponded to the experimental results, providing partial insights into the varying binding potencies among these compounds.
Antcin-B significantly reduced SARS-CoV-2 3CL Pro enzymatic activity in vitro. To validate the results obtained from molecular docking and MD simulation, an in vitro enzymatic inhibitory assay was performed. 3CLPro enzyme inhibitory effects of antcins were screened at 20 µM concentration, as indicated by the MD, and the docking results, which are showed potent inhibition at a lower concentration. However, the inhibitory potency of antcin-C, citronellol, and limonene showed weaker interaction at a lower concentration. Therefore, higher concentrations of 40 µM, 100 µM, and 100 µM, respectively, were used. GC376 (100 nM) was used as positive drug control. 3CLPro enzyme inhibitory assay revealed that compared with the control group (no inhibitor), treatment with GC376, antcin-A, antcin-B, antcin-H, antcin-I, antcin-M, and citronellol were significantly inhibited 3CLPro activity by 96.72%, 25.7%, 96.39%, 41.17%, 54.74%, 66.54%, and 37.7% respectively (Fig. 6a). Whereas, antcin-C (-18.5%) and limonene (0.7%) failed to inhibit 3CLPro activity (Fig. 6a). Indeed, among the tested antcins, antcin-B exhibited a highly significant reduction in 3CLPro activity, which is highly comparable with GC367. This finding is consistent with antcin-B’s low binding energy, strong interaction with target residues, and stable interaction during MD simulation. Next, a dose-dependent study determined the optimal concentration to inhibit maximum 3CLPro enzymatic activity. Subsequently, a dose-dependent analysis with increasing concentrations (0.3125, 0.625, 1.25, 2.5, 5, 10, and 20 µM) of antcin-B was conducted. The results revealed that antcin-B exhibited the maximum inhibitory activity against 3CLPro at a concentration of 2.25 µM, surpassing the effectiveness of the positive drug control, GC376. Notably, all antcins (A, B, H, I, and M) exhibited inhibitory activity against 3CLPro, except antcin-C. Conversely, limonene did not demonstrate significant inhibitory activity against 3CLPro.