Tackling the SARS-CoV-2 main protease using hybrid derivatives of 1,5-disubstituted tetrazole-1,2,3-triazoles: an in silico assay

In regard to the actual public health global emergency and, based on the state of the art about the ways to inhibit the SARS-CoV-2 treating the COVID19, a family of 1,5-disubstituted tetrazole-1,2,3-triazoles, previously synthesized, have been evaluated through in silico assays against the main protease of the mentioned virus (CoV-2-M Pro ). The results show that three of these compounds present a more favorable interaction with the selected target than the co-crystallized molecule, which is a peptide-like derivative. It was also found that also hydrophobic interactions play a key role in the ligand-target molecular couplings, due to the higher hydrophobic surfaces into the active site. Finally, a pharmacophore model has been proposed based on the results below, and a family of 1,5-DT derivatives has been designed and tested with the same methods employed in this work. It was concluded that the compound with the isatin as a substituent (P8) present the higher ligand-target interaction, which makes this a strong drug candidate against COVID19, Abstract 60 In regard to the actual public health global emergency and based on the state of the art about the 61 ways to inhibit the SARS-CoV-2 treating the COVID19, a family of 1,5-disubstituted tetrazole-62 1,2,3-triazoles, previously synthesized, have been evaluated through in silico assays against the 63 main protease of the mentioned virus (CoV-2-M Pro ). The results show that three of these 64 compounds present a more favorable interaction with the selected target than the co-crystallized 65 molecule, which is a peptide-like derivative. It was also found that also hydrophobic interactions play a key role in the ligand-target molecular couplings, as a higher number of hydrophobic 67 surfaces make up the active site. Finally, a pharmacophore

117 target (considering the electrostatic, hydrophobic and hydrogen bond interactions) leading to the 118 to design of a novel family of molecules that can potentially inhibit the CoV-2-M Pro .
Computational Methods

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The Cartesian coordinates from the selected target, CoV-2-M Pro , were obtained from the protein 124 data bank (PDB code: 6lu7), which was one of the first crystallized structures of the main protease 125 of the SARS-CoV-2 virus. Furthermore, the Chimera package was used to add charges, remove 126 solvents and correct residues of the target structure (Pettersen et al. 2004).
127 128 Moreover, the 1,5-disubstituted tetrazole-1,2,3-triazoles and the co-crystallized compound (into 129 the CoV-2-M Pro structure), see Fig. 1, which are considered as ligands, were modeled using the 130 Avogadro software (Jin et al. 2020) and charged with the Chimera package (Pettersen et al. 2004). 131 However, to obtain a better approach, the ligands were optimized at the UFF level (Rappe et al. 132 1992) using the Gaussian 09 (G09) package (Frisch et al. 2009). Note that, the UFF method was 133 employed due to the good distance and angle bonds it provides to organic molecules, as well as 134 the low computational cost. Finally, the pharmacophore model was developed using the ZINCPharmer server (Koes & 145 Camacho 2012), considering the obtained properties obtained throughout the whole study. It is 146 noteworthy to mention that the proposed novel inhibitors underwent the same process as the 1,5-147 disubstituted tetrazole-1,2,3-triazoles and were evaluated using the selected target with the above 148 method. Manuscript to be reviewed Chemistry Journals Analytical, Inorganic, Organic, Physical, Materials Science 156 and interacted with the catalytic triad residues, such interaction will be explained in the discussion 157 section.  Table 1 shows the hydrogen bond energies, the electrostatic interactions, and the LE values 160 obtained for the selected ligands. Additionally, Fig. 3 shows the active site of the target, with the 161 co-crystalized ligand and two of the best ligands interacting. The hydrogen bond and electrostatic interactions between the selected target and the ligand 1e, 167 which is the compound with the most favorable ligand-target interactions, are depicted in Fig. 4. 168 The principal interactions are those with histidine residues, as well as one with serine and 169 glutamine.   185 The proposed pharmacophore model is shown in Fig. 6, and consists of ten principal components: 186 two hydrophobic fragments (Hy, depicted in green color), one aromatic fragment (Ar, colored in 187 blue color), three hydrophobic-aromatic fragments (Hy-Ar, represented in purple color), two 188 hydrogen donor fragments (HD, depicted in gray color) and two hydrogen acceptor fragments 189 (colored in orange color).  Fig. 8A shows that that molecule P8 prefers to interact in the right side of the 198 molecule in a similar manner as the co-crystallized ligand and 1e compounds. The structure of 199 compound P8 is located in the deep of the active site.

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Finally, Fig. 9 shows the molecules 1e and P8 in the pharmacophore model, and reveals the 202 occupied space by these molecules into the pharmacophore model. To evaluate the best ligand docked in the selected target, CoV-2-M Pro , the MolDock score energy 210 was considered as a parameter of measurement. Furthermore, the ligand efficiency (LE = 211 Energy/No. of heavy atoms) was used to determine with better precision the ligand-target binding 212 strength. This parameter gives the energy provided per atom in the ligand-target interaction, 213 making it a better way of comparison between ligands with different number of atoms, see Table  214 1.   Table 1, demonstrating that the VdW the limiting energy in 221 obtaining a better ligand-target interaction for this kind of systems. In other hand, best ligands 222 show the most favorable VdW energies. However, the state of the art regarding this protease 223 indicates that the hydrogen bond interactions are one of the most important energies, especially 224 with the amides of the catalytic triad residues (Gly143, Cys145, and Ser144) (Zhang et al. 2020). 225 Note that table 1 includes compounds P1-10, which are the designed potential inhibitors presented 226 in this work and will be boarded in section 4.4  Fig. 3 shows first the co-crystallized molecule (a peptide like derivative) which directly interacts 238 with the catalytic triad and part of the molecule dock perfectly in almost the whole cavity. 239 Furthermore, Fig. 3B depicts the two best ligands interacting in a similar manner to the co-240 crystallized molecule, but filling the right site of the computed cavity. This behavior might 241 explain the most favorable interactions seen when comparing them with the co-crystallized 242 molecule, see Table 1.  Contrary to the co-crystallized ligand, compound 1e does not cross the cavity space in the right 264 site. However, in interacts in the deep site of the cavity, docking less in the left site of the surface. 265 Note that ligand 1e presents higher L.E., than the co-crystallized ligand, see Table 1. Therefore, it 266 is clear that the presence of aromatic and hydrophobic rings in both molecules is essential and key 267 for better interactions. The hydrophilic interactions are practically negligible.  Based on the obtained results and considering the pharmacophore model, a series of ten 1,5-284 disubstituted tetrazole-1,2,3-triazoles have been proposed as inhibitors of the CoV-2-M Pro , which 285 are depicted in Fig. 7. As shown in Table 1, the P8 and P10 designed molecules present the more 286 favorable interactions with the selected target as they show a more negative LE value than the 287 other evaluated compounds. Note that, six of the ten designed molecules exhibit better interaction 288 with the CoV-2-M Pro than the co-crystallized molecules, which is the reference molecule. 289 290 On the other hand, the compound P8 has an isatin scaffold (1H-indole-2,3-dione) as part of its 291 structure, which is considered a privileged structure given its broad biological and pharmacological 292 activity. Some of which include antibacterial, anticancer, antitubercular, antimalarial, antifungal, 293 anticancer, anti-HIV, and in general antiviral (Varun et al. 2019). Analyzing the bio-active possess 294 of compound P8, it is seen that it promotes an intramolecular stabilization due to two stacking 295 interactions: one with the triazole ring and the other with the benzene ring, face-to-edge, and face-296 to-face, respectively.
297 298 Fig. 8 shows the principal hydrophobic interactions between P8 and the Cov-2-M Pro , depicted in 299 blue surfaces. These results can be explained by the higher quantity of rings in the P8 structures, 300 which could promote hydrophobic interactions. 301 302 In the case of the hydrogen bond interactions, molecule P8 interacts not only with the catalytic 303 triad, specifically with the Ser144 and Cys145, and presents a higher number of interactions with 304 other residues that include Ser1 and Asn142. The last one promotes a higher H bond energy than 305 compound 1e, see Table 1. In fact, (Liu et al. 2020b) mention that hydrogen bond interactions 306 play a key role in the ligand-target interaction as highlighted in the state of the art. Moreover, the 307 electrostatic interactions between P8 and the selected target take place at the residues His41, 308 His163, Glu166, and His172 residues, which in terms of Elstat energy, do not promote a favorable 309 interaction (To understand better the behavior of the electrostatic interactions as a function of the 310 different moieties in the studied molecules, the molecular electrostatic potential surfaces are 311 depicted in Supplemental Files). On the other hand, molecule 1e, which was previously synthesized by some of us, and the designed 314 compound P8 were evaluated into the pharmacophore model and analyzed through the segments 315 docked with the proposed structure. Fig. 9A shows the molecule 1e in the pharmacophore model, 316 which reveals that this molecule needs some components to complete all the pharmacophore 317 fragments. Specifically, it needs an aromatic moiety, as well as an HD and HA fragments in the 318 top of the molecule. 319 320 321 Finally, Fig. 9B depicts the P8 structure into the pharmacophore model and shows that this 322 molecule only an Hy and one HD fragments in order to complete all the requirements. Analyzing 323 the results, it is clear that to obtain some better molecules that could inhibit the Cov-2-M Pro it is 324 necessary to have a system that includes some rings in their structure. Also, the right side is the 325 more important site of the cavity, as long as the size of the molecule does not overpass the size of 326 the cavity.

V. Conclusions
330 331 A family of compounds previously synthesized by some of us was tested to inhibit the protein 332 Cov-2-M Pro , the results show that three of these compounds present a more favorable interaction 333 with the selected target than the co-crystallized molecule, which is a peptide-like derivative. 334 Moreover, although the fact that hydrogen bond interactions are mentioned in the state of the art 335 about the selected protease, it can also be found that the electrostatic interactions and main the 336 hydrophobic interactions play a key role in the ligand-target molecular couplings. 337 338 At the same time, the results reveal that a molecule can couple into the active site, which presents 339 higher hydrophobic surfaces. Thus, in the quest to develop potential candidates it is essential to 340 synthesize some molecules with a higher number of aromatic rings in their structures. Note that 341 the residues of the active site interact in a stronger way with the best coupled ligand.

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Finally, a pharmacophore model has been designed and used to propose a new family of 1,5-344 disubstituted tetrazole-1,2,3-triazoles derivatives. These compounds are potential candidates to be 345 synthesized as a perspective of this work. Based on the obtained results, the best ligands were 346 coupled with the pharmacophore model, highlights the importance of the isatin moiety. Also, the 347 pharmacophore model revealed that derivatives bearing the isatin substituent have a higher 348 potential in the design of new drugs against the SARS-Cov-2. Hydrophobic and stacking 349 interactions also play a key role in the design of new drug candidates to treat the COVID19.