CHA-12 as a multi-target inhibitor
Leukotrienes are broncho-constrictors whose pharmacological effects mimic the pathological changes in asthma. The biological activity of leukotrienes is mediated by the activation of cysteine leukotrienes (CysLT1 and CysLT2), leading to bronchoconstriction, mucus secretion, and asthma-like conditions. Accordingly, CysLT1 (LTD4) receptor antagonists are considered as important therapeutic agents in the treatment of lung and respiratory diseases such as asthma.
Results of the current study revealed that the hit compound CHA-12 has a magic affinity towards all tested targets. Interestingly, CHA-12 was first introduced as a CysLT1 antagonist in 1997 in a study by Mariel E. Zwaagstra et al 99. Among more than sixty compounds synthesized in that study, CHA-12 showed the best CysLT1 antagonistic activity in both in vitro and in vivo assays. In fact, patients infected with COVID-19 suffer from acute inflammation in the lung and respiratory system mostly due to the cytokine storm and having this important pharmacological activity of CHA-12 greatly increases the chance of CHA-12 to be a lead compound for treating COVID-19 complications in the respiratory system.
Impressive results of the virtual screening of the 757 chalcone-based compound library over 14 host and virus-based molecular targets and also the MM/PB(GB)SA analysis indicated that CHA-12 is a unique compound and has the highest affinity towards the 3CLpro and PLpro active sites. As shown in Fig. 9, CHA-12 fits well into the 3CLpro active site. Presence of the phenyltetrazole moiety in the structure of this compound structurally makes a partial similarity to losartan, an angiotensin II receptor blocker (ARB) reported to have remarkable effect for the treatment of COVID-19 (Fig. 10) 100–104. In a recent HTS study performed by Zhengnan Shen et al, candesartan as an ARB was identified as one of a few compounds that showed inhibitory effects against SARS-CoV-2 PLpro, out of about 15,000 screened compounds 105. The phenyltetrazole moiety of CHA-12 makes crucial interactions in the active site of the viral targets (especially PLpro) which are discussed later in the following.
The most important interactions of CHA-12 in the active site of 3CLpro are shown in Fig. 9. Totally, hydrogen bonds were found to be the main part of the binding affinity. Essentially, several hydrogen bondings were observed between the bound conformation of CHA-12 and the 3CLpro active site residues. In fact, two hydrogen bonds are formed by the interaction of His41 with the quinoline nitrogen and oxygen atom of the oxymethylene chain; one hydrogen bond exists between Glu166 and the NH-tetrazole, another hydrogen bonding between Thr190 and phenolic hydroxyl group and finally, one hydrogen bonding also exists between Gln192 and the carbonyl oxygen atom. In addition, Pi-sulfur interactions of Met49 to the quinolone and phenyl rings and a Pi-sigma interaction of Thr25 with the pyridine ring of quinoline moiety and also a Pi-donor H-bond interaction of Gln189 with the phenyl ring of the chalcone backbone contribute to the stronger binding of CHA-12 in the 3CLpro active site.
Evaluation of the CHA-12 interactions at the PLpro active site shows that the tetrazole moiety forms two key hydrogen bonds with Lys157 and Gln269 residues, as well as a Pi-anion interaction with the Glu167. A hydrogen bonding also exists through phenolic hydroxyl group with Tyr264 and Tyr268 has three interactions of type Pi-Pi stacked, Pi-Pi T-shaped and amide-Pi stacked with the phenyl and quinoline moieties of CHA-12. There is also a Pi-alkyl interaction between Pro248 and the phenyl ring of CHA-12 (Fig. 9).
Therefore, reported relaxant effects of CHA-12 on pulmonary smooth muscles established by in vitro and in vivo assays 99, along with its prominent predicted inhibitory effects on the all SARS-CoV-2 targets supports the discovery of a valuable compound for the treatment of COVID-19.
Chalcones bearing sulfonamide moiety
CHA-378 and CHA-297 having a sulfonamide moiety in their structure are also interesting compounds showed relatively acceptable results in the active site of the 3CLpro and PLpro targets. The CHA-378 generates strong interactions in the active sites of most HBATs including p38 MAPK (rank 10, docking score: -9.0 kcal/mol), Cathepsin L (rank 10, docking score: -8.0 kcal/mol), CDK2/CyclinA (rank 5, docking score: -10.1 kcal/mol), CDK9/cyclinT1 (rank 4, docking score: -10.4 kcal/mol), RBD2 (rank 1, docking score: -9.4 kcal/mol), RBD4 (rank 4, docking score: -9.8 kcal/mol), and both SARS-CoV-2 targets 3CLpro (rank 4, docking score: -8.7 kcal/mol) and PLpro (rank 10, docking score: -9.1 kcal/mol) (Table 2).
Structurally, CHA-378 consists of two structural moieties: a chalcone scaffold and a moiety containing sulfonamide functional group. This compound seems to be an interesting hybrid of hirsutenone (a well-known SARS 3CLpro and PLpro inhibitor 106) and compound A (Fig. 11). The latter was first synthesized in 2005 by Woo Duck Seo et al. as an α-glucosidase inhibitor. In their study, among all synthesized compounds, this ligand showed the best α-glucosidase inhibitory activity with IC50 = 0.4 µM 107. Interestingly, in a recent study by Spencer J. Williams and Ethan D. Goddard-Borger in which they highlighted α-glucosidase inhibitors as host cell N-glycosylation pathway blockers with high potential for the treatment of COVID-19 108.
In a recently published study, a large-scale screening of electrophile and non-covalent fragments was performed by Alice Douangamath et al., against the SARS-CoV-2 3CLpro through combined mass spectrometry (MS) and X-ray method 11. Evaluations of this study showed that the active site of the SARS-CoV-2 3CLpro has four subsites namely S1, S1’, S2 and S3. The S3 subsite has been reported to be explored and occupied by the sulfonamide group of the inhibitors and interacts with hydrogen bonding to Gln189. This space consists of His164, Met165, and Asp187, which is exposed to the solvent. So, the fragments located in this space can be oriented to the solvent. The crystal structure of 3CLpro protein bonded with compound A has recently been released in Protein Data Bank with PDB Code of 5RF8. This compound is able to create specific interactions in the S3 subsite. In a combined X-ray crystallography and fragment screening method, Charlie Nichols et al. reported that this fragment could interact well in the active site of the P38 MAPK target (PDB code: 5R9P) 109. The presence of a structural moiety similar to this fragment (compound A) in the structure of CHA-378 (Fig. 11) can be one of the important reasons for creating remarkable interactions in the active site of the p38 MAPK target (rank 10, -9.0 kcal/mol) in our screening results. Moreover, p38 MAPK was recently highlighted as one of the most promising targets for the treatment of COVID-19 18.
The sulfonamide moiety has frequently been found in the structure of both the reported 3CLpro and PLpro inhibitors. In 2008, Arun K. Ghosh and co-workers developed small molecules with potent inhibitory activity against SARS-CoV 3CLpro, some of which contain the sulfonamide functional group (Figue 11, compound C) 110. In a recently published study, Armin Welker and co-workers also introduced some chalcone-amide-based small molecules with inhibitory activities against SARS-CoV/SARS-CoV-2 PLpro, which contain a sulfonamide moiety (Figue 11, compounds D, and E) 111. The mentioned experimental results together with our computational findings indicate that compounds CHA-297 and CHA-378 could be specific compounds with the remarkable potential to inhibit SARS-CoV-2.
The types and interacting residues for the CHA-378 and its analogue CHA-297 in the active site of PLpro and 3CLpro are displayed in Fig. 12 and Tables 7 and 8. The key interactions created by CHA-378 at the 3CLpro active site are: a conventional hydrogen bond (Leu141 with 3-OH of catechol ring), a Pi-donor H-bond (Asn142 with catechol ring), four Pi-alkyl interactions (Met49, Met165, Cys145, and His41), Pi-sigma interaction (Gln189), and two Pi-cation and Pi-Pi stacked interactions of His41 with phenyl ring of sulfonamide moiety. There is also an unfavorable acceptor-acceptor interaction of Ser144 and Leu141 with the 4-hydroxy group of the catechol moiety. This unfavorable interaction is distracting in the active site of the 3CLpro and reduces its binding energy in the gas phase (Table 3, ΔGgas). Finding that presence of the two hydroxy groups in the catechol moiety is not accepted, hence the existence of only one hydroxyl group by selecting the most similar CHA-297 in our compound library should be a rational decision.
As shown in Table 3, the MM/PB(GB)SA results indicated that the affinity of CHA-297 is higher than CHA-378 by 6.5 and 3.8 kcal/mol calculated by the GB and PB respectively towards the 3CLpro active site. Investigating the mode of binding of CHA-297 to the 3CLpro active site showed that the key hydroxyl group discussed earlier has formed a hydrogen bond with Asn142 without any unfavorable interactions (Fig. 12). Interestingly, it was found that the sulfonamide moiety occupied the S3 subsite and hydrogen bonded to Gln189 which has been proved by a recent study to be a specific site for the binding of the sulfonamide containing 3CLpro inhibitors 11.
CHA-378 and CHA-297 were also investigated in the active site of the PLpro since the primary results of the docking screening indicated them as PLpro inhibitors as well (Table 2). In summary, NH of the sulfonamide moieties in both CHA-378 and CHA-297 creates hydrogen bond with Glu167 in the active site of the PLpro. On the other hand, oxygen atoms of the sulfonamide moiety of CHA-297 formed hydrogen bond interactions with Gln269. Similarly, Gln269 and Leu162 residues formed the same interactions Pi-sigma (Gln269) and amide-Pi stacked (Leu162) with the A-ring of the chalcone backbone in both CHA-297 and CHA-378. Tyr264 is another key residue that creates the same interactions in the PLpro active site for both CHA-297 and CHA-378 including a conventional hydrogen bond with the carbonyl group of the chalcone moiety, and a Pi-Pi T-shaped interaction with the catechol and phenolic rings of CHA-378 and CHA-297 respectively. However, the results of the MM/PB(GB)SA calculations indicated that these two compounds are weak PLpro inhibitors and can not be considered as PLpro hit compounds for further study.
Active ligands selective towards the 3CLpro
The biphenyl urea scaffold is a recently identified fragment introduced by Alice Douangamath and co-workers that can generate specific interactions in the active site of the SARS-CoV-2 3CLpro enzyme 11. As the researchers well characterized, the S1 subsite of the 3CLpro-active site revealed the two key residues His163, which is interacting with the pyridine nitrogen or with nitrogen-containing heterocycles, and Glu166 which is a key amino acid in the formation of hydrogen bonds to the amide or ureide substructures of the 3CLpro-inhibitors. In Fig. 13, the chemical structure of some of the biaryl urea small molecules (compounds B and E) and their isosteric amide-analogues (compounds F and G) with their corresponding PDB codes which were recently released as selective 3CLpro inhibitors are displayed. Also, the structural similarity of the reported biaryl urea compounds with some of our ureide-chalcone hybrid structures existed in our library were found to be as hit compounds selective against 3CLpro (see Table 2) are displayed in Fig. 13.
The key interactions of the co-crystal compound B in the active site of the 3CLpro (PDB code 5R83) are shown in Fig. 14A. Correspondingly, NH and oxygen atoms of the ureide moiety in compound B form two important conventional hydrogen bonds with the residues Arg188 and Glu166. Investigating the binding mode of the ureide-chalcone hybrid structures CHA-233, CHA-236, CHA-383, CHA-384, CHA-392, and CHA-397 and interacting residues within the 3CLpro active site are shown in Fig. 14B. As discussed earlier, these compounds were previously synthesized and reported as antimalarial agents 112. The spatial orientation of these ureide-chalcones in the active site of the 3CLpro is precisely the same and the ureide substructure formed two conventional hydrogen bonds by the interaction of Arg188 and the two NH atoms (Fig. 14B). These hydrogen bonding interactions cause the urea to be oriented exactly at a specific location adjacent to the Arg188 (Fig. 15). These ureide-chalcone hybrid structures have a high selectivity to 3CLpro compared to PLpro, and significantly produce stronger interactions than compound B (as standard) at the SARS-CoV-2 3CLpro active site.
Among the evaluated 3CLpro-selective ureide-chalcones, CHA-384 generates the highest affinities in the active site of the virus and host-based targets compared to the other congeners. The rankings predicted for CHA-384 against the different virus and host-based targets (see Table 2) is as follows: CDK2/CyclinA (rank 7, docking score: -9.2 kcal/mol), ERK2 (rank 2, docking score: -9.9 kcal/mol), DHODH (rank 2, docking score: -12.7 kcal/mol), BRD4 (rank 7, docking score: -9.7 kcal/mol), sigma-1 receptor (rank 3, docking score: -11.6 kcal/mol), and 3CLpro (rank 2, docking score: -8.9 kcal/mol). In addition to the conventional hydrogen bonds mentioned above, the most important interactions of CHA-384 in the active site of the 3CLpro are as follows: Pi-Alkyl interactions of Met49 and Met165 residues with A-phenyl ring of the chalcone backbone, and Pi-Alkyl interaction of Pro168 with phenyl ring of the ureide moiety. Moreover, the chlorine atom of the ureide moiety generates two alkyl interactions with Pro168 and Ala191 residues. The Pi-sigma interaction of the B-phenyl ring with Thr25, and a Pi-Pi Stacked interaction of the A-phenyl ring with His41 are other interactions formed by CHA-384 in the active site of the 3CLpro. These interactions are shown comparatively for CHA-384 and its congeners in Fig. 14B.
Selective ligands towards the SARS-CoV-2 PLpro
A long time after the emergence of the SARS-CoV pandemic in 2002, structure-based design approaches for the development of PLpro inhibitors were unsuccessful due to the insufficiency of the target structural information. For the first time in 2008, Ghosh and co-workers were able to identify two selective SARS-CoV PLpro inhibitors by performing an extensive high throughput screening on 50,080 compounds 113. Interestingly, GRL0617 (Fig. 16A) with a chalcone-amide backbone is a well-known compound generated from the study of Ghosh et al, and currently it is used for the design and development of the SARS-CoV/SARS-CoV-2 PLpro inhibitors as a standard and lead compound. Herein, to validate the assessments and better understanding the interactions, GRL0617 was used as a standard.
Interestingly, CHA-7, CHA-11, CHA-37, CHA-44, and CHA-177 showed superior docking scores among our chlacone-based compound library against the SARS-CoV-2 PLpro and host-based targets, and also CHA-37 was found to have the highest affinity towards the PLpro among them (Table 2). The ranking and docking energy score of CHA-37 at the active sites of the host and viral-based targets are as follows: p38 MAPK (rank 2, -9.4 kcal/mol), Cathepsin L (rank 4, -8.3 kcal/mol), CDK1 (rank 3, -9.3 kcal/mol), CDK2/CyclinA (rank 9, -9.6 kcal/mol), CDK9/cyclinT1 (rank 7, -9.9 kcal/mol), ERK2 (rank 4, -9.5 kcal/mol), DHODH (rank 9, -12.0 kcal/mol), CK2 alpha' (rank 1, -11.4 kcal/mol), BRD2 (rank 5, -8.8 kcal/mol), BRD4 (rank 1, -10.4 kcal/mol), and sigma-1 receptor (rank 2, -11.7 kcal/mol), 3CLpro (rank 12, -8.3 kcal/mol) and PLpro (rank 3, -9.5 kcal/mol). Although, the calculated binding affinity of CHA-37 in the active site of the PLpro was calculated to be lower than GRL0617 by the PB and GB methods, it still shows more affinity than the co-crystal ligand of the 7JN2 PDB code (see Table 4). Interestingly, its calculated binding free energy of -19.20 kcal/mol by the PB method indicated its higher affinity towards the PLpro compared to the standard inhibitor hisutenone (-16.52 kcal/mol).
As displayed in Fig. 16B and Table 8, CHA-37 created important interactions in the active site of the PLpro. The benzotriazole moiety of this compound forms key interactions, including Pi-alkyl with Pro247 and Pro248 residues, one amide-Pi stacked interaction resulting from the interaction of triazole fraction with Asn267, and one Pi-Pi T-Shaped interaction with Tyr268.
Similarly, the naphthyl ring of the GRL0617 creates three Pi-alkyl interactions with Pro247 and Pro248 residues, and also two Pi-Pi T-shaped interactions with Tyr268. In addition, the formation of three conventional hydrogen bonds by the carbonyl group with Tyr268, amide-NH with Asp164, and the amino group of GRL0617 with Gly163, help to strengthen the binding of this compound in the active site of the SARS-CoV-2 PLpro. 2D and 3D interactions of CHA-37 and GRL0617 in the active site of the SARS-CoV-2 PLpro enzyme are shown in Fig. 16.
Structural evaluation of the selective PLpro ligands shows outstanding similarities. In general, these selective PLpro compounds have unsubstituted and flat nitrogen-containing aromatic groups on ring B of the chalcone backbone (Fig. 17A and 17B). Correspondingly, quinoxaline ring in CHA-11, benzo-[1, 2, 3]triazole in CHA-37, and benzimidazole in CHA-44 in position 2 or 3 on the B-ring are a main motif for binding. As indicated in the structure of GRL0617, the same position is occupied by a fused phenyl ring with the B-ring of the chalcone backbone. Interestingly, in a recent structure-based drug design attempt, extensive SAR evaluations have led to a chalcone-amide isoster bearing a 2-thienyl at position 3 of ring B for producing the strongest derivatives against SARS-CoV-2 PLpro (Fig. 17A, structure H) 105. The meta- and para-fluorophenyl substitutions in positions 2 and 3 of the B-ring also existed in the structure of CHA-7 and CHA-177 which were found to have high docking scores in our compound library. These substitutions are also found in the structure of high affinity SARS-CoV PLpro inhibitors ever reported which confirms and validates our results (Fig. 17C, compounds L and M) 114. Therefore, a fused and nitrogen containing aromatic ring or a fluorophenyl ring substituted at position 3 of the chalcone B-ring (or chalcone-amide) is useful for increasing the inhibitory effect of compounds against the PLpro enzyme.
Tables 7 and 8 comparatively summarize all the predicted interactions related to the most promising CHA-12, CHA-37, CHA-378, and CHA-384 along with the appropriate standards baicalein and GRL0617 in the active site of the SARS-CoV-2 3CLpro and PLpro.
In addition to having strong affinity of CHA-37 in the PLpro active site (rank 3, docking score = -9.5 kcal/mol), it is interesting that this ligand also creates remarkable interactions in the 3CLpro active site with the rank of 12 among the 757 chalcone-based compound library. Accordingly, we examined also the interactions of this compound in the active site of both the viral targets PLpro and 3CLpro.
Benzotriazole for selective 3CLpro inhibition
One of the important reasons for creating strong interactions of CHA-37 in the 3CLpro active site could also be attributed to the presence of the benzotriazole group. This moiety has been observed in the structure of the previously reported potent 3CLpro inhibitors, as in representative compounds I, J, and K (Fig. 17) 115,116.
Comparison of the interactions of CHA-37 with compound J, a highly potent SARS-CoV 3CLpro inhibitor (IC50 = 51 nM), in the SARS-CoV-2 3CLpro active site shows that benzotriazole moieties of both compounds are occupying exactly the same space, and mostly interact with the same residues His41, Cys44, and Met49 (Fig. 19). The strong affinity of CHA-37 towards the 3CLpro binding space was further confirmed by the MM/PB(GB)SA calculations (Fig. 5 and Table 3). The results showed that CHA-37 has higher affinity towards the 3CLpro active site than the standard inhibitors baicalein (-30.93 kcal/mol) and hirsutenone (-24.12 kcal/mol) which was evidenced by its binding free energy of -31.09 kcal/mol in the GB method.