key: cord-0908465-swmkkdya
authors: Santos, Lucianna H.; Kronenberger, Thales; Almeida, Renata G.; Silva, Elany B.; Rocha, Rafael E. O.; Oliveira, Joyce C.; Barreto, Luiza V.; Skinner, Danielle; Fajtová, Pavla; Giardini, Miriam A.; Woodworth, Brendon; Bardine, Conner; Lourenço, André Luiz; Craik, Charles S.; Poso, Antti; Podust, Larissa M.; McKerrow, James H.; Siqueira-Neto, Jair L.; O’Donoghue, Anthony J.; da Silva Júnior, Eufrânio N.; Ferreira, Rafaela S.
title: Structure-based identification of naphthoquinones and derivatives as novel inhibitors of main protease Mpro and papain-like protease PLpro of SARS-CoV-2
date: 2022-01-05
journal: bioRxiv
DOI: 10.1101/2022.01.05.475095
sha: 6c28762c7163840079da7cc8f73d2df9fa201679
doc_id: 908465
cord_uid: swmkkdya

The worldwide COVID-19 pandemic caused by the coronavirus SARS-CoV-2 urgently demands novel direct antiviral treatments. The main protease (Mpro) and papain-like protease (PLpro) are attractive drug targets among coronaviruses due to their essential role in processing the polyproteins translated from the viral RNA. In the present work, we virtually screened 688 naphthoquinoidal compounds and derivatives against Mpro of SARS-CoV-2. Twenty-four derivatives were selected and evaluated in biochemical assays against Mpro using a novel fluorogenic substrate. In parallel, these compounds were also assayed with SARS-CoV-2 PLpro. Four compounds inhibited Mpro with half-maximal inhibitory concentration (IC50) values between 0.41 µM and 66 µM. In addition, eight compounds inhibited PLpro with IC50 ranging from 1.7 µM to 46 µM. Molecular dynamics simulations suggest stable binding modes for Mpro inhibitors with frequent interactions with residues in the S1 and S2 pockets of the active site. For two PLpro inhibitors, interactions occur in the S3 and S4 pockets. In summary, our structure-based computational and biochemical approach identified novel naphthoquinonal scaffolds that can be further explored as SARS-CoV-2 antivirals.

transmission from human to human is undoubtedly the main source of contagion, which 61 occurs mainly through droplets, hand contact, or contact with contaminated surfaces [5] . 62

To control the spread of this pandemic virus, biosecurity and hygiene measures are now 63 worldwide applied [6] . Despite the rapid development and emergency authorization of 64 vaccines, viral escape mutants have emerged, and SARS-CoV-2 infections remain a 65 concern for the global community. Therefore, there is a continuing need to discover 66 structural frameworks for drugs that can be employed against . 67 Drug development efforts have targeted the SARS-CoV-2 main protease (Mpro) 68 also known as 3-chymotrypsin-like protease (3CLpro) or non-structural protein 5 (nsp5) 69 [8, 9] . Mpro is an essential cysteine protease that cleaves the precursor replicase 70 polyprotein in a coordinated manner [10] , to generate at least 11 non-structural proteins 71 [11] . As a target, Mpro is conserved among other coronaviruses, and has no closely 72 related human homolog [12] [13] [14] . Therefore, it has been intensively investigated as a drug 73 target for SARS and Middle East Respiratory Syndrome (MERS) [15] [16] [17] [18] were studied and evaluated according to their potential to act as anti-SARS-CoV-2. Imine 154 derivatives were also targeted in our studies and were placed in group 6 [67] . 155 Finally, groups 7 and 8 are formed by hydrazo, imidazole, and oxazole derivatives 156 [108] [109] [110] [111] . The compounds in these groups were prepared from the quinones described 157 above and represent our attempt to study quinone-derived heterocyclic compounds with 158 biological activity against various microorganisms and their effectiveness against the 159 virus that causes COVID-19. Prior to biochemically evaluating the compounds against Mpro, we designed a 287 fluorescent-quenched peptide substrate with the sequence ATLQAIAS that corresponds 288 to the P4 to P4ʹ amino acids of the nsp7-nsp8 cleavage site and the dash representing the 289 scissile bond. This substrate was chosen because the sequence most closely matches the 290 consensus sequence for all 11 viral polypeptide cleavage sites (Figure 5A and B) [126] . 291 ATLQAIAS was flanked by 7-methoxycoumarin-4-acetyl-L-lysine on the N-terminus, 292 dinitrophenyl-L-lysine on the C-terminus. The peptide contains several non-polar amino 293 acid residues and therefore two d-Arginine residues were added on the N-terminus to 294 increase solubility. Using a concentration range of 3 µM to 250 µM, the KM for this 295 substrate was calculated to be 52.1 µM ± 14.4 µM. 296 297

We evaluated the 24 hit compounds from our virtual screen in a biochemical assay 300 using recombinant SARS-CoV-2 Mpro. The enzyme was pre-incubated with each 301 compound at 10 µM and then assayed with the fluorogenic peptide substrate. To avoid 302 detecting aggregators as false positives [127, 128] , our assay was performed in the 303 presence of 0.01% Tween 20. Additionally, we evaluated the absorbance of MCA 304 fluorescence by the compounds, to make sure the observed enzyme inhibition was not an 305 artifact of fluorescence, another common cause of false positives in enzyme assays [129] . 306 From this screen, three 1,4-naphthoquinones derivatives, 379, 382, and 415, fully 307 inhibited Mpro, while two quinone-based 1,2,3 triazoles, 191 and 194, had 50% or more 308 inhibition. 668 was insoluble in assay buffer and was therefore eliminated from further 309 analysis, while the remaining compounds had inhibition profiles of less than 50% (Table  310 1). The most potent compounds were subsequently evaluated at a concentration range of 311 10 µM to 9.7 nM and the half-maximal inhibitory concentration (IC50) was calculated to 312 be 66 µM ± 22 for 191, an ortho-quinone-based 1,2,3 triazole, 5 µM ± 0.15 for 415, 0.63 313 µM ± 0.04 for 379, and 0.41 µM ± 0.015 for 382 (Table 1 and Figure 5) . The IC50 values observed in these two assay conditions were similar, with slightly lower 345 IC50 values upon preincubation (0.42 µM ± 0.02 upon incubation vs 0.80 µM ± 0.06 346 without incubation for 382 and 5.0 µM ± 0.2 upon incubation vs 16 µM ± 1 without 347 incubation for 415) (Figure 6A and B) , while for the positive control GC373 the IC50 was 348 ten-fold lower upon preincubation (0.003 µM ± 0.001). A dilution experiment was also 349 performed, to check whether the compounds were irreversible. We incubated the 350 inhibitors and Mpro at high concentrations and then diluted the incubation mixture, 351 resulting in inhibitor concentrations 10-fold lower than their apparent IC50. In this assay, 352 an irreversible inhibitor will maintain approximately 10% of enzymatic activity, while a 353 rapidly reversible inhibitor will dissociate from the enzyme to restore approximately 90% 354 of enzymatic activity following the dilution event [130, 131] . When this was performed 355 with Mpro and GC373, a covalent Mpro inhibitor, the enzyme remained inhibited upon 356 dilution. The same behavior was observed for compound 415 suggesting that this 357 inhibitor is an irreversible covalent inhibitor (see Figure 7 for the proposed binding 358 mechanism). However, when the same test was carried out with compound 382 enzyme 359 activity returned after dilution ( Figure 6C ). This suggested that the inhibition by 382 is 360 reversible. 361 experiments (data shown as spheres or squares for each experiment), in which the 366 compounds were pre-incubated with Mpro prior to substrate addition (black) and without 367 preincubation with the compounds (purple). Reversibility assay (C). After preincubation 368 of Mpro with compounds, at higher concentrations, the sample was diluted, and product 369 formation was monitored for 120 minutes. Compound 382 reduced the enzymatic reaction 370 rate by 26% compared to vehicle control (red), while the compound 415 reduced product 371 formation by 100%, and this activity was not restored over a 2h period post dilution, as 372 observed for the covalent inhibitor GC373 (black). 373

To gain insights into the proposed binding mode of our Mpro inhibitors and guide 374 future optimization efforts, we conducted docking and MD studies with compounds 382 375 and 415, representatives from two inhibitor scaffolds discovered. Our simulations 376 considered the 415 ligand covalently bound, given the proposed reaction mechanism 377 ( Figure 7A) , to both monomers in the Mpro dimer, and 382 freely. For the free 378 simulations, however, the loss of interactions with E166 resulted in ligands being expelled 379 from one of the binding sites within a few nanoseconds (~200 ns) of simulation 380 (Supporting Information Table S2 ). Our analysis is focused on the other binding site, that 381 retained the ligand with stable interactions along the analyzed trajectory. 382

For both ligands, the most representative binding modes observed in the MD 383 simulations (Figure 7A and 7B) retain key interactions proposed based on docking with 384 Glide (Supporting Information Figure S45 ). However, compound 415 showed a more 385 conserved binding mode throughout the trajectory, being well represented by a single 386 pose, in which the nitro group interacts with the S1 pocket and the 1,4 naphthoquinone 387 interacts with the catalytic H41 and S2 subsite residues ( Figure 7B ). On the other hand, 388 the higher variability in the orientation of compound 382 led to four clusters with 389 frequency between 17.5 and 31.7% (Supporting Information Figure S48 ). Overall, the 390 1,4-naphthoquinone ring of ligand 415 occupies the S1 pocket, but fluctuations in the ring 391 orientation reflect on varied positions for the phenyl substituents. In the most populated 392 cluster ( Figure 7C ), the methoxyphenyl substituents occupy the S1ʹ and S2 subsites. 393

Compounds 382, and 415 display stable polar contacts (hydrogen bond and water 394 bridges) with G143 and S144 in the S1 pocket and π-cation or π-π interactions with the 395 sidechain of H41. These interactions were more frequent in the covalent simulations. The 396 ligands also display stable polar interactions with the main-chain nitrogen from E166 and 397 electrostatic contacts with its side-chain ( Figure 7D and 7E) , a residue that adopts a stable 398 conformation due to an interaction between its sidechain and the S1 from the other 399 protomer (S1*). Hydrophobic interactions to M49 and M165 from the S2 pocket, are 400 seldomly observed for these inhibitors and frequent interactions with the side-chain of 401 C145 was seen for the covalent inhibitor. conditions the hit naphthoquinone compounds were tested in three concentrations (24 517 µM, 6 µM, and 1.4 µM) and showed no significant antiviral activity dissociated from host 518 cytotoxicity (Supporting Information Figure S51) . We decided to test some compounds 519 in serial dilution starting at 1 µM and in infected human-derived HeLa cells expressing 520 ACE2 in addition to infected Vero cells. For HeLa-ACE2 cells, remdesivir was more 521 potent with EC50 of 40 nM, however, cell cytotoxicity was also noted at concentrations 522 above 2.4 µM. At lower concentrations the naphthoquinone compounds had no 523 significant antiviral activity up to 1 µM (Supporting Information Figure S52 ). Therefore, 524

it is important to further study the mechanism of action to understand the cytotoxicity and 525 decouple from the direct antiviral activity. 526 In this study, we used computational and biochemical approaches to evaluate a 530 library of quinones to find inhibitors against SARS-CoV-2 proteases. The wealth of 531 structural information from Mpro and PLpro allowed us to generate patterns of common 532 protein-ligand interactions, which were helpful in two stages of our computational 533 analysis. First, the selection of computational hits was guided by protein-ligand 534 interactions frequently observed in Mpro crystallographic complexes. Thus, we 535 prioritized compounds that interacted with conserved water molecules, S1 and S2 536 residues, filling one or more of the subsites with minimum solvent exposure. This strategy 537 was successful as, among 24 compounds selected for inhibitory assays, three molecules 538 with two different scaffolds were confirmed as Mpro inhibitors with low micromolar or 539 

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