The proteases from SARS-CoV-2, i.e., Mpro and PLpro, are important targets for the development of antivirals against COVID-19. The functional group 1,2,4-thiadiazole was demonstrated to inhibit cysteine proteases, such as papain and Cathepsins, and it is found in a new class of cephalosporin FDA-approved drugs: Ceftaroline Fosamil, Ceftobiprole, and Ceftobiprole Medocaril. In this work, we investigate the interactions of these drugs and their metabolites with the SARS-CoV-2 proteasesby molecular docking and density functional theory (DFT) calculations and propose a mechanism of inhibition as well. Our results indicate that the PLpro enzyme could be a better target for these drugs than the Mpro, suggesting that these compounds and metabolites can be tested inin vitro/vivo assays to confirm their inhibitory action. In addition, the data here reported can help in the design of new potential drugs against COVID-19.
Research Article
In silico studies of Mpro and PLpro from SARS-CoV-2 and a new class of Cephalosporin drugs containing 1,2,4-thiadiazole
https://doi.org/10.21203/rs.3.rs-1396565/v1
This work is licensed under a CC BY 4.0 License
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Cephalosporins
Drug repurposing
Computational Docking
1
2
4 thiadiazoles
DFT
MD
The global respiratory pandemic COVID-19 (Corona Virus Disease 2019) is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which remains afflicting millions of people. Although the majority of infected people (~ 80%) are asymptomatic or present mild symptoms(David A. Berlin et al., 2020; Gandhi et al., 2020; JA, 2020), the severe cases, which require hospitalization, are still increasing. These cases can evolve to pneumonia, kidney failure, and central nervous system symptoms (encephalitis, seizures) and death(David A Berlin et al., 2020; H et al., 2020; Harapan et al., 2020; Jeong et al., 2020).
With an important therapeutic significance, the cysteine-proteases from coronavirus (MERS-CoV, SARS-CoV, and SARS-CoV-2) have been considered potential targets for the drug repositioning and development(He et al., 2020; JAC et al., 2020; Jin et al., 2020; Lin et al., 2018; Rathnayake et al., 2020).
The main protease (Mpro, or 3C like protease (3CLpro)) and papain-like protease(PLpro)are essential for virus replication owing to their post-translational action on pp1a and pp1ab polyproteins processing. The catalytic systems of the Mpro and PLpro, adyad and a triad, respectively, are responsible for the peptide hydrolase activity, mainly promotedby the cysteine (Cys) residues(Anirudhan et al., 2021; Francés-Monerris et al., 2020).
Since the beginning of the pandemic, an extensive effort has been done in the search for SARS-CoV-2 proteases' inhibitors(Jin et al., 2020).For instance, the organochalcogen drugs Ebselen, Disulfiram, and Tideglusib, have been demonstrated to inhibits the Mpro and PLpro from SARS-CoV-2in vitro(Jin et al., 2020); these outcomes have also been analyzed and confirmed in silico(Lobo-Galo et al., 2020; Nogara et al., 2021a). Among the ~ 10,000 investigated molecules, only one compound containing the 1,2,4 thiadiazole functional group, i.e., the Tideglusib, was able to inhibit the Mpro(Jin et al., 2020). Despite the inhibitory mechanism of Tideglusib was not investigated in detail, its 1,2,4 thiadiazole moiety has an electrophile center at the sulfur atom, which is involved in the proteases inhibition, such as Papain and Cathepsins B, L, and K Fig. 1(JC et al., 2002; Leung-Toung et al., 2005, 2003). Sarkar et al. recently demonstrated that the compound RRA2, that contain the 1,2,4-thiadiazole functional group, had significant mycobactericidal activity against the intracellular active stage Mycobacterium bovis BCG and Mycobacterium tuberculosis with minimum inhibitory concentration (MIC) values of 2.3 and 2.0 µg/mL, respectively(Sarkar et al., 2021).
The S atom behaves like an electrophilic center, while the thiol (-SH) of the Cys proteases attacks the sulfur ring to form a disulphide bond with concomitant ring opening(Leung-Toung et al., 2005, 2003; Vega-Teijido et al., 2014). However, in the study of Jin et al., the authors did not comment about the in silico interaction or the in vitro inhibition of Sars-Cov-2 Mpro by other molecules containing the 1,2,4-thiadiazole moiety.
Based on the fact that Tideglusib inhibits the SARS-CoV-2 Mpro in vitro, possibly via the above-mentioned electrophilic interaction of 1,2,4-thiadiazole, a systematic search of compounds was performed using the Drug Bank database in order to increase and optimize the potential of the antiviral drugs, focusing on drugs previously approved by the FDA (Food and Drug Administration).
Recently, in silico studies, using approaches from the Korea Chemical Bank (KCB-DR) drug reuse database, have indicated some putative inhibitors of Mpro, including Ceftaroline Fosamil (a drug containing 1,2,4-thiadiazole functional group)(Kumar et al., 2021) Fig. 1. The virtual screening, molecular dynamics (MD) simulations, and binding-free energy approaches demonstrated the interaction between Ceftaroline Fosamil, forming hydrogen bonds with important active site residues in Mpro, such as His41. However, the authors did not elaborate on the possible mechanism of inhibition and the distance of the interaction with the cysteine residue (Cys 145) in the active site of the enzyme was not investigated.
Our search for drugs containing this 1,2,4-thiadiazole functional group showed two approved drugs and one in the experimental phase, i.e., Ceftaroline Fosamil, Ceftobiprole, and Ceftobiprole Medocaril, respectively Fig. 2. They are a new generation of broad-spectrum cephalosporins in late-stage development with activity against methicillin-resistant Staphylococcus aureus (MRSA)(JE, 2013; Laudano, 2011). The presence of the 1,2,4-thiadiazole moiety has been reported to facilitate the antibiotic permeation inside Gram-negative bacteria, and the transpeptidase activity(Laudano, 2011; Zhanel et al., 2009). In addition, these drugs are in use for the treatment of human respiratory tract infections and pneumonias with good efficacy (JE, 2013). With therapeutic significance, recent pharmacokinetic studies have indicated that intravenous administration of Ceftaroline Fosamil, Ceftobiprole and Ceftobiprole Medocaril has been associated to concentrations reaching milligrams per liter of their respective active metabolites in plasma and epithelial lining fluid (ELF)(Kiem and Schentag, 2008; Riccobene et al., 2016; Rodvold et al., 2009; Torres et al., 2016). One of the advantages of this study is precisely the repurposing of drugs already approved for clinical use, since their toxicity and required concentration levels have already been elucidated (Murthy and Schmitt-Hoffmann, 2008; Shirley et al., 2013).
In this work, we perform in silico molecular docking analysis, density functional theory (DFT) calculations and molecular dynamics (MD) to propose new Mpro and PLpro inhibitors, as well as, to explain the mechanisms of enzyme inhibition at the molecular and atomic levels. This latter analysis is fundamental to assess the role of the distinct molecular moieties for improved and rational drug design.
2.1. 1,2,4-thiadiazole containing drugs and metabolites
To find previous FDA approved drugs containing 1,2,4-thiadiazole functional group, a search was made on Drug Bank, www.go.drugbank.com(Wishart et al., 2018). The Ceftaroline Fosamil, Ceftobiprole and their respective metabolites were found as approved drugs, while the Ceftobiprole Medocaril is still in the experimental list. They were all considered in our in silico studies to verify if they might be Mpro and PLpro potential inhibitors.
2.2. Docking simulations
AutoDockVina was used for the docking simulations(Trott and Olson, 2010), with exhaustiveness of 50, according to a previous study (Nogara et al., 2021a). The Mpro and PLpro crystallographic structures were obtained from the Protein Data Bank (PDB) with the codes 6LU7 and 7JN2, respectively. Water, ions, ligands, and other molecules were removed from the protein structures; then, the hydrogen atoms were added using the CHIMERA program, followed by 100 steps of energy minimization (Pettersen et al., 2004). The Mpro gridbox of size 25 x 35 x 25 Å was centred on the active site (-14.04, 17.44, 66.22). For the PLpro, the docking grid box was centred on the active site (39.64, 30.68, 1.66; size: 20 x 20 x 20 Å) and in the Zn binding site (82.40 x 26.32 x -0.62; size: 20 x 20 x 20Å). The three-dimensional model of Ceftaroline Fosamil, Ceftobiprole, Ceftobiprole Medocaril and their metabolites, were created with Avogadro and MOPAC (PM6 method)(Hanwell et al., 2012; Stewart, 2016, 2007), using the dielectric constant of water (78.4), and taking into account the physiological pH equilibrium (7.0-7.4) determined by the Marvin Skecth 17.21.0, ChemAxon program (see Supporting Information (SI) Figures S17-19(“ChemAxon - Software Solutions and Services for Chemistry & Biology,” n.d.). This approach used in the optimization of the molecules under analysis aims aproducing the conditions closest to the real environment. In addition, both Z and E isomers of the drugs Ceftaroline Fosamil and Ceftobiprole Medocaril were considered in this study; they are due to the oxime functional group. These details are very important and can affect the predicted binding pose in molecular docking simulations. For each molecule, the 20 best conformers (in terms of ∆G) were analysed in the Discovery Studio Visualizer. The conformers of 1,2,4-thiadiazole containing drugs and metabolites which displayed the best interaction with the Cys residues from Mpro and PLpro (in terms of S···S distances and ∆G) were highlighted. Specifically, the distance between the S atom (ligand) to the S atom (Cys) was considered an indicator of potential covalent bond formation between 1,2,4-thiadiazole compounds and metabolites with the enzymes.
In total, 14 molecules were tested, derived from the drugs Ceftaroline Fosamil, Ceftobiprole and Ceftobiprole Medocaril, including their isomers and protonated forms in their predominant states between pH 7 to 7.4, SI Figure S17-19. Among them, 10 will be discussed in this article; the others are in the supplementary material section. Two conformers were chosen, i.e., the conformer with the largest negative binding energy and the shorter S∙∙∙S interaction.
2.3. Density functional theory (DFT) calculations;
All density functional theory (DFT) calculations were carried out using the Amsterdam Density Functional (ADF) program(“Amsterdam Modeling Suite”Baerends et al., 2019.; Velde et al., 2001). Scalar relativistic effects were taken into account using the zeroth-order regular approximation (ZORA)(Van Lenthe et al., 1994). The OLYP density functional was used, in combination with the TZ2P basis set, according to the literature (Bortoli et al., 2016). The softness (σ = 1 / η; η = [E(LUMO) - E(HOMO)] / 2) was computed according to LoPachin (2012)(LoPachin et al., 2012), using the Hirshfeld charges (Hirshfeld, 1977), which have a good overall reactivity prediction performance (Liu, 2015; Wang et al., 2019).
2.4. Molecular dynamics;
Molecular dynamics simulations were run for two compounds, namely Ceftaroline Fosamil and Ceftaroline Fosamil metabolite, employing AMBER2021(Case et al., 2021). The complexes were treated using the AMBER ff14SB force field for the protein residues and the generalized AMBER force field (GAFF) to define the ligands’ parameters. To simulate the Zn2+ atom present in PLpro the four cysteine residues directly bonded to zinc and the zinc atom itself were modelled employing the Zinc AMBER Force Field (ZAFF)(Peters et al., 2010). The structures were solvated in an octahedral box of TIP3P water molecules. Starting from the best pose obtained via the docking procedure and after energy minimization, heating to 310 K at constant volume and temperature was performed over 60 ps using the Langevin thermostat.
Afterwards equilibration at constant temperature (310 K) and pressure (1 bar, Berendsen barostat) was conducted for 60 ps using weak restraints (2 kcal mol− 1 Å−2) on the protein-ligand complex and then for 2 ns without any restraint, followed by 200 ns production runs from the MD trajectories, 1000 frames taken at 0.2 ns intervals were extracted and employed in the calculation of RMSDs and RMSFs.
For a better presentation of the results, the docking data about Ceftaroline Fosamil isomers (Z,E)1, Ceftaroline Fosamil Metabolite Z, E, Ceftobiprole, Ceftobiprole Medocaril isomers (Z,E)1, and Ceftobiprole Metabolites isomers Z,E in the conformation with the shorter S···S distance are presented in the main text, while the data about the best energy conformer have been included in the SI. This choice is based on the most relevant conformations in the chosen pH (7,0–7,4) range; the table with the percentages is Table S2. CeftarolineFosamil isomers (Z,E)2 and Ceftobiprole Medocaril isomers (Z,E)2 are shown in the SI.
Starting from the proposed inhibition mechanism involving the sulfur atom present in the 1,2,4-thiadiazole heterocycle and the thiol groups present in the Cys- enzymes Fig. 2. The in silico studies were carried out with the new class of cephalosporins and their respective metabolites, with the purpose of studying their binding poses, interactions, distances between Cys S∙∙∙S (1,2,4-thiadiazole), and proposing new drugs focusing on the inactivation of the active sites of the proteases of SARS-CoV-2, Mpro, PLpro, and PLpro Zn binding site Fig. 3.
3.1.1,2,4-thiadiazole containing drugs and metabolites interaction with M pro
Mpro is the mainly integrant to the proteolytic processing of SARS-CoV-2 polyproteins, remaining conserved in coronaviruses(Alves et al., 2020). Its function is cleaving both polyproteins 1a and 1ab into 16 distinct proteins, forming the viral replication complex (Fang et al., 2008). As with other cysteine proteases, the active sites contain a Cys-His catalytic dyad in charge of the peptide bond hydrolysis at specific sites of a polypeptide chain(Brocklehurst et al., 1987). The interaction of His41 with electrophiles decreases the pKa of its N1 imidazole atom, which will accept more easily the proton from the thiol group of Cys145. Consequently, upon deprotonation, the nucleophilicity of Cys145 residue will increase considerably(Tong et al., 2021). In this way, covalent blockage of the thiol/thiolate moiety of Cys145 will inhibit the Mproproteolytic function.The binding poses obtained from the docking studies focusing on the Mpro active site demonstrate that the sulfur atom of 1,2,4-thiadiazoles drugs and metabolites interacts with the thiol/thiolate group of Cys145 Fig. 4A-J.
The Ceftaroline Fosamil isomers (Z,E)1, showed similar bond posture and S∙∙∙S interaction, while the binding poses of the metabolites E1 and Z1 and S∙∙∙S distances were different (3.9Å and 4.7Å, respectively) suggesting that the phosphate group can interfere in the binding pose Fig. 4A-D. For the Ceftobiprole drug isomers comparable binding poses are predicted, but different distances with the Cys145, with the (Cys)S∙∙∙S(thiadiazole) interaction being shorter in the E1 isomer (5.2Å) than in the Z1 (4.8 Å) Fig. 4E-F. In order to analyze the isomers of the Ceftobiprole metabolite, a similar binding pose of thiadiazole was observed. However, Ceftobiprole metabolite Z1 (4.5 Å) presented a shorter S∙∙∙S interaction than the isomer E1 (5.2 Å) Fig. 4G-H. For the Ceftobiprole metabolite isomers, an equal binding pose between Cys145 and S(thiadiazole) was observed; however, Ceftobiprole metabolite Z1 (5.0Å) presented a longer interaction than the isomer E1 (4.6Å) Fig. 4I-J. The analysis of the S∙∙∙S interaction shows that the E1 isomer of the metabolite from the Ceftaroline Fosamil presents the shortest distance Table 1, suggesting the formation of an adduct with Cys145.
In addition, it is important to note that the H-bonds with the Ser144, His163, Gln189, Asn142and Cys145 residues, help to stabilize the Mpro-ligand complexes. As well as, the π-alkyl interaction between the 1,2,4-thiadiazole ring and the Cys145 lateral chain may have great value, as a recently in silico study have been demonstrated (Kumar et al., 2021). Among the conformers with the largest negative binding energy Table 2, only Ceftaroline Fosamil Metabolite Z, Ceftobiprole E, Ceftobiprole Metabolite Z, and Ceftobiprole Medocaril isomer E1 showed S···S interaction; however, their distances were higher than the conformers with the shortest distances Table 1, Figure S5-S7.
A similar H-bonds pattern was observed for these conformers, with distances around 1.8-3.0 Å with Thr190, Ser144, His163, Thr26, Asn142, Glu166, and Cys145.
Table 1. Predicted binding free energies (∆G, kcal∙mol-1) between Mpro and PLpro and PLpro Zn binding site, with 1,2,4-thiadiazole containing drugs and S···S interaction distances.
|
|
aMpro |
|
bPLpro |
|
cPLpro |
Zn |
|
|
Molecule |
∆G |
dist. (Å) S*∙∙∙S |
∆G |
dist. (Å) S*∙∙∙S |
∆G |
dist. (Å) S*∙∙∙S |
|
|
(Cys 145) |
(Cys 111) |
(Cys 192) |
|||||
|
Ceftaroline Fosamil Z1 |
-7.3 |
4.1 |
-5.9 |
3.6 |
-5.0 |
4.8 |
|
|
Ceftaroline Fosamil E1 |
-6.6 |
4.0 |
-6.3 |
3.8 |
-5.2 |
4.8 |
|
|
Ceftaroline Fosamil Metabolite Z |
-7.5 |
4.7 |
-6.3 |
4.0 |
-5.5 |
4.4 |
|
|
Ceftaroline Fosamil Metabolite E |
-5.8 |
3.9 |
-5.7 |
3.7 |
-5.1 |
4.7 |
|
|
Ceftobiprole Z |
-6.5 |
4.8 |
-5.4 |
3.9 |
-5.2 |
4.4 |
|
|
Ceftobiprole E |
-7.6 |
5.2 |
-5.5 |
5.6 |
-4.7 |
5.4 |
|
|
Ceftobiprole Medocaril Z1 |
-7.4 |
5.0 |
-5.9 |
5.4 |
-5.9 |
4.4 |
|
|
Ceftobiprole MedocarilE1 |
-8.0 |
4.6 |
-6.0 |
3.4 |
-5.4 |
4.1 |
|
|
Ceftobiprole Metabolite Z |
-7.4 |
4.5 |
-5.4 |
4.0 |
-5.2 |
4.7 |
|
|
Ceftobiprole Metabolite E |
-7.1 |
5.2 |
-5.7 |
3.9 |
-4.4 |
4.7 |
|
S*∙∙∙S indicates the distance interaction (in Å) of the electrophile center of the ligand (i.e., the sulfur atom of the 1,2,4 thiadiazole heterocycle with the sulfur atom of the cysteynil residues of MPro. Distance (in Å) of the thiol from aCys145, bCys111, and cCys192 to the ligand of the S from the 1,2,4-thiadiazole heterocycle. The green, yellow, and red colors indicate a favorable, intermediate, and less favorable interaction.
Table 2. Predicted binding free energies (∆G, kcal∙mol-1) between Mpro and PLpro and PLpro Zn binding site, with 1,2,4-thiadiazole containing drugs with the largest negative ∆G and S···Sinteraction distances.
|
|
aMpro |
|
bPLpro |
|
cPLpro |
Zn |
|
Molecule |
∆G |
dist. (Å) S*--S (Cys 145) |
∆G |
dist. (Å) S*--S (Cys 111) |
∆G |
dist. (Å) S*--S (Cys 192) |
|
Ceftaroline Fosamil Z1 |
-7.5 |
- |
-6.4 |
4.5 |
-5.4 |
- |
|
Ceftaroline Fosamil E1 |
-7.0 |
- |
-6.3 |
3.8 |
-5.8 |
4.8 |
|
Ceftaroline Fosamil Metabolite Z |
-7.6 |
- |
-6.6 |
5.1 |
-5.5 |
4.4 |
|
Ceftaroline Fosamil Metabolite E |
-6.9 |
4.0 |
-6.3 |
- |
-5.5 |
4.8 |
|
Ceftobiprole Z |
-7.4 |
- |
-6.3 |
- |
-5.3 |
4.7 |
|
Ceftobiprole E |
-7.6 |
5.2 |
-6.1 |
- |
-5.7 |
- |
|
Ceftobiprole Medocaril Z1 |
-8.2 |
- |
-6.1 |
- |
-5.9 |
- |
|
Ceftobiprole Medocaril E1 |
-8.4 |
6.0 |
-6.3 |
- |
-5.7 |
- |
|
Ceftobiprole Metabolite Z |
-7.5 |
5.8 |
-5.4 |
4.0 |
-5.4 |
4.7 |
|
Ceftobiprole Metabolite E |
-7.6 |
- |
-6.9 |
- |
-4.8 |
4.8 |
S*∙∙∙S indicates the distance interaction (in Å) of the electrophile center of the ligand (i.e., the sulfur atom of the 1,2,4 thiadiazole heterocycle with the sulfur atom of the cysteynil residues of Mpro. Distance (in Å) of the thiol from aCys145, bCys111, and cCys192 to the ligand of the S from the 1,2,4-thiadiazole heterocycle. The green, yellow, and red colors indicate a favorable, intermediate, and less favorable interaction.
3.2. 1,2,4-thiadiazole containing drugs and metabolitesinteraction with PLpro
In PLpro, the Cys111 from the catalytic triad contains the nucleophilic center that directly participates in the cleavage of the peptide bond of pp1a and pp1ab polyprotein from SARS-CoV-2, and the His272 and Asp286 residues participate as an acid-base pair that promotes the thiol deprotonation of Cys111. The resulting thiolate has enhanced nucleophilicity(Anirudhan et al., 2021; Ismail et al., 2021). Thus, blocking the thiol moiety of Cys111 is an important strategy to inhibit PLpro.
The binding poses obtained from the docking studies focusing on the PLpro active site demonstrate that the sulfur atom of 1,2,4-thiadiazole containing drugs and metabolites is able to interact with the thiol group of Cys111residue Fig. 5A-J.
The Ceftaroline Fosamil isomers (Z,E)1 show bonding poses and S∙∙∙S interaction similar to those observed in the Mpro active site for the Z (3.6Å) andfor the E isomer (3.8Å). In contrast, their metabolites Z and E interact differently, and the distance of the S∙∙∙S interactions is more favorable in the E (3.7Å) than in the Z isomer (4.0Å) Fig. 5A-D.
The Ceftobiprole isomers interact with different binding poses and S∙∙∙S interaction distances with the active site of PLpro, where the Z1 isomer (3.9Å) has a shorter distance than the E1 (5.6Å) isomer Fig. 5E-F. In the Ceftobiprole metabolite, the shortest interaction is observed for the isomer E1 (3.9Å), this being the best interaction here computed for this protease Fig. 5G-H. For the Ceftobiprole Medocaril, a shorter interaction is observed in the isomer E1 (3.4Å) than in the Z1 (5.4 Å). In fact, in terms of distance between the sulfur atoms of the ligand and the enzyme, the E1 isomer exhibits the best interaction Fig. 5I-J.
In view of the proposed mechanism for the inhibition of the proteases depicted in Fig. 1, it is important to note that the H bonds between His272 and Cys111 are critical for stabilizing the PLpro-ligand complexes. Indeed, the docking data here presented, confirm the importance of the interaction between His 272 and Cys111. As observed for the Mpro, an apparent critical interaction is the π-π bondstacking between the aromatic 1,2,4-thiadiazole ring and the Cys111 Fig. 5. For the best conformer, (in terms of thermodynamic stability), the drugs and metabolites exhibiting the shortest distances between (Cys145)S∙∙∙S(1,2,4-thiadiazole), are Ceftaroline Fosamil (Z,E)1, Ceftobiprole Z, and Ceftobiprole Metabolite Z. Similar pattern of H bond interactions is observed for the energetically most stable conformers, with distances ranging from 1.8–3.3Å, between amino acids surrounding the active site (for instance, Ans109, His272 and Cys111). The binding poses for these interactions are shown in the SI Figures S8-10.
3.3. 1,2,4-thiadiazole containing drugs and metabolitesinteraction with PLpro Zn binding site
While the active actives of Mpro and PLpro have only one Cys as a target, the Zn site of the PLprois composedby a Zn (II) ion coordinated to four Cys residues (Cys189, Cys192, Cys224 and Cys226). This type of Cys-rich motif structure is found in many proteins, i.e. in zinc finger containing proteins(Abbehausen, 2019; M et al., 2017), and it is a potential molecular target for therapeutic interventions. Theoretically, the inhibition of the PLpro function by the oxidation of the cysteynil residues binding Zn ion by organochalcogens (such as the 1,2,4 thiadiazole containing molecules) has not been elucidated yet.
In fact, the enzyme inhibition can occur via the ejection of a zinc ion at the PLpro Zn site of SARS-CoV-2. Accordingly, the recent study of Sargsyan et al.,(Sargsyan et al., 2020) has indicated that the safe drugs ebselen and disulfiram can covalently bind to cysteine residues of the Zn binding site of the PLpro. In the present study, the binding poses obtained from the docking simulations focusing on the Zn site demonstrate that the sulfur atom of the 1,2,4-thiadiazole containing drugs and metabolites indeed interact with the thiol group of cysteynil Cys192 Fig. 6A-J.
The Ceftaroline Fosamil isomers (Z,E)1 exhibit similar binding poses and distances between the sulfur atoms from the 1,2,4 thiadiazole moiety and the Cys192 close to S∙∙∙S (4.8Å), while the metabolite isomers binding pose and S∙∙∙S distances are different, ranging from (4.7Å) in E and (4.4Å) in the Z isomer Fig. 6A-D.
The Ceftobiprole drug isomers show distinct binding poses and S∙∙∙S distances. The interaction with Cys192is shorter in the Z1(4,3Å) than in the E1 isomer (5.3Å) Fig. 6E-F. In the case of Ceftobiprole metabolite, a similar behavior is observed. Identical distances were obtained with the Z and E isomers (4.7Å) Fig. 6G-H. Regarding the Ceftobiprole Medocaril isomers, the partner of the interaction poses are different; the observed S∙∙∙S interaction distances in the Z (4.4Å) and E isomer (4.1Å) are similar Fig. 6I-J.
It is important to note that the H-bonds with Thr225 and Cys192 help to stabilize the PLpro Zn-ligand complexes, which are here described, and may be of great value in the mechanism of inhibition of this site, possibly promoting the ejection of the zinc ion, after the nucleophilic attack to Cys192, as suffragated by previous studies(Sargsyan et al., 2020). Considering the conformers with the largest negative ΔG, Ceftaroline Fosamil (E)1, Ceftaroline Fosamil Metabolite (Z), Ceftobiprole (Z), and the experimental drug Ceftobiprole Medocaril (Z,E)1 demonstrateoptimal S∙∙∙S interactions (Table 2 and Figure S11-S13).
For the best conformer (in terms of ΔG) with the PLpro Zn binding site Table 2, the drugs and metabolites show (Cys192)S∙∙∙S(1,2,4thiadiazole) distances ranging between 4.8 and 5.1Å). The binding poses are included in the SI Figures S14-16. Similar H bonds are observed for this energetically best favored conformer, with distances around 2.0-3.4Å, including amino acids around the binding site as, Gln195, Thr225 and Pro223.
3.4 Reaction between a thiolate and 1,2,4-thiadiazoles
According to the docking studies, the 1,2,4-thiadiazole ring might be the reactive center of the antibiotic drugs. Thus, we hypothesized that this moiety might react with the catalytic or structural Cys residues forming a stable adduct via a disulfide bond. We studied the reactivity and computed the reaction energies modeling the process with 1,2,4-thiadiazole ring (tdz) and a methylthiolate (MeS−) (low molecular weight thinks are often used as a simple model of Cys residue or other biological thiols like GSH(Madabeni et al., 2021, 2020; Nogara et al., 2021b) running DFT calculations. The 3,5-dimethyl-1,2,4-thiadiazole (tdzMe2)and 3-methyl-1,2,4-thiadiazol-5-amine (tdzMeN) were used as models of the antibiotic drugs. Furthermore, the reactivity of the corresponding protonated molecules ([tdzHMe2]+and [tdzHMeN]+) were considered too.
To better understand the reactivity, Hirshfeld charges were computed and the softness analysis was done (Table 3). The results suggest that the reactions of MeS− with protonated thiadiazoles ([tdzHMe]+ and [tdzHMeN]+)are more favorable than those with the neutral ones.Also according to the Hard and Soft, Acids and Bases (HSAB) theory, soft bases (MeS−)prefer to react with soft acids ([tdzHMe]+ and [tdzHMeN]+)(LoPachin et al., 2012). In addition, the S partial charge analysis suggests that this atom is more electrophilic in the protonated than in neutral form.
| Phase | Reactants | S charge | HOMO | LUMO | Softness |
|---|---|---|---|---|---|
| gas | MeS− | -0.737 | 1.741 | 4.062 | 0.861 |
| tdzMe2 | 0.150 | -6.293 | -1.804 | 0.445 | |
| [tdzHMe2]+ | 0.322 | -11.976 | -7.652 | 0.462 | |
| tdzMeN | 0.114 | -5.534 | -1.298 | 0.472 | |
| [tdzHMeN]+ | 0.271 | -10.922 | -6.923 | 0.500 | |
| water | MeS− | -0.847 | -4.776 | -0.375 | 0.454 |
| tdzMe2 | 0.184 | -6.458 | -1.965 | 0.445 | |
| [tdzHMe2]+ | 0.381 | -7.495 | -3.090 | 0.454 | |
| tdzMeN | 0.130 | -5.662 | -1.421 | 0.471 | |
| [tdzHMeN]+ | 0.307 | -6.4611 | -2.462 | 0.500 | |
| softness: σ = 1 / η; hardness: η = [E(LUMO) - E(HOMO)] / 2. Level of theory: (COSMO)-ZORA-OLYP/TZ2P. | |||||
As shown in Table 4, the reaction between the protonated [tdzHMe2]+ and MeS− gives an adduct with a disulfide bond and in an open ring conformation (Entry A and B). In the gas phase, the reaction energy is more negative than in water, because the charged reactants are much less stabilized in the gas phase than in water (Hamlin et al., 2018; Serdaroğlu, 2011). We studied this reaction also using the cysteinate as a nucleophile (C and D), and the energy values in gas and condensed phase also present this large difference.
In the gas phase, using the neutral thiadiazole (tdzMe2) as substrate we obtaineda three-center intermediate (TCI) [tdzMe-SMe]−, instead of the open ring product [tdzMe2-SMe]−which was recovered in water (E and F). The TCI-[tdzMe2-SMe]− presents negative reaction energy while the [tdzMe2-SMe]− formation in water shows positive reaction energies, suggesting an endergonic process.
Finally, we used the [tdzHMeN]+ model, in which the methyl group at position 5 of the 1,2,4-thiadiazole was replaced by an amine group (in similar way as found in the antibiotic drugs), to compute the reaction energies (G and H). As previously observed, there is a significant difference between the energies in gas and condensed phase. Anyway, they are both energetically favorable. The reaction with the neutral tdzMeN molecule also was found to be an endergonic process (I), suggesting that the protonation of the 1,2,4-thiadiazole ring is essential for the reaction.
In this sense, theoretical data from the [tdzHMe2]+ and [tdzHMeN]+ are in nice agreementwith the hypothesis reported in literature, where it is described that the thiolateformof cysteine enzymes can attack the S atom from protonated thiadiazoles forming a disulfide bond with concomitant ring-opening, leading to the enzyme inhibition (Leung-Toung et al., 2005, 2003).
Table 4. Reaction energies. Level of theory ZORA-OLYP/TZ2P.
3.5 Molecular Dynimics simulation of PLpro with Ceftaroline Fosamil and metabolite isomer Z compund;
MD simulations were performed on two selected systems. i.e. PLpro with Ceftaroline Fosamil and metabolite isomer Z, respectively, results of the MD simulations show two different scenarios. The calculations on the PLpro complex with Ceftaroline Fosamil isomer Z(PLpro-Ceftaroline Fosamil) clearly showed that this structure is not suitable for an efficient inhibition because, even if docking scores indicated it as a potential target, after 200 ns of MD the interactions between Ceftaroline Fosamil and the inhibition site are not strong enough to maintain the small molecule in position. In fact, already after 50 ns the ligand is not bonded anymore to the protein structure, and it is moving freely in the simulation box. This is reflected from the very high RMSF attributed to the Ceftaroline Fosamil molecule, which is calculated to be 36.1 Å (Fig. 7A, Ceftaroline Fosamil corresponds to the last residue in the RMSF graph). During the dynamics, there are two brief windows in which the ligand shows interaction with two different regions of the protein. From approximately 60 to 90 ns it is found in proximity of Gln212 and Gln218 and from 190 ns until the end of the simulation around Leu122 as it can be seen from the RMSD graph Fig. 7A that shows lower fluctuations in these intervals. Longer simulations and further analysis in prompted to assess if any of these sites could be relevant to the inhibitory action of Ceftaroline Fosamil (isomer Z).
Conversely, the MD simulations on the PLpro-Ceftaroline Fosamil Metabolite isomer Z (PLpro-Ceftaroline Fosamil metabolite ) show that this ligand tends to remain the same region throughout the simulation as shown by the structures extracted at 100 and 200 ns Fig. 7B and reflected in the RMSD fluctuations which are noticeably lower than those of CF in PLpro-Ceftaroline Fosamil and in the RMSF value for Ceftaroline Fosamil metabolite (3.6 Å) which was found to be comparable to the other residues in the protein. Interestingly, during the dynamics there are two sudden changes in conformation of Ceftaroline Fosamil metabolite at approximately 25 and 175 ns which reflect the abrupt changes in RMSD values. The structure, which is initially found to resemble that of a linear alkane, moves towards a more spherical geometry around 25 ns as the outermost parts bend inward toward the center of the molecule. This configuration is seen for approximately 150 ns after which another distention towards a more linear structure is seen. These three conformations (linear/spherical/linear) can be seen in Fig. 7B at 0 100 and 200 ns respectively. Nevertheless, during whole simulation Ceftaroline Fosamil metabolite maintain it interaction with the initial binding site effectively making it a potential inhibitor.
In this work we have studied the mechanism of inhibition of 1,2,4-thiadiazole containing drugs and their metabolites, combining molecular docking and quantum chemistry calculations. Our results suggest that the PLpro enzyme from SARS-CoV-2 might be a better target for the cephalosporin drugs and their metabolites containing 1,2,4-thiadiazoles functional group than Mpro. These conclusions are based on the interactions, binding poses, and distances (1,2,4-thiadiazole)S∙∙∙S(Cys111). Specifically, the sulfur atom in the thiadiazole ring from the Ceftaroline Fosamil isomers and its Z1 active metabolite showed an adequate interaction with the thiol group of Cys111 from PLpro active site, as well the isomer metabolites of the drug Ceftobiprole and the isomer E1 of the experimental drug, Ceftobiprole Medocaril. This suggests that the inhibition might be covalent. In fact, through DFT calculations, we demonstrate that the adduct formation is energetically favorable. In this way, the use of these cephalosporin drugs and their metabolites might be an option in the search for antivirals against COVID-19.In addition, the approved drugs Ceftaroline Fosamil and Ceftobiprole are widely used in cases of pneumonia and respiratory tract infections(Riccobene et al., 2016; Rodvold et al., 2009; Torres et al., 2016).
We also demonstrate that Cephalosporin drugs and their metabolites, containing the 1,2,4 thiadiazole moiety, have potential against COVID-19 and may bind to the SARS-CoV-2 Mpro and PLpro. Only Ceftobiprole Metabolite isomer Z is able to interact with the Cys residues from Mpro and PLpro showing the best S∙∙∙S interaction and the largest negative energy, indicating it is a potential inhibitor. Structural modifications of this molecule might be a promising strategy to find new antivirals. Overall, these data suggest that these compounds, as well as their derivatives containing the 1,2,4 thiadiazole ring, should be tested in in vitro and in vivo trials to confirm the inhibitory and pharmacological potential.
Thus, in addition to their antibacterial effects, Ceftaroline Fosamil, Ceftobiprole, and Ceftobiprole Medocaril may be also effective inhibitors of the thiol-containing enzymes of the SARS-CoV-2.
Acknowledgements:
The authors would like to thank the financial support by Coordination for Improvement of Higher Education Personnel CAPES/ PROEX (n° 23038.005848/2018-31; n°0737/2018). J.B.T.R., P.A.N., and C.P.D. were funded by CAPES (Edital 09-88887.505377/2020-00; 88887.511828/2020-00; 88887.512045/2020-00; 88887.512885/2020-00); F.B.O. was funded by CAPES (Edital 88887.354370/2019-00).
M.B. and L.O. acknowledge the C3P high performance computing facility of the Department of Chemical Sciences of the University of Padova (Padova, Italy) and the cloud@CNAF resources (project Insight on Nitrogen Chalcogen Interaction, INCIpit, p. i.: M.B.) granted by the CNAF center of the Italian institute of Nuclear Physics (INFN, Bologna, Italy) for the computational infrastructure.
Funding:
Coordination for Improvement of Higher Education Personnel CAPES/ PROEX (n° 23038.005848/2018-31; n°0737/2018). J.B.T.R., P.A.N., and C.P.D. were funded by CAPES (Edital 09-88887.505377/2020-00; 88887.511828/2020-00; 88887.512045/2020-00; 88887.512885/2020-00); F.B.O. was funded by CAPES (Edital 88887.354370/2019-00).
Calculations have been carried out using CINECA computational facilities thanks to the ISCRA C project PROSIT2, P.I.: L.O.
Conflicts of interest:
The authors declare no conflicts of interest.
Availability of data and material:
Data and materials are freely accessible.
Code availability:
Not applicable.
Author’s contributions:
Authors Cássia Pereira Delgado, Dr. Pablo Andrei Nogara and Dr. João Batista da Rocha, performed the Docking contributions and repurposing research.
The authors Dr. Laura Orian and Dr, Pablo Andrei Nogara contributes to DFT calculations and data analysis. The author Dr. Marco Bortoli contributes to MD calculations and data analysis.
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