Pharmacophore of the co-crystal ligand N3
As illustrated in Figure 1, Mpro monomer has three domains: domain Ⅰ (6 antiparallel β-sheet), domain Ⅱ (6 antiparallel β-sheet) and domain Ⅲ (α-helixes, which are closely related to proteolytic activity), and a long loop connects domains Ⅱ and Ⅲ. A highly conserved substrate-binding pockets (with a Cys145-His41 catalytic dyad) located in a cleft between domains Ⅰ and Ⅱ, suggesting the antiviral inhibitors targeting this site should have broad-spectrum anti-coronavirus activity .
As shown in the diagram of Figure 1, a covalent bond between the Sγ atom of Cys145 and the Cβ of the vinyl group is formed, which means the Michael Addition that is critical in the catalytic mechanism has occurred . The lactam functional group at P1 site inserts into the subsite S1 and forms a hydrogen bond with His163, while the functional group Leu at P2 site inserts deeply into the hydrophobic subsite S2 . The functional group Val at P3 site is solvent-exposed tolerating a variety of functional group substitutions. The functional group Ala at P4 side is in a hydrophobic pocket. P5 site makes van der Waals interactions with Pro168, Thr190, and Ala191, while the hydrophobic aromatic ring of N3 forms van der Waals contacts with Thr24 and Thr25. Besides, N3 forms multiple hydrogen bond interactions with the active site residues, helping to lock the inhibitor inside the binding pocket, which determines the inhibition of the enzyme as well as the coronavirus replication .
Mpro, which is highly conserved among all coronavirus, is a good target for the development of a single antiviral agent or in combination with other potential therapies to provide an effective first line of defense against all coronavirus-associated diseases . The co-crystal structure of SARS-CoV-2 Mpro complexed with N3 is a good model for identifying inhibitor lead through in silico screening.
In silico screening by AutoDock Vina
For validation of docking simulation, N3 was re-docked into Mpro. The described docking workflow allowed top-ranked and reproduced binding conformation which was close to those of the 6LU7 co-crystal structure (checked by PyMOL, RMSD of 1.126 Å). According to AutoDock Vina, binding affinity ≤ -0.0 kcal/mol means the receptor and ligand could automatically bind together. In this study, molecule with binding affinity ≤ -8.5 kcal/mol was treated to be potential based on recent reports on in silico screening of SARS-CoV-2 Mpro inhibitors .
All the 135 ‘old’ drug structures, biological activities, targets, and top-ranked binding affinities were summarized (Supporting information Table. S1). In which, 6 molecules including anti-HIV drug (raltegravir), antibacterial drugs (cefonicid, cefoperazone, minocycline), and antidiabetic drugs (canaglifozin, glyburide) showed high affinities (≤ -8.5 kcal/mol) as well as interesting binding conformations (bound to the Mpro active site and formed interesting interactions with key residues). In particular, the antibiotic minocycline, an inhibitor of bacterial ribosomal rRNA, showed the highest binding affinity (-9.6 kcal/mol) compared with N3 (-7.7 kcal/mol). The results indicated that these small molecular drugs might be Mpro inhibitors of SARS-CoV-2.
Minocycline is an FDA-approved, second-generation tetracycline class antibiotic with an established safety profile that has been used in clinic for more than 30 years. It acts selectively binding to the 16S rRNA, inhibiting the binding of RNA to ribosomes, and interferes with protein synthesis . The main treatment conditions of minocycline were both gram-positive/negative bacterial infections and the more recent multidrug resistant Acinetobacter baumannii . The immune imbalance and bacterial infection often appear in the later stages of COVID-19 progression, the efficacy of antiviral drugs might remain unsatisfactory or insufficient . The antibiotics and glucocorticoid were sometimes administered according to the clinical characteristics and physicians’ discretion .
Re-docking of N3 and minocycline by Discovery Studio
To gain further validation of the docking simulation, re-docking of known ligand with the target and comparison of docking results generated by different software are academically consensus. From the CDOCKER results generated by Discovery Studio, N3 (Figure 2A) formed conventional hydrogen bonds with residues Phe140, His163, His164, Glu166, Gln189, and Thr190. The isoxazole group formed Pi-Alkyl interaction with Ala191 and Pro168, and hydrophobic aromatic ring formed van der Waals' forces with residues Thr24, Thr25, Leu27 and Cys145. In addition, N3 molecule forms covalent bonds with multiple residues of Mpro. The docking results were closely consistent with the co-crystal structure (checked by PyMOL, RMSD of 1.650 Å), indicating that the CDOCKER docking model was validated and suitable for in silico screening of Mpro inhibitors.
For minocycline (Figure 2B), it contains multiple hydrophilic groups which formed conventional hydrogen bonding networks with key residues Phe140, Gly143, Cys145, His164, and Glu166 in the active site. The hydrophobic aromatic rings formed van der Waals' forces with multiple amino acid residues of Mpro. It is commonly accepted that covalent bond formed between the Cys145-His41 catalytic dyad and the designed compound would increase the Mpro inhibition potency, resembling the intermediate during substrate cleavage . Beside the hydrogen bond between Cys145 and 2-carboxamide, a critical Pi-Cation formed between His41 and 4-dimethylamino group, which strongly supported minocycline as a Michael Addition acceptor binding with the exact catalytic site to inhibit Mpro. These results indicated that the multiple especially critical interactions stabilized minocycline-Mpro in a low energy state, which was required for Mpro selection and antiviral activity.
Recent evidence suggested that the precise site of interaction between minocycline and cellular RNA molecules could be double-stranded RNAs (dsRNAs), which have been observed as intermediates of the viral replication of positive-stranded viruses, such as SARS, the aberrant induction of inflammatory cytokines/chemokines in case of SARS infection was mostly activated by dsRNA intermediates [19, 20]. In addition, the robust viral replication and delayed IFN-γ signaling accompanying the initial steps of SARS seem to be consequence of the coronavirus ability to initially evade the host dsRNA-sensors [21, 22]. Therefore, early administration of dsRNA-binding minocycline might reduce the risk of SARS-CoV-2.
Structure-affinity relationship of minocycline
By referring to the literatures from PubMed, Elsevier, Springer, and Google Scholar, a 44-compound (in which 21 compounds were clinical drugs) small-scale database of minocycline analogues was established. After docking simulation, the chemical structures and top-ranked binding affinities of the analogues were summarized (Supporting information Table. S2).
Indeed, minocycline showed a promising highest binding affinity among all the 44 analogues. Structures containing the main octahydrotetracene-2-carboxamide skeleton were analyzed and the structure-affinity relationship was summarized (Figure 3). Carbonyl functional groups should be kept and the middle hydroxyl group might be better if changed to be carbonyl. Furthermore, the terminal 2-carboxamide could be modified with moderate (not too long) moiety. On the 4,7-bis(dimethylamino) side, 4-dimethylamino group is critical for the high affinity, and the S-stereochemistry of C4 is better than the R-stereochemistry, which was also verified from the previous binding mode investigation that it could form the key covalent bond with His41.
Docking simulation and the structure-affinity relationship study found critical covalent bond formed between the active Cys145-His41 catalytic dyad and minocycline, which helped us to better understand why the functional groups as well as the tetracycline skeleton could be suitable for the Mpro active-site binding and interaction.
In vitro activity assays of the SARS-CoV-2 Mpro inhibition
As is shown in Figure 4, minocycline showed a dose-dependent Mpro inhibitory activity with IC50 of 5mM. Minocycline is an FDA-approved, second-generation tetracycline class antibiotic with an established safety profile, and is used in pharmacological conditions of both bacterial/mycoplasma infections. In spite of this, minocycline appears to have broad-spectrum antiviral activities: reducing West Nile Virus titers in brain-derived cell types, reducing Japanese encephalitis-induced damage in neuronal cells inhibiting H7N9 replication in human lung epithelial cells, and attenuating pathogenic immune responses during infection with human and simian immunodeficiency virus (HIV/SIV) [23, 24-27]. Moreover, based on molecular docking and dynamic studies, minocycline was proposed as potential antiviral therapy against Congo Crimean hemorrhagic fever virus to inhibit the binding of virus to host nucleoprotein . In a randomized controlled trial of dengue hemorrhagic fever patients, compared with standard-of-care, combination therapy with doxycycline (analogue of minocycline) significantly decreased the TNF and IL-6 levels, and mortality .
Tetracycline inhibiting pro-inflammatory cytokines and matrix metalloproteinases plays a key role in coronavirus acute infection and is involved in chemokine activation and in tissue destruction [30, 31]. Of note, this immunomodulatory effect seems to be dsRNA-mediated . Besides, minocycline attenuates T cell and microglia activity to impair cytokine production in T cell‐microglia interaction . Severe COVID-19 patients were more likely to develop neurological symptoms . ACE2 (the functional receptor for SARS-CoV-2) is present in multiple human organs including nervous system and skeletal muscle . Due to the small size and lipophilic nature, minocycline might cross into tissue compartments with potentially therapeutic concentrations.
The in vitro validation result suggesting that the inhibitory activity against SARS-CoV-2 Mpro of minocycline might be beneficial in addition to other well-known mechanisms. Further, minocycline could be used as interesting lead to design analogs that can more potently and selectively inhibit SARS-CoV-2 Mpro to improve its antiviral activity and avoid the unwanted adverse effects associated with other mechanisms.