In Silico Screening, In Vitro M pro Inhibitory, and Adjunctive Therapy Value of Minocycline for the Treatment of COVID-19

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are being investigated for the treatment of COVID-19, and the drugs prevent viral replication through various mechanisms, including blocking SARS-CoV-2 3-chymotrypsin-like protease (3CLpro, also named as M pro ) and RNA-dependent RNA polymerase (RdRp), causing viral mutations.Besides, the previous study found that the combination of brequinar (BRQ), a dihydroorotate dehydrogenase (DHODH) inhibitor, and dipyridamole (DPY), a remedial pathway inhibitor, expresses a strong synergistic antiviral activity to treat SARS-CoV-2 [3].As SARS-CoV-2 mutant strains continue to emerge, new therapeutic entities are still needed to be discovered.
Nonstructural proteins (NSPs) are involved in SARS-CoV-2 RNA transcription and translation, protein synthesis, protein processing and modifcation, virus replication, and host infection [4].In NSPs, M pro is automatically cleaved from polyproteins to produce mature enzymes and further cleaves downstream NSPs at 11 sites to release NSP 4-16 [5].Te structure and catalytic mechanism of SARS-CoV M pro allows it as a promising target for anti-coronavirus drug development.To develop an anti-SARS-CoV-2 drug from scratch is theoretically time consuming [6].We can test existing broad-spectrum antivirals for their metabolism, used dosages, efcacy, and side efects.However, the side efects of broad-spectrum antivirals should not be underestimated [7].Because "old drugs" have been prepared, the medication has sufcient experience, and the safety and pharmacokinetic parameters are well known, screening for SARS-CoV-2 therapeutic candidates from existing clinical "old drugs" is always a good strategy [8,9].From this strategy, Masitinib was recently discovered as a broad coronavirus 3CL inhibitor that blocked SARS-CoV-2 virus replication [10].
In this study, we established a small-scale "old drug" database (clinical drugs being used in Ordos Central Hospital) according to Lipinski's rule of 5 and conducted in silico screening of M pro inhibitors by molecular docking.Binding afnity and interaction as well as structure-afnity relationship were analyzed to better understand the potentiality.Te in vitro M pro inhibition test was conducted based on established methods.Finally, literature support for the potentiality of anti-SARS-CoV-2 and treatment of COVID-19 was reviewed and analyzed.Tis study will provide contribution to the transient ongoing infectious diseases.

Pharmacophore Study of the Co-Crystal Ligand N3.
Te crystal structure of SARS-CoV-2 M pro in complex with designed ligand N3 (2.1 Å resolution, PDB code: 6LU7) was determined by Professors Zihe Rao and Haitao Yang's research team from Shanghai Tech University [11].Te protein coordinates of the M pro used in this study were donated by Zihe Rao et al. in Jan 28th, 2020.Based on the structure, key helixes/loops, amino acid residues, and hydrophobic interactions in the binding site were investigated.Te pharmacophore of N3 was summarized and was used as a control in the following in silico study.

Drug Database Establishment.
According to Lipinski's RULE of 5, we established a small-scale database including 135 drugs clinically being used in Ordos Central Hospital [12].Requirement-reached drug 2D structures were drawn by ChemDraw Professional 17.0 software (CambridgeSoft Corporation, Cambridge, MA, USA).Te 2D structures of candidates were converted into 3D structural data by Chem3D ultra 17.0 software (CambridgeSoft Corporation, Cambridge, MA, USA), and all structures of the ligands were energy-minimized.

Molecular Docking by AutoDock
Vina.We applied a workfow for molecular docking which was described in our previous work [13][14][15].Te chain B (co-crystal ligand N3 in 6LU7) and chain C (water molecules) were deleted, and chain A was prepared for docking within the molecular modeling software package Chimera 1.10.2(National Institutes of Health, Bethesda, MD, USA) [16].Adding polar hydrogens and Kollman charges, Gasteiger computing and grid box parameters defning were done using MGL tools 1.5.6 (Te Scripps Research Institute, La Jolla, CA, USA) [17,18].
All the ligands were set as fexible, and the receptor was set as rigid.Docking calculations were performed using AutoDock Vina 1.1.2software (Te Scripps Research Institute, La Jolla, CA, USA) [19].A search grid box was set to cover the whole surface of M pro protein to collect all possible orientations and conformations of the ligand paired with the protein (including compounds outside the active site).For which, the center was set as center_x � −23.982, center_y � 12.114, center_z � 57.466, and the size was set as size_x � 58, size_y � 78, size_z � 66. Spacing angstrom was set as 1.000, and the exhaustiveness was set as 100.Te default settings and the AutoDock Vina scoring function were applied.
Totally, 9 binding modes were generated by AutoDock Vina for each compound, and the mode (even outside the active site) with the highest binding afnity was selected as the most predictable.Visual investigation and analysis of ligand-protein interactions were performed using PyMOL V.1.5(Schrodinger LLC, New York, NY, USA).

Re-Docking by Discovery
Studio.Te 3D protein structure of M pro was defned as the receptor and optimized by hydrogenation, dehydration, and removing redundant residues.Location of N3 in the cocrystal was defned as the active site with a radius as 13.890841 covering the binding region.Te X, Y, and Z centers were set as −10.797, 12.536, and 68.905, respectively.Molecular structures of ligands were prepared and converted to 3D structures.Te molecular docking was performed using CDOCKER tool.-CDOCKER_ENERGY and -CDOCK-ER_INTERACTION_ENERGY were used to score the interaction between the receptor and ligand.Discovery Studio (DS) 2022 software (BIOVIA, San Diego, CA, USA) was used for the docking, visualization, and analysis [20].

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Journal of Clinical Pharmacy and Terapeutics 2.5.Molecular Dynamics (MD) Simulation.MD approach is widely used to assess atoms' behavior, structural stability, and study the conformational changes on atomic level.Herein, after the molecular docking, MD simulation was performed on the compounds with best afnity by DS.Te complexes were minimized using the CHARMM force feld.During the solvation process, default waterbox size options were selected, and the waterbox size was adjusted to match the protein's size using a rectangular waterbox type with an edge distance of 7.0 Å.To neutralize the system and to achieve a NaCl concentration of 0.145 M, 20 Na + ions and 17 Cl − ions were added to the complex.A total of 3,000 steps of energy minimization were performed using the steepest descent method.Subsequently, the minimized system was equilibrated and run for 20 ps at a constant temperature of 300.00 K, followed by a 10 ns production run.Basic parameters for trajectory analysis such as root mean square deviation (RMSD), root mean square fuctuation (RMSF), and hydrogen bond (H-bond) interactions were analyzed for each protein-ligand complex.

Structure-Afnity Relationship Study.
Considering the generated information is relatively limited, investigation of the promising drug analogs will provide information for further study such as structure modifcation.After the in silico screening and the re-docking study, the analogs of the promising drug were collected by referring to literatures from PubMed, Elsevier, Springer, and Google Scholar.Ten, oneby-one docking of the analogs targeting on M pro was performed.Based on the analogs' binding afnities, the structureafnity relationship of the promising drug was summarized.

In Vitro Activity Assays of the SARS-CoV-2 M pro
Inhibitors.Te inhibition rate of "old drug" on M pro enzyme was measured using the 2019-nCoV M pro inhibitor screening kit (P0312S, Beyotime Biotechnology, Shanghai, China) according to the manufacturer's instructions.Tis kit was monitored at excitation of 340 nm and emission of 490 nm wavelengths on a microplate multimode reader by fuorescence resonance energy transfer.Te percentage inhibition was calculated.

Literature Review of Promising Drug.
After the in silico and in vitro study, we further questioned whether the promising "old drug" possessed documented biological activities associated with the pathological changes in COVID-19 condition.We searched PubMed, Elsevier, Springer, and Google Scholar for articles describing SARS-CoV-2 virus, COVID-19 condition, and the biological changes during the drug use.Te condition of COVID-19 and the drug efect on pathological changes were summarized.

Results and Discussion
3.1.Pharmacophore of the Co-Crystal Ligand N3.As illustrated in Figure 1, M pro monomer has three domains: domain I (6 antiparallel β-sheet), domain II (6 antiparallel β-sheet), and domain III (α-helixes, closely related to proteolytic activity), and a long loop connects domains II and III.A highly conserved substrate-binding pocket (with a Cys145-His41 catalytic dyad) is located in a cleft between domains I and II, suggesting the antiviral inhibitors targeting this site should have a broadspectrum anti-coronavirus activity [11].
As shown in Figure 1, a covalent bond between the Sc atom of Cys145 and the Cβ of the vinyl group is formed, supporting the critical Michael addition in the catalytic mechanism [5].Te lactam of P1 site inserts into the subsite S1 and forms a hydrogen bond with His163, while Leu of P2 site inserts deeply into a hydrophobic subsite.Te Val of P3 site is exposed to solvent, tolerating a variety of functional group substitutions.Te Ala of P4 is in a hydrophobic pocket.P5 site makes van der Waals interactions with Pro168, Tr190, and Ala191, while the aromatic ring forms van der Waals contacts with Tr24 and Tr25.N3 forms multiple hydrogen bonds with the active site residues, locking the inhibitor inside the binding pocket, which determines the inhibition of the enzyme as well as the coronavirus replication [11].
M pro , 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 efective frst line of defense against all coronavirus-associated diseases [16].Te cocrystal structure of SARS-CoV-2 M pro complexed with N3 is a good model for identifying inhibitor lead through in silico screening.

In Silico Screening by AutoDock Vina.
For docking validation, N3 was re-docked into M pro .Te described docking workfow 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).In this study, a molecule with binding afnity ≤−8.5 kcal/mol was treated to be potential based on recent reports on in silico screening of SARS-CoV-2 M pro inhibitors [21].
All the 135 "old" drug structures, biological activities, targets, and top-ranked binding afnities were summarized (Supporting Information Table.S1).In which, 6 molecules including anti-HIV drug (raltegravir), antibacterial drugs (cefonicid, cefoperazone, and minocycline), and antidiabetic drugs (canaglifozin and glyburide) showed high afnities (≤−8.5 kcal/mol) as well as interesting binding conformations (bound to the M pro active site and formed interesting interactions with key residues).In particular, the antibiotic minocycline, an inhibitor of bacterial ribosomal RNA, showed the highest binding afnity (−9.6 kcal/mol) compared to N3 (−7.7 kcal/mol).Tese small molecular drugs might be M pro inhibitors of SARS-CoV-2.
Minocycline is a second-generation tetracycline antibiotic with an established safety profle that has been used in clinic for more than 30 years.It selectively binds to the 16S rRNA, inhibiting the binding of RNA to ribosomes, and interferes with protein synthesis [22].Te main treatment conditions of minocycline were Gram-positive/negative bacterial infections and the more recent multidrug-resistant Acinetobacter baumannii [23].For minocycline (Figure 2(b)), it contains multiple hydrophilic groups which formed conventional hydrogen bonding networks with key residues Phe140, Gly143, Cys145, His164, and Glu166 in the active site.Te hydrophobic aromatic rings formed van der Waals' forces with multiple amino acid residues of M pro .It is commonly accepted that the covalent bond formed between the Cys145-His41 catalytic dyad and the designed compound would increase the M pro inhibition potency, resembling the intermediate during substrate cleavage [5].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 M pro .Tese results indicated that the multiple especially critical interactions stabilized minocycline-M pro in a low energy state, which was required for M pro selection and antiviral activities.

MD Simulation.
Te ligand-binding status in the physiological milieu was predicted by MD simulation.Te MD simulation of M pro -N3 and M pro -minocycline complexes was performed for 10 ns.RMSD is calculated considering the proteins' backbone with respect to the initial conformations [24].Te RMSD values of the M pro -N3 complex remained constant (∼3 Å) from 0-4 ns and reached another plateau state after 5 ns and maintained the deviation below 2.5 Å. Te RMSD value of M pro -minocycline complex was found to be stable without signifcant deviation which was maintained below 3.5 Å (Figure 3(a)).Tese results preliminarily showed that there was no major deviation or conformation adjustment as the interaction of these ligands with the protein is stable.Te RMSF was used for quantifying local changes/amino acid fuctuations along the protein chain [25].Fluctuations for each of the individual amino acid of the target protein in case of N3 and minocycline were observed from the RMSF values.Te average fuctuation of the amino acid residue for M pro -N3 complex was 5.26 Å, and that was 6.85 Å for the M pro -minocycline complex (Figure 3(b)).Less RMSF fuctuation indicates more interaction with the active site [26].Subsequently, we investigated the persistence and variability of H-bond interactions by plotting H-bond thermograms.As shown in Figure 4, the H-bond interactions changed dynamically at diferent times throughout the simulation, but the total number of the interactions remained relatively stable.Tis indicated that the H-bond interactions in the M pro -minocycline complex were highly persistent and stable.

Structure-Afnity Relationship of Minocycline.
By referring to the literatures from PubMed, Elsevier, Springer, and Google Scholar, a database of minocycline analogs (44-compound, in which 21 compounds were clinical drugs) was established.After molecular docking, the chemical structures and top-ranked binding afnities of the analogs were summarized (Supporting Information Table.S2).
Indeed, minocycline showed a promising highest binding afnity among all the 44 analogs.Structures containing the main octahydrotetracene-2-carboxamide skeleton were analyzed and the structure-afnity relationship was summarized (Figure 5).Carbonyl functional groups should be kept, and the middle hydroxyl group might be better if changed to carbonyl.Furthermore, the terminal 2-carboxamide could be modifed with moderate (not too long) moiety.On the 4,7-bis(dimethylamino) side, 4-dimethylamino group is critical for the high afnity, and the S-stereochemistry of C4 is better than the R-stereochemistry, which was also verifed from the previous binding mode investigation that it could form the key covalent bond with His41.Docking simulation and the structure-afnity relationship study revealed that critical covalent bond formed between minocycline and the Cys145-His41 catalytic dyad of M pro , which helped us to better understand why the functional groups as well as the tetracycline skeleton could be suitable for the M pro active site binding.

In Vitro Activity Assays of the SARS-CoV-2 M pro
Inhibition.As shown in Figure 6, minocycline showed M pro inhibitory activity in a dose-dependent manner with IC 50 of 5 mM.Te in vitro validation result suggests that the inhibitory activity against SARS-CoV-2 M pro of minocycline might be benefcial in addition to other well-known mechanisms.Furthermore, minocycline could be used as an interesting lead to design analogs that can more potently and selectively inhibit SARS-CoV-2 M pro to improve its antiviral activity and avoid the unwanted adverse efects associated with other mechanisms.
Minocycline has been used in pharmacological conditions of both bacterial and mycoplasma infections.Moreover, 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 immunodefciency virus (HIV/SIV) [27][28][29][30][31]. 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 [32].
In a randomized controlled trial of dengue hemorrhagic fever patients, compared with standard-of-care, combination therapy with doxycycline (analog of minocycline) signifcantly decreased the TNF and IL-6 levels and mortality [33].Tetracycline inhibiting proinfammatory cytokines and matrix metalloproteinases plays a key role in coronavirus acute infection and is involved in chemokine activation and in tissue destruction [34,35].Of note, this immunomodulatory efect seems to be dsRNA-mediated [36].
A variety of clinical data revealed that the infammatory factor storm (IFS) existed and played a critical role in severe or fast-progressive COVID-19 condition.In the later phase, the level of ESR, CRP, IL-6, and D-dimer increased and the counts of lymphocytes, total T cells, CD 4+ T cells, and CD 8+ T cells were dramatically reduced, while patients in declining period presenting decreased levels of TNF-α, IFNc, IL-6, and IL-10 and restored counts of T cell [21,63,64,85].In severe COVID-19 patients, the elevation of the CRP level and white blood cell count might be accompanying with bacterial infection, and antibiotics were usually prescribed [49].As shown in Table 1, the levels of  broad-spectrum biological markers especially TNF-α, IFNc, IL-6, and IL-10 associated with minocycline administration were opposed to those of COVID-19 condition (both severe and nonsevere), which was strongly supported by in vivo and in vitro data.Infammatory responses triggered by viral infection play a crucial role in pulmonary pathology severity, suppressing the IFS to reduce lung infammation is a valuable treatment method [86].High doses of glucocorticoid were widely applied during the outbreaks of SARS to suppress lung infammation and immune response; however, it appeared to be associated with side efects [87][88][89].Talidomide (immunomodulatory and anti-infammatory agent) in combination with antiviral drugs and low-dose glucocorticoid was reported the protective efect on lung injury and immunological stress caused by COVID-19 [90].Chloroquine was included in the 6th version of Diagnostic and Treatment Protocol for COVID-19 in China due to the in vitro anti-SARS-CoV-2 results and in vivo antiinfammatory activity.Hydroxychloroquine, which is chemically and biologically similar but safer than chloroquine, had been included in the local diagnostic and treatment guidelines for COVID-19 (Shanghai, China) [91].In a two-year randomized controlled trial on early seropositive rheumatoid arthritis patients, minocycline achieved better antiinfammatory outcomes than hydroxychloroquine [60].Te immune imbalance and bacterial infection often appear in the later stages of COVID-19; the efcacy of antiviral drugs might remain unsatisfactory [92].Te antibiotics and glucocorticoid were sometimes administered according to the clinical characteristics and physicians' discretion [59].
Moreover, minocycline was reported to attenuate T cell and microglia activity to impair cytokine production in T cell-microglia interaction [78].Angiotensin converting enzyme 2 (ACE2), the function receptor for SARS-CoV-2, is present in multiple human organs [93].Cytokine release leads to serious complications in the cardiovascular system, digestive system, and central nervous system (CNS) in COVID-19 patients [48].Because of its high lipophilicity and small size, minocycline can cross the blood-brain barrier and accumulate in cerebrospinal fuid (CSF) and CNS cells, resulting in benefcial efects on CNS diseases.
Besides, 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 positivestranded viruses, the aberrant induction of infammatory cytokines/chemokines in case of SARS infection was mostly activated by dsRNA intermediates [36,94].In addition, the robust viral replication and delayed IFN-c signaling accompanying the initial steps of SARS seem to be consequence of the coronavirus ability to initially evade the host dsRNA sensors [95,96].Terefore, early administration of dsRNA-binding minocycline might reduce the risk of SARS-CoV-2.

What Is New and Conclusion
In conclusion, from in silico screening of 135 clinical drugs targeting on SARS-CoV-2 M pro , minocycline, inhibitor of bacterial ribosomal rRNA, showed interesting binding afnity (−9.6 kcal/mol).Critical hydrogen bonding with the Cys145-His41 catalytic dyad and hydrophobic interactions were found between minocycline and M pro active sites.During the 0-10 ns MD simulations, molecular dynamics stability of M pro -minocycline complex was also demonstrated to be close to M pro -N3 complex with persistent H-bond interactions.Te structure-afnity relationship explained the conformational suitability of minocycline.Minocycline showed in vitro M pro inhibitory activity with IC 50 of 5 mM.Literature review found that minocycline had both in vitro and in vivo broad-spectrum antiviral as well as anti-infammatory activities, and the levels of a broad spectrum of biological markers during minocycline administration were opposed to those of COVID-19 condition.Tese fndings suggested that minocycline, a safe, inexpensive, and readily available antibiotic, could be considered as an adjunctive therapeutic option for severe and fast-progressive COVID-19 patients.Tis study shed a new light on an adjuvant treatment strategy for this viral disease.Limitations of this study include the short duration of the MD simulations, which were performed for only 10 ns, resulting in only a brief understanding of the conformational changes of M pro -minocycline complex.

3. 3 .
Re-Docking of N3 and Minocycline by Discovery Studio.To further validate the docking result, re-docking and comparison of docking results generated by diferent software are academically consensus.From the CDOCKER results generated by Discovery Studio, N3 (Figure 2(a)) formed conventional hydrogen bonds with residues Phe140, His163, His164, Glu166, Gln189, and Tr190.Te isoxazole group formed pi-alkyl interaction with Ala191 and Pro168.Te aromatic ring formed van der Waals' forces with residues Tr24, Tr25, Leu27, and Cys145.In addition, N3 molecule forms covalent bonds with multiple residues of M pro .Te docking results were closely consistent with the co-crystal structure (checked by PyMOL, RMSD of 1.650 Å).

Figure 1 :Figure 2 :Figure 3 :
Figure 1: N3 bound in M pro active site and the diagram of the interactions in the coco-crystal [11].P1, P1′, P2, P3, P4, and P5 sites of N3 are indicated; 2Fo-Fc density map is shown around N3 molecule in blue mesh, C145-A in yellow mesh, and water in blue mesh.Te key residue is shown in stick, hydrogen bond is shown in black dashed line, and water is shown as red sphere.

Table 1 :
Comparison of the efect of COVID-19 and minocycline on selected biomarkers.