From each highly populated chemical class obtained from the HiTSet, we analyzed the lead compound showing the highest docking score. For these compounds, we have elucidated and discussed their binding mode into Mpro protease active site and predicted their binding energy as well as their IC50 (Table 2).
4.2.1 Benzene and substituted derivatives
This chemical class is the largest in terms of number of compounds. The most active compound holds a fluorobenzene moiety and presented a docking score of –10,6 Kcal/mol which corresponded to a predicted IC50 of 0.6 μM derived from the simple linear regression we have obtained before. This compound, referred to as HBF–0259, was reported as an inhibitor of hepatitis B virus surface antigen (HBsAg) secretion [25]. The visual inspection of its binding mode to Mpro protease revealed multiple contacts to the residues of the active site of types, HB (Leu–141, Gly–143, Ser–144, Cys–145), VDW (Met–49, His–163), and π-sigma (Leu–27, His–41, Met–165) (Figure 4-a). Further optimization of the potency of this compound could be carried out through diverse substitutions on the phenyl groups attached to the pyrimidine core. The substitutions could target new interactions with other amino acids in the active site for enhanced affinity to the protease.
4.2.2 Quinolines and derivatives
The best compound from this class, a triazine derivative, resulted in a docking score of –10.8 Kcal/mol which corresponds to a predicted IC50 of 0.57 µM. This compound, named Capmatinib, is a potent inhibitor of c-Met kinase with antineoplastic activity [26]. It showed dense network of contacts within the Mpro protease active site including HB (Ser–144, Thr–25), VDW (Cys–44, Gln–189, Ser–46, His–172, His–163, Leu–141, Leu–27), π-Alkyl (Met–49, Cys–145, Thr–26) and π-π T-shaped to His–41 (Figure 4-b). Different potency optimization possibilities are available on the quinoline moiety as well as on the Fluro-phenyl moiety.
Table 2. Predicted most active compounds from the HitSet chemical classes.
Compound
|
Chemical Class
|
Direct Parent Class
|
Predicted Binding Energy (Kcal/mol)
|
Stdev Binding Energy (Kcal/mol)
|
Predicted IC50 (µM)
|
HBF-0259
|
Benzene and substituted derivatives
|
Fluorobenzenes
|
-10,60
|
1,10
|
0.60
|
Capmatinib
|
Quinolines and derivatives
|
Quinolines and derivatives
|
-10.80
|
1.17
|
0.57
|
Arcyroxocin B
|
Indoles and derivatives
|
Hydroxyindoles
|
-10.80
|
0.72
|
0.57
|
DR396
|
Benzopyrans
|
Xanthenes
|
-11.30
|
0.52
|
0.51
|
Pazinaclone
|
Carboxylic acids and derivatives
|
Beta amino acids and derivatives
|
-11.50
|
1.03
|
0.49
|
Fumiquinazoline C
|
Diazanaphthalenes
|
Quinazolines
|
-11.60
|
0.95
|
0.48
|
57
|
Diazinanes
|
N-arylpiperazines
|
-10.30
|
0.67
|
0.64
|
SR-3029
|
Azoles
|
Phenylimidazoles
|
-11.20
|
0.76
|
0.52
|
AMG-579
|
Organooxygen compounds
|
Aryl-phenylketones
|
-10.50
|
0.49
|
0.61
|
Silydianin
|
Flavonoids
|
Flavones
|
-11.1
|
0.69
|
0.53
|
4.2.3. Indoles and derivatives
In this chemical class, the best two compounds (Score = –10.9 Kcal/mol) are straurosporine like compounds which are potent non-specific inhibitors of kinases but present high toxicity. For this reason, we choose to analyse the binding mode of the third best compound presenting a docking score of –10.8 Kcal/mol (predicted IC50 = 0.57 uM) and which is referred to as Arcyroxocin B. This alkaloid was isolated from two fungal species Arcyria denudata and Arcyria obvelata and showed cytotoxicity against Jurkat cells [27], an immortalized line of human T lymphocyte cells that are used to study acute T cell leukemia. When docked into the active site of Mpro protease, Arcyroxocin B atoms established multiple HB contacts (Cys–145, Gly–143, Ser–144, Leu–141, His–163, Glu–166), VDW interactions (His–41, Phe–140, Met–165), and π-Alkyl interaction with Cys–145 (Figure 4-c). The potency of this compound can be further optimized through substitutions on the phenyl groups of the two indole moieties.
4.2.4. Benzopyrans
Two compounds from this chemical class, fluorescein 5-maleimide and DR396 a benzoic acid derivative compound, presented docking scores of –11.3 Kcal/mol corresponding to predicted IC50 of 0.5 µM. We choose to study the binding mode of DR396 since it has a reported biological activity as an apoptotic DNase γ inhibitor [28]. In fact, DR396 showed several interactions with the protein amino acids of type HB (Tht–190, Gln–192), Halogen (Glu–166, Phe–140, Gly–143, Asn–142), VDW (Arg–188, Leu–167, Ser–144, Cys–145, His–41, Met–49, Gln–189), and π-interactions (Met–165) (Figure 4-d). Chemical diversifications for optimizing the potency of this compounds could take place at different positions of the molecule on the benzopyran core as well as on the phenyl group of the benzoic acid.
4.2.5. Carboxylic acids and derivatives
In this chemical class of selected hits, the best compound showed a docking score of –11.5 Kcal/mol and a predicted IC50 of 0,48 µM. This compound referred to as Pazinaclone (DN–2327), is an anxiolytic drug [29]. The evaluation of the binding mode of pazinaclone in the Mpro protease active site showed that it is interacting with several protein amino acids through HB (Gly–143, Cys–145, Ser–144, Asn–145, Glu–166), VDW (Leu–27, Leu–141, His–163, His–164), Alkyl interaction with Met–165, and π-interactions with His–41 (Figure 4-e). For optimization purposes, chemistry could take place with diverse substitutions on the naphtyiridine group.
4.2.6. Diazanaphthalenes
Two compounds from this chemical class, fumiquinazoline C and H, presented a docking score of—11,6 Kcal/mol corresponding to predicted IC50 of 0.47 µM. These two molecules are produced by Aspergillus fumigatus, a marine fungus that makes a series of fumiquinazoline peptidyl alkaloids [30]. As the compounds are very similar, we reported only the binding of fumiquinazoline C whose derivatives were reported to have antitumor activity [38]. Fumiquinazoline C predicted binding mode revealed that the atoms of this compounds are in close contact with different protein residues through HB (His–41, His–163, Glu–166, Cys–145), VDW interactions (Leu–141, His–172, His–163, His–164, Met–165), and π-interactions (pi-Alkyl with Pro–168 and Cys–145, π-π T-Shaped with His–41) (Figure 4-f).
4.2.7. Diazinanes
The best compound in terms of docking score from this class shows predicted values for binding energy of –10.3 Kcal/mol and an IC50 of 0.63 uM. It was reported to have an antagonist activity against 5-HT receptor [31]. Compound 57 presented a high density of interactions within the Mpro protease active site. In fact, it was able to make HB contacts to four amino acids (Gly–143, His–163, Leu–141, Ser–144), VDW interactions four amino acids (Met–49, Phe–140, His–164, Met–165), and π-interactions to three amino acids (Glu–166, His–41, Cys–145) (Figure 4-g). In terms of possibilities of chemistry in order to optimize its potency, substitutions are possible in the piperazine group, as well as the naphthalene group and the indazole core.
4.2.8. Azoles
The azoles chemical class showed compound SR–3029 with the highest docking score of –11.2 Kcal/mol and a predicted IC50 of 0.52 µM. SR–3029 was reported to be highly selective casein kinase 1 Delta /1 Epsilon inhibitor with potent antiproliferative properties [32]. The docked pose of SR–3029 in the Mpro protease active site showed some interactions of types fluorin-HB (Cys–44, Glu–166, Phe–140), multiples VDW interactions (Met–49, His–41, Leu–27, Thr–25, Thr–45, His–172), and π-interactions (Cys–145, Gly–143, Leu–141, Ser–144) (Figure 4-h). Possible structural diversifications could be done on fluoro-phenyl group or the non-substitued positions on the benzodiazole core.
4.2.9. Organo-oxygens
The best compound of this chemical class is AMG–579 which showed a docking score of –10,5 Kcal/mol and a predicted IC50 of 0,6 µM. AMG–579 is a potent, selective, and efficacious inhibitor of phosphodiesterase 10A [33]. AMG–579 pose in the Mpro active site showed different interactions with the protein amino acids: HB (Thr–26, Ser–144), VDW (Leu–27, His–163, Leu–141, Asn–142), π-interactions (pi-Alkyl to Pro–168 and Cys–145), and alkyl interaction with Cys–145 (Figure 4-i). This molecule can be further optimized for its potency by chemical modifications on the amid moiety, the phenyl group from the benzodiazole and the central phenyl group.
4.2.10. Exceptional chemical class
Few compounds showed good docking scores but did not obey the different filtering rules such as Lipinski’s rules. We choose to select the best of these compounds to study its binding mode to Mpro protease. One of these compounds is silydianin which showed a docking score of –11,1 Kcal/mol and a predicted IC50 of 0,53 µM. Silydianin, an isomer of Silymarin extracted from the plant Silybium marinum, can control the main symptoms of asthma through modulating immune system responses [34]. Silydianin pose in the Mpro active site showed different interactions with the protein amino acids: HB (Thr–190, Gln–192, His–164, Ser–144, Leu–141, Phe–140, Glu–166), VDW (Met–49, His–163, Cys–145, His–41, Arg–188), π-interactions (π -Sigma to Gln–189, π-Alkyl to Pro–168), and alkyl interaction with Cys–168 (Figure 4-j).
Overall, and for all the analyzed hits from the different chemical classes, it appeared that some amino acids within the Mpro active site were the key residues entering in close interactions with the different ligands. In fact, four amino acids were involved in HB contacts with ligands very frequently. These amino acids concerned Ser–144, Cys–145, Glu–166, Gly–143. They were involved in HB interactions with at least 4 ligands. Four amino acids established VDW interaction with at least 4 analyzed ligands (His–163, Leu–141, Leu–27, Met–49). These VDW interactions are the predominant type of interactions that the majority of ligands have established with the protein. Three amino acids showed π interactions with at least 3 ligands (Cys–145, His–41, Met–165). These amino acids could be targeted during the design a new of optimized Mpro inhibitors.
As we have considered in our binding mode analysis only one representative from each chemical, other compounds from the same class could investigated also for experimental inhibition screening against the Mpro protease. This will provide interesting data to generate structure-activity relationships maps to be used for optimizing the activity of the molecules of interest.