Small-Molecule Thioesters as SARS-CoV-2 Main Protease Inhibitors: Enzyme Inhibition, Structure–Activity Relationships, Antiviral Activity, and X-ray Structure Determination

The main protease (Mpro, 3CLpro) of SARS-CoV-2 is an attractive target in coronaviruses because of its crucial involvement in viral replication and transcription. Here, we report on the design, synthesis, and structure–activity relationships of novel small-molecule thioesters as SARS-CoV-2 Mpro inhibitors. Compounds 3w and 3x exhibited excellent SARS-CoV-2 Mpro inhibition with kinac/Ki of 58,700 M–1 s–1 (Ki = 0.0141 μM) and 27,200 M–1 s–1 (Ki = 0.0332 μM), respectively. In Calu-3 and Vero76 cells, compounds 3h, 3i, 3l, 3r, 3v, 3w, and 3x displayed antiviral activity in the nanomolar range without host cell toxicity. Co-crystallization of 3w and 3af with SARS-CoV-2 Mpro was accomplished, and the X-ray structures showed covalent binding with the catalytic Cys145 residue of the protease. The potent SARS-CoV-2 Mpro inhibitors also inhibited the Mpro of other beta-coronaviruses, including SARS-CoV-1 and MERS-CoV, indicating that they might be useful to treat a broader range of coronaviral infections.


■ INTRODUCTION
The etiological agent of the current human coronavirus disease 2019 (COVID-19) is the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). It is a global health concern due to its ability to rapidly transmit from person to person and evade human immune surveillance. 1−4 As of 25 February 2022, SARS-CoV-2 has infected around 430 million individuals globally, resulting in approximately 6 million deaths. 5 The severe disease generally occurs in the elderly or those with pre-existing medical conditions. There are more than 20 different SARS-CoV-2 vaccines in use, all of which have been shown to be effective. However, because vaccine hesitancy persists globally and breakthrough infections are prevalent in vaccinated people, efficient antiviral therapeutics are required. Furthermore, SARS-CoV-2 is constantly evolving, and vaccination effectiveness will continue to diminish as mutations accumulate. 6,7 Because of their faster transmission capacity and higher mortality, 8,9 the B.1.617.2 (Delta) and B.1.1.529 (Omicron) variants have emerged as prominent in the current pandemic. 8−10 According to new findings, available COVID-19 immunizations are less effective against the Delta and Omicron variants, and persons who have been vaccinated are still at risk of infection. 11,12 The only direct-acting antivirals approved for emergency use are remdesivir and nirmatrelvir. 13−15 Remdesivir is an RNAdependent RNA polymerase (RdRp) inhibitor that is given intravenously to individuals with COVID-19 infection. 13,14 Nirmatrelvir is a peptidomimetic M pro inhibitor commercialized under the brand name Paxlovid in combination with ritonavir. 15 SARS-CoV-2 is one of many beta-coronaviruses in the Coronaviridae family, also including SARS-CoV-1 and Middle East respiratory syndrome coronavirus (MERS-CoV) that were responsible for the SARS and MERS epidemics in 2003 and 2012, respectively. 16−18 The RNA genome sequence of the SARS-CoV-2 virus offers an ideal starting point for drug discovery and the development of efficient COVID-19 treatments. 19,20 Coronaviruses are enveloped, single-stranded, nonsegmented positive-sense RNA viruses featuring the largest viral RNA genomes known to date. The genome of SARS-CoV-2 encodes two large polyproteins: pp1a (∼450 kDa) and pp1ab (∼750 kDa). These are extensively processed by two viral proteases, the papain-like protease (PL pro ) and the main protease (M pro , also called 3CL pro ), producing 16 nonstructural proteins (NSPs), including the RNA-dependent RNA polymerase (RdRp), that are involved in viral replication and transcription. 21,22 Due to the indispensable role of M pro in viral replication and its high conservation among related pathogenic coronaviruses such as MERS-CoV and SARS-CoV-1, it represents a promising target for the development of broad-spectrum antiviral therapy. 16,17,23,24 Moreover, M pro is unique in recognizing the substrate specificity and cleaves at the glutamine position. Because human proteases lack this property, it may be possible to develop selective protease inhibitors that are not toxic to humans. Only one human protease, kallikrein-3, has a comparable substrate specificity as M pro for cleavage on the Cterminal side of glutamine. Because kallikrein-3 is a serine protease expressed solely in the prostate gland, 25 inhibitors affecting the cysteine protease M pro are unlikely to target it, and if they do, inhibition will be restricted to the prostate gland.
So far, a variety of SARS-CoV-2 M pro inhibitors have been reported that can be classified into covalently and noncovalently acting agents. 26−31 The active-site M pro contains a catalytic dyad where Cys145 functions as a nucleophile and His41 serves as a catalytic general base. Covalent inhibitors, including small molecules and peptidomimetics, bear different warhead groups and act through a reaction with the active-site cysteine, producing a covalent bond with the inhibitor. 32 −36 In continuous efforts to develop SARS-CoV-1 and SARS-CoV-2 therapeutics, we and others have developed a wide variety of substrate-derived peptidomimetic and nonpeptidic smallmolecule inhibitors targeting the main protease. 23,24,28−38 In particular, a series of novel chloropyridyl ester derivatives (I and II, see Figure 1) displayed potent SARS-CoV-2 M pro inhibitory activities. 37,39 The positions of an ester moiety and other substituents on the indole ring affected SARS-CoV-2 M pro inhibitory potency and antiviral activity. 37,39 The indole carboxylate scaffold is thought to be important for binding to the M pro active site. The unique benzothiazolyl ketone derivative (III, see Figure 1), which was first reported as a SARS-CoV-1 M pro inhibitor, 24 also has a significant antiviral and SARS-CoV-2 M pro activity. 38   In the present study, a virtual screening of the Tubingen Kinase Inhibitor Collection (TuKIC), containing >10,000 proprietary kinase inhibitors, was performed. Based on the results from this screen, we report the design and synthesis of a novel class of thioesters and discuss their structure−activity relationships. The compounds were tested for inhibition at recombinant SARS-CoV-2 M pro employing a recently developed robust assay, 37 and enzyme inhibition kinetics were measured to elucidate the compounds' mechanism of inhibition. For potent inhibitors, antiviral activity was determined. Moreover, the cocrystallization of selected compounds with SARS-CoV-2 M pro was performed to obtain insights into their molecular interactions with the target. The SARS-CoV-2 M pro inhibitors were additionally tested against M pro from other pathogenic beta-coronaviruses, namely, SARS-CoV-1 and MERS-CoV, which they also inhibited with high potency.
■ RESULTS AND DISCUSSION Virtual Screening. The 3D structure of SARS-CoV-2 M pro in association with multiple inhibitors is accessible for in silico screening to discover novel lead structures. We virtually screened our in-house TuKIC library against M pro . This collection contains over 10,000 compounds that target a nucleophilic cysteine residue in a variety of kinases. Starting from the M pro 3D structure, 40 we employed a pipeline combining docking with short molecular dynamics (MD) simulations of 200 ns to yield 15 selected hit molecules, which were subsequently tested for their SARS-CoV-2 M pro inhibitory activity (Supporting information, Table S1).
All compounds were initially screened at 10 μM against SARS-CoV-2 M pro , and three compounds, inhibiting M pro by 50% or more, were chosen to determine concentrationdependent inhibition. Inhibitors IV and V showed moderate potency against M pro , while VI had a K i value of 6.5 μM ( Figure  2). Its proposed binding mode suggests that the imidazo [4,5d]pyrrolo [2,3-b]pyridine unit occupies the S2 pocket, displaying a stable Π−Π interaction with His41. The structure-dependent activity analysis of these three inhibitors suggests that the acrylonitrile warhead was slightly preferred for M pro inhibition. Additionally, these inhibitors are selective JAK-3 inhibitors with very high potencies. 41 The inhibition of JAK-3 is a promising approach for the treatment of COVID-19 as it reduces the inflammatory/cytokine storm that is one of the major factors for the organ damage that leads to death. 42 Indeed, many JAK inhibitors are already in advanced clinical trials for the treatment of COVID-19. 43 Therefore, inhibitors targeting the viral main protease and JAK would not only have direct antiviral effects but also beneficially suppress the overproduction of cytokines induced by viral infection.
Some disulfides including IPA-3 and JX-06 were identified as hit molecules with IC 50 values of 2.35 and 4.03 μM ( Figure 2). IPA-3 was reported as a non-ATP competitive, allosteric, and reversible covalent inhibitor of dysregulated p21-activated kinase 1 (Pak1), 44 which has been linked to oncogenesis. In previous studies by us and others, disulfiram was reported as a SARS-CoV-2 M pro inhibitor with IC 50 values ranging from 7 to 9 μM. 37,45 JX-06, a morpholine derivative, was discovered as a hit molecule with a lower IC 50 value than disulfiram. JX-06 was the first covalent 3-phosphoinositide-dependent protein kinase-1 (PDK1) inhibitor forming a reversible mixed disulfide with a conserved cysteine residue of the target. 46 We also discovered that a maleimide derivative of indole-2-carboxamide (LN5535) inhibited M pro with an IC 50 of 2.08 μM.
The active site of M pro is divided into four subsites (S1′, S1, S2, and S3), each of which accommodates four fragments (P1′, P1, P2, and P3) of peptidomimetic inhibitors. 23,24 However, particularly for irreversible small-molecule inhibitors, the efficacy is frequently determined by the strength of the warhead group even when the four sites are not fully occupied. 23,24 The co-crystal structure of M pro in a complex with an indole chloropyridyl ester derivative showed the acylation of the active site Cys145 to form a thioester intermediate with the indole moiety oriented toward the S2 pocket ( Figure 3a). 39 To design a novel class of inhibitors, we docked our two screening hits, IPA-3 and LN5535, at the Mpro active site (PDB ID: 7RC0) and observed that they share a similar orientation to the indole chloropyridyl ester derivative. Subsequently, we designed a novel class of thioesters by inserting variable cyclic systems from IPA-3 and the (hetero)aryl thiols from LN5535, as shown in Figure 3B. Subsequently, we designed a novel class of thioesters by inserting variable cyclic systems from IPA-3 and the (hetero)aryl thiols from LN5535, as shown in Figure 3B. We proposed that the designed new inhibitors may have a similar binding mode to that of the indole chloropyridyl ester derivatives. 39 A series of (hetero)aromatic thioesters were developed with different thiolate residues to be accommodated in the S1′ pocket. The electronic properties of (hetero)aromatic P1′ moieties may alter the reactivity of the thioester toward Cys145. As P2 fragments, a broad range of mono-cyclic or bicyclic rings with various physiochemical characteristics was investigated. This resulted in 40 novel thioesters that were evaluated against SARS-CoV-2 M pro .
Next, the combination of P2 and P1′ moieties was investigated (Scheme 2). The P2 indole was kept constant, as in our previous strategy. The thioesters 3w−z were obtained (48−84%) by reacting 1a or 1l with three pyrimidine-2-thiols (2k−m) under the same reaction conditions. Alternatively, pyrimidine-2-thiols were maintained and combined with various carboxylic acids (1i−r) to produce the products 3aa−ag, most  SARS-CoV-2 M pro Inhibition Assay. SARS-CoV-2 M pro was expressed and purified as previously described. 37 SARS-CoV-2 M pro activity assays were performed using a recently developed fluorogenic substrate (Boc-Abu-Tle-Leu-Gln-AMC), 38 and compounds were initially screened at a high concentration of 10 μM. For compounds showing M pro inhibition by at least 50%, full concentration−inhibition curves were determined with at least eight different inhibitor concentrations, and the respective product formation rates were observed during the first 10 min of the enzymatic reaction. IC 50 values were calculated by nonlinear regression analysis. For inhibitors that showed a time-dependent inhibition, the secondorder rate constant k inac /K i was determined by monitoring the effects of five different inhibitor concentrations on the product formation rate for 60 min (see Table 1).
Structure−Activity Relationships. The indole-2-thioesters with 4-chlorobenzene (3a) and 2,4-dichlorobenzene (3b) did not show any inhibition of SARS-CoV-2 M pro . Surprisingly, replacing the aryl ring with a heteroaromatic ring system such as 1,3,4-thiadiazole in 3c (IC 50 0.143 μM) resulted in potent  inhibitory activity against M pro . This inhibitor was kinetically characterized as an irreversible SARS-CoV-2 M pro inhibitor, and the second-order rate constant of inactivation, k inac /K i , was 10,700 M −1 s −1 (K i = 0.0744 μM) (3c, see Figure S1). 5-Methyl substitution of the 1,3,4-thiadiazole ring slightly reduced the inhibitor potency (3d, IC 50 0.624 μM). A positioning scan of the 1,3,4-thiadiazole thioester residue connected to the indole core was performed to study the chemical space of the indole thioesters. When compared to inhibitor 3d, which bears the thioester moiety at position 2 of the indole residue, all of the regioisomeric thioesters (3e−h) displayed an increased inhibitory activity against M pro , with the thioester moiety at position 6 being the most potent of all isomeric inhibitors (3g, IC 50 0.0547 μM). The k inac /K i for inhibitors 3d and 3e were not calculated due to considerable M pro reactivation within 1 h; for 3f (K i = 0.393 μM), 3g (K i = 0.186 μM), and 3h (K i = 0.250 μM), they are 3369, 5850, and 3960 M −1 s −1 , respectively. Methylation of the indole nitrogen resulted in a 2-fold increase in inhibitory activity (3i, IC 50 0.349 μM) compared to 3d, along with a k inac /K i value of 7170 M −1 s −1 (K i = 0.295 μM), demonstrating that the free NH is not necessary (Table 1).
The bioisosteric substitution of the indole moiety in 3d by a benzofuran ring resulted in a more potent inhibitor, 3p, with a k inac /K i value of 5190 M −1 s −1 (K i = 0.0357 μM). Next, we continued modifying the benzofuran ring of 3p. For example, the replacement of benzofuran by 5-bromofuran marginally reduced the affinity (3q), whereas 5-phenylfuran (3r, IC 50 0.0344 μM) and 5-(phenylethynyl)furan derivatives (3s, IC 50 0.250 μM) showed stronger inhibitory activity than 3p; in particular, compound 3r had a 14-fold higher potency. The k inac / K i values were not determined for 3q and 3r, but for 3s, it was found to be 5200 M −1 s −1 (K i = 0.0344 μM). The inhibitory activity of the trifluoromethyl analog of 3t dropped, demonstrating once again that a 5-CF 3 -1,3,4-thiadiazole thioester was not beneficial for M pro inhibition. The furan ring in 3r was exchanged for pyrrole, resulting in the inhibitor 3u, which had a lower potency but still showed inhibitory activity in the low micromolar range. The k inac /K i value for 3u is 5070 M −1 s −1 (K i = 0.605 μM). Potency was somewhat increased by replacing the indole ring in 3d by a naphthalene ring system. The resulting inhibitor, 3v, had an IC 50 value of 0.454 μM. The k inac /K i value for 3v is 6960 M −1 s −1 (K i = 0.0211 μM).
In the next attempt, we addressed the ring size of the thiolate portion. The replacement of 1,3,4-thiadiazole in 3d with pyrimidine led to the most potent inhibitor, 3w, of the present study. It has an IC 50 value of 0.0114 μM and a k inac /K i value of 58,700 M −1 s −1 (K i = 0.0141 μM) (3w, see Figure S4), making it a suitable scaffold for further development. The introduction of one methyl group into the 4-position of the pyrimidine ring marginally reduced inhibitory activity (3x, IC 50 0.0876 μM (k inac /K i = 27,200 M −1 s −1 , K i = 0.0332 μM), see Figure S5), while 4,6-dimethyl substitution decreased it significantly (3y, IC 50 2.03 μM). These findings imply that the thioester with an unsubstituted pyrimidine is the best leaving group for covalent M pro inhibition of the present series.
With these new warheads, we explored another part of the molecules. Initially, we incorporated a 6-ethoxy group into the indole, resulting in 3z that had no inhibitory activity. When indole was exchanged for 5-phenylfuran resulting in inhibitor 3aa, M pro inhibition was equipotent to that of 3w with an IC 50 value of 0.0181 μM, but noticeable M pro reactivation within 1 h was observed. Compound 3ab, in which 5-(phenylethynyl)furan was combined with a 6-dimethylpyrimidine thioester group, displayed stronger M pro inhibitory activity (IC 50 of 0.288 μM) than 3y; however, k inac /K i could not be calculated.
Finally, we installed a benzothiazole (in 3ah, K i = 0.0726 μM, see Figure S7) in place of a pyrimidine (in 3w), achieving an IC 50 of 0.117 μM along with a high k inac /K i value of 14,200 M −1 s −1 (K i = 0.0726 μM) (3ah, see Figure S7), indicating that this bicyclic thioester is also well suitable. Following this discovery, the M pro inhibitory activity of a number of bicyclic benzothiazole thioesters was studied. Inhibition was lost when indole was replaced with benzofuran. However, the introduction of 5- Inhibitors showed time-dependent inhibition. Progress curves in the presence of five different inhibitor concentrations were followed over 60 minutes and analyzed by nonlinear regression using the equation is the product concentration, v i is the initial rate, k obs is the observed first-order rate constant, and d is the offset. b The second-order rate constant, k inac /K i , was determined by plotting k obs versus [I] and nonlinear regression using the equation k obs = (k inac × [I])/([I] + K i × (1 + [S]/K m )). The deviation of each data point from the calculated nonlinear regression was less than 10%. c n.d.: not determined. d Compounds displayed time-dependent inhibition, but progress curves bent upward, which indicated that the inhibitor was covalently bound to the enzyme and was slowly released, hence restoring the enzyme activity. Consequently, the inhibitor concentration decreased over time due to protease-catalyzed cleavage. Such compounds were only characterized by their IC 50 values. e Evaluation was not possible as progress curves were linear. The compound might have inhibited the enzyme noncovalently as k obs did not increase with increasing inhibitor concentration. phenylfuran (3aj, IC 50 0.166 μM), 5-(4-Br-phenyl)furan (3ak, IC 50 0.255 μM), benzothiophene (3al, IC 50 0.190 μM), 5phenylthiophene (3am, IC 50 0.125 μM), or quinoline (3an, IC 50 0.089 μM) resulted in a similar inhibitory activity as compound 3ah, although none of these modified compounds outperformed 3ah. The concentration−inhibition curves for selected compounds are shown in Figure 4.
Cytotoxicity and Antiviral Activity against SARS-CoV-2. Cytotoxicity. Selected M pro inhibitors were further investigated for their antiviral activity in Calu-3 and Vero 76 cells. It is worth mentioning that the cytotoxicity of test compounds reduces cell virion production, which is commonly misunderstood as antiviral activity. Therefore, we first carefully checked the cytotoxicity of M pro inhibitors in both cell lines at a high concentration of 10 μM. As outlined in Figure S8, none of the tested compounds exerted cytotoxicity. Antiviral Activity. Based on the promising inhibitory activity of the thioesters against SARS-CoV-2 M pro , we evaluated the antiviral efficacy of selected potent inhibitors against SARS-CoV-2 in vitro. Calu-3 and Vero 76 cells were infected with SARS-CoV-2 to evaluate each of these M pro inhibitors. The Calu-3 cell line, which is derived from human lung, is the most extensively used cellular model for assessing antiviral efficacy against respiratory pathogens in vitro. Whereas in Calu-3 cells, SARS-CoV-2 enters the cells in a TMPRSS2-dependent manner, Vero cells barely express TMPRSS2 and viral entry is mediated by cathepsins. 47 Cells were incubated with 10 μM of each inhibitor 1 h prior to infection and 24 h post-infection (p.i.) at a multiplicity of infection (MOI) of 0.01. Viral titers of Calu-3 and Vero 76 cell culture supernatants were evaluated by titration on Vero E6 cells 24 h p.i. and are given as plaque-forming units (PFU) per milliliter (see Figure S9). As positive controls, we used remdesivir and I, an established SARS-M pro inhibitor. 37,39 In Figures S10 and S11, the viral titers of SARS-CoV-2 derived from Calu-3 or Vero 76 cells incubated with 10-fold serial dilutions (10−0.001 μM) of each inhibitor or DMSO (solvent control) 1 h prior and 24 h post infection (p.i.) are shown. Based on these results, dose-dependent inhibition curves were generated for all active inhibitors that exhibited EC 50 values ranging from 0.0383 to 4.05 μM in Calu-3 ( Figure 5, see Figure  S12 for compounds 3f, 3g, 3aa, 3ae, and 3af) and 0.00458 to 0.642 μM in Vero 76 cells ( Figure 6), respectively.
In Vero 76 cells, compounds 3c, 3i, and 3x were confirmed to act as anti-SARS-CoV-2 agents (see Figure S11). With an EC 50 value of 0.00458 μM, 3i was the most potent compound identified in this study. The antiviral activity of 3c was maintained in Vero 76 cells with an EC 50 of 0.641 μM, whereas the activity of 3x was higher when measured in Vero 76 cells as compared to Calu-3 cells, showing an EC 50 of 0.185 μM. Remdesivir and I, tested in the same system as control compounds, exhibited EC 50 values of 0.0026 and 0.00958 μM, respectively. All other compounds that showed activity as antiviral agents in Calu-3 cells (3f-h, 3l, 3r, 3v, 3w, 3aa, 3ae, 3af, and 3ah) did not display antiviral activity in Vero 76 cells at the highest test concentration of 10 μM. This might be attributed to the poor compound permeability in Vero cells or high expression of the efflux transporter P-glycoprotein; many recent reports have revealed such variances. 48−50 Concentration− response curves for 3c, 3i, 3x, remdesivir, and I in Vero76 cells are shown in Figure 6.
Broad-Spectrum Effect against SARS-CoV-1 M pro and MERS-CoV M pro . M pro sequences for SARS-CoV-2, SARS-CoV-1, and MERS-CoV are known to have a high similarity in the catalytic domain, which is responsible for protein cleavage. Due to the promising M pro inhibition and antiviral activity against SARS-CoV-2, we investigated the inhibitory activities of selected compounds on the main proteases of the human-pathogenic coronaviruses SARS-CoV-1 and MERS-CoV as well. Compounds were tested against recombinant SARS-CoV-1 M pro and MERS-CoV M pro at a high concentration of 10 μM. The classical fluorescence resonance transfer (FRET) assay was used. 51−53 Concentration-dependent inhibition curves for compounds that achieved at least 50% inhibition against the relevant M pro enzyme were determined to calculate IC 50 values (see Table 2 for IC 50 values and Figures S13 and S14 for concentration− inhibition curves). GC373 54 (see Table 2) was employed as a positive control for both M pro inhibitory activities.
At 10 μM, 3c, 3h, 3s, 3v, 3w, 3x, and 3af displayed more than 50% inhibition of SARS-CoV-1 M pro . Among all of the examined compounds, 3w demonstrated the highest inhibitory activity, with an IC 50 value of 0.0613 μM. 3af had submicromolar inhibitory activity (IC 50 0.253 μM), whereas 3c (IC 50   (IC 50 1.65 μM), and 3y (IC 50 1.52 μM) displayed inhibitory activities in the micromolar range. Compounds 3f, 3k, 3l, 3ae, and 3ah did not inhibit SARS-CoV M pro , although they did inhibit SARS-CoV-2 M pro . This is possibly due to the difference in shape and size between the two M pro enzymes. 55 Concentration−response curves for 3v (IC 50 Figure S13. We identified many compounds to inhibit the MERS-CoV M pro activity in the submicromolar to nanomolar range (see  Table 2). With an IC 50 value of 0.0792 μM, compound 3aa was discovered to be one of the most effective representatives. The rank order of potency at MERS-CoV M pro is as follows: MERS-CoV M pro was not inhibited by compounds 3ah and 3ae. Concentration−inhibition curves for 3c, 3e, 3f−i, 3k, 3l, 3s, 3v, 3w, 3x, 3aa, and 3af are shown in Figure S14.
Thus, these thioesters represent a novel class of broadspectrum inhibitors that target M pro of SARS-CoV-1, SARS-CoV-2, and MERS-CoV, with the highest potency against SARS-CoV-2 M pro followed by MERS-CoV M pro and SARS-CoV-1 M pro .
X-ray Structures of SARS-CoV-2 M pro Bound with Inhibitors. To understand the binding interactions of the inhibitors with the protease, co-crystal structures of SARS-CoV-2 M pro in complex with the broad-spectrum M pro inhibitors 3w and 3af were determined at a resolution of 2.3, and 1.5 Å, respectively ( Figure 7). Table S2 summarizes the X-ray data and refinement statistics for both complexes. Figure 7 shows that the protease-catalyzed cleavage of the inhibitors produced a thioester-type enzyme−inhibitor complex. The remaining fragment of the compounds was covalently bound to the catalytic Cys145, while the pyrimidine thiolate acted as a leaving group and departed from the active site. The indole ring of compound 3w and the quinoline ring of 3af mainly formed hydrophobic interactions with surrounding residues including His41, Met165, Asp187, Arg188, and Gln189 ( Figure 7A,B). The catalytic His41 together with Met165 functioned as a clamp to catch the aromatic indole and quinoline ring of the inhibitors. In the complex structure of 3w with SARS-CoV-2 M pro , Met49 also contributed to the hydrophobic interactions with the indole ring, while this interaction was absent in the complex structure of 3af due to the unclear electron density was missing in the loop where Met49 is located. The superposition of the two complex structures revealed that the quinoline of 3af was shifted toward His41 compared to the position of the indole ring of 3w, leading to a cooperative movement of His41 and of Met165. Such a movement of the quinoline ring might sterically clash with Met49 and thus cause a disorder of the loop containing Met49. Overall, the co-crystal structures provide a mechanistic insight into the inhibition of SARS-CoV-2 M pro by two pyrimidine thioesters in which the catalytic Cys145 is acylated by the indolecarbonyl or quinolone-carbonyl group, which are well positioned in a relatively hydrophobic binding pocket.
Molecular Modeling of 3w and 3af with SARS-1-CoV M pro and MERS-CoV M pro . Docking studies of compounds 3w and 3af into M pro of SARS-CoV-1 and MERS-CoV followed by short molecular dynamics (MD) simulations (5 × 200 ns) reveal a stable π−π interaction with His41 (31−58% of the analyzed simulation time) for both proteins and a specific hydrogen bond interaction between MERS-CoV M pro -Q167 and the nitrogen of the indole ring for 3w, pointing toward the solvent. Several polar contacts with the S2 pocket support the binding mode: M49, M165, and H41 (numbering of SARS-CoV-1 M pro ). By comparison, 3af stabilizes a water molecule within the site, with its extra H-bond acceptor group. It is important to highlight that during our simulations, a classical force-field model was adopted that disregards potential changes in the ionization state of side chains. This is especially relevant for His41 that could further display a π-charge interaction with either the indole or the quinoline ring, yielding a more stable interaction (see Figure  S15).
Glutathione Stability. We tested the stability of selected potent M pro thioester inhibitors against excess glutathione (GSH) as a physiological nucleophile to identify the inherent activity toward thiols under physiological conditions. These experiments revealed that inhibitors are stable with low inherent activity under the assay conditions (5 mM GSH at pH 7.4) (see a representative example, 3an, in Figure S16). This indicates that the M pro inhibition of thioesters is dependent not on their chemical reactivity toward GSH but rather on the specific reactivity toward catalytic Cys145 at the active site of M pro , as shown by the X-ray structures of SARS-CoV-2 M pro bound with inhibitors in Figure 7.

■ CONCLUSIONS
The M pro of SARS-CoV-2 is an important target since it is involved in the coronavirus life cycle. We developed, synthesized, and tested novel low-molecular-weight thioesters that act as inhibitors of M pro of SARS-CoV-2, the virus that is causing the current COVID-19 pandemic. A number of compounds showed low nanomolar M pro inhibitory activity with an irreversible inhibition mechanism. The co-crystal structure of M pro with 3a and 3af, two of the most potent inhibitors, validated the proposed binding mechanism. In cellbased experiments, several compounds exhibited excellent antiviral activity. The SARS-CoV-2 M pro inhibitors were additionally shown to inhibit M pro of the related pathogenic coronaviruses SARS-CoV-1 and MERS-CoV, indicating that they are potential candidates for further development as broadspectrum anti-coronaviral agents.
■ EXPERIMENTAL SECTION General Experimental Procedures. All commercially available reagents and solvents were used as received. Reactions sensitive to air or moisture were performed under an atmosphere of argon and/or in anhydrous solvents. Anhydrous solvents were purchased from Acros Organics (AcroSeal). Unless stated otherwise, extracts were dried over sodium sulfate prior to filtration. Thin-layer chromatography was performed on TLC Silica Gel 60F254 aluminum sheets provided by Merck, with detection at 254 and 366 nm. Flash chromatography was carried out on an Interchim PuriFlash XS420 flash chromatography system and Grace Davison Davisil LC60A 20e45 mm silica or Merck Geduran Si60 63−200 μm. Mass spectra were recorded on an Advion DCMS interface (ESI voltage: 3.50 kV, capillary voltage: 187 V, source voltage: 44 V, capillary temperature: 250°C, desolvation gas temperature: 250°C, gas flow rate: 5 L/min N 2 ), with elution of the spots with MeOH. High-resolution mass spectrometry (HRMS) for the final compound was measured by the mass spectrometry department, Institute of Organic Chemistry, Eberhard Karls University Tuebingen, on a Bruker maXis 4G ESI-TOF instrument. The instrument was run in ESI+ mode, and the settings were as follows: nebulizer gas of 1.2 bar, gas flow of 6.0 L/min, source temperature of 200°C, capillary voltage of 4500 V, endplate offset of −500 V, and m/z range from 80 to 1000. NMR spectra were measured on Bruker Avance 400 or 600 NMR spectrometers. The spectra were calibrated on the deuterated solvents, and chemical shifts (d) are stated relative to tetramethylsilane in ppm. The solvent used was deuterated chloroform (d 3 ) and deuterated DMSO (d 6 ). Melting points were measured on a melting point apparatus (Mettler Toledo MP70, hand method, temperature from 60 to 360°C, heating rate 20.0°C/min) and are uncorrected.
Synthesis of Thioesters 3a−z and 3aa−an. POCl 3 (1.37 mmol, 1.1 equiv) was added to a solution of pyridine (1.37 mmol, 1.1 equiv) and corresponding carboxylic acids (1a−s, 1.25 mmol, 1.0 equiv) in dry DCM (10 mL), and the solution was stirred for 10−15 min at room temperature to generate carboxylic acid chlorides in situ. The appropriate thiols (2a−n) were added first followed by pyridine (2.0 mmol, 1.6 equiv). TLC with UV detection was used to monitor the   reaction. The mixture was put into ice water and extracted with DCM (2 × 25 mL) when the reaction was completed after 12 h. After washing with 1 N HCl (20 mL) and 30−40% NaOH solutions, the combined organic layers were brined (20 mL). The solution was then dried over Na 2 SO 4 , filtered, and evaporated until it was completely dry. The resulting residue was purified by silica gel chromatography using 10− 20% EtOAc in hexane to afford the desired products.
The SARS-CoV-2 M pro protein structure was previously prepared from the PDB ID: 5R82 40,56 using the Protein Wizard Preparation tool, with standard options, and MD simulations for equilibration as described. Molecular docking was carried out with Glide SP (version 9.1). 57 Grids were centered at the central point of the active site residues G143, C145, M49, and H41. The box was 12 Å long in each direction. The top 500 hits were visually inspected by observing the interaction with relevant residues within P1 and P2. Whenever mentioned, covalent docking as performed using CovDock 58 using the C145 as the anchor and nucleophilic addition to the double bond as the reaction type and generating up to 10 poses for each ligand. Poses were selected according to the docking score and relevant interactions. Selected poses underwent short MD simulations (200 ns, data not shown). Ligands that remained within the pocket during the simulated time were further selected for experimental testing.
Potential Binding Mode of Compounds in MERS-CoV and SARS-CoV-1 Main Proteases. The ligands 3w and 3af were covalently docked to the MERS and SARS-CoV-1 M pro models to study their binding mode. 3D structural models M pro from MERS (PDB ID: 4YLU), SARS-Cov-1 (7LMJ), and SARS-CoV-2 were generated, filling the missing residues in the C-terminal portion, using Prime. 59,60 The generated models then underwent preparation using PrepWizard (Maestro 2021.4).
MD Simulations and Trajectory Analyses. The minimized structures were submitted to MD simulation for further refinement. Selected docking poses were further validated by MD simulation, where ligand stability within the proposed pocket and its interactions were evaluated. MD simulations were carried out using the Desmond engine 61 with the OPLS4 force-field. 62 The simulated system encompassed the protein−ligand complex, a predefined water model (TIP3P) 63 as a solvent, and counterions (Na + or Cl − adjusted to neutralize the overall system charge). The system was treated in a cubic box with periodic boundary conditions specifying the shape and the size of the box as 13 Å distance from the box edges to any atom of the protein. RESPA integrator time steps of 2 fs for bonded and near and 6 fs for far were applied. Short-range Coulombic interactions were performed using a time step of 1 fs and a cutoff value of 9.0 Å, whereas long-range Coulombic interactions were handled using the Smooth Particle Mesh Ewald (PME) method. 64 Desmond Relaxation Protocol. Simulations were run in NPT ensemble, with a temperature of 310 K (Nose−Hoover thermostat) and pressure of 1.01325 bar (Martyna−Tobias−Klein barostat).
Results of simulations, in the form of trajectory and interaction data and RMSD and RMSF values, are available on the Zenodo repository (code: 10.5281/zenodo.6303511). MD trajectories were visualized, and figures were produced using PyMOl v.2.5 (Schrodinger LCC, New York, NY, USA). For each ligand, simulations at least five independent 200 ns replicas were carried out. Protein−ligand interactions were determined using the Simulation Event Analysis pipeline implemented in Maestro (Maestro v2021.4) with standard settings.
SARS-CoV-2 M pro Assay. The SARS-CoV-2 main protease (M pro ) was expressed and purified according to a previously published procedure. 37 In brief, a codon-optimized cDNA sequence encoding the M pro enzyme (accession no.: MN908947.3, ORF1ab polyprotein residues 3264−3569) with an N-terminal M pro autocleavage site and a C-terminal His 10 tag linked via an HRV 3C protease cleavage site was inserted into the bacterial expression vector pGEX-6P-1. After transformation of BL21 Escherichia coli bacteria (NEB, Ipswich, USA), cells were grown in an LB medium supplemented with ampicillin (100 μg/mL). Subsequently, IPTG (final concentration 1 mM) was added to induce recombinant gene expression until an OD 600 of 0.5 was reached. After growing the bacteria at 30°C for 3−4 h, cells were sedimented at 4000g and 4°C for 10 min. Cells were then resuspended in cold PBS supplemented with 5 mM imidazole (pH 7.4). Subsequently, the cells were lyzed on ice using a Sonoplus HD2070 (Bandelin, Berlin, Germany) and centrifuged at 48,000g and 4°C for 30 min in collect the supernatant. The His-tagged M pro enzyme was purified using HisPur Ni-NTA spin columns (Thermo Fisher Scientific, Waltham, USA). For the native M pro enzyme, the His tag was removed by an HRV 3C protease (Merck, Darmstadt, Germany) overnight at 4°C . Subsequent disposal of the GST-tagged HRV 3C protease was performed by a Glutathione Spin Column (Thermo Fisher Scientific, Waltham, USA). The collected flow through contained the native M pro enzyme that was subsequently used for pharmacological assays. For the initial inhibition experiments at a single concentration, crude cell extracts containing the M pro enzyme were used. For the subsequent pharmacological characterization of hit compounds to determine IC 50 , K i , and k inac /K i values, we employed the purified His-tagged M pro .
SARS-CoV-2 M pro activity assays were performed as previously established. 37 Frozen recombinant His-tagged SARS-CoV-2 M pro was thawed and immediately used for the enzyme assays that were performed on a Pherastar FSX plate reader (BMG Labtech, Offenburg, Germany) at 37°C with an excitation wavelength of 360 nm and an emission wavelength of 460 nm. Black half area 96-well plates with a flat bottom were obtained from Greiner Bio-One (Kremsmunster, Austria). The assay buffer contained 50 mM 3-(N-morpholino)propanesulfonic acid (MOPS, pH 7.2), 10 mM NaCl, 1 mM EDTA, and 0.01% Triton X-100. The fluorogenic substrate Boc-Abu-Tle-Leu-Gln-AMC 37 diluted in the assay buffer was added to the test compounds, and the mixture was preincubated at 37°C for 5 min. The proteolytic cleavage of the substrate was started by addition of the enzyme (e.g., 400 ng Histagged M pro diluted in the assay buffer), and the fluorescence increase was followed for 10 or 60 min, respectively. The final protein concentration of SARS-CoV-2 M pro was adjusted to a slope increase of approximately 2000/min within the initial 10 min. The final substrate concentration was 50 μM, which approximately corresponds to its K m value. 37 The final DMSO concentration was 4%. The product formation rate of the uninhibited control was set at 100%. For the determination of concentration−inhibition curves, at least eight different inhibitor concentrations were investigated to observe the respective product formation rates during the first 10 min. IC 50 values were calculated by nonlinear regression. For inhibitors showing timedependent inhibition, the second-order rate constant k inac /K i was determined by monitoring the effects of five different inhibitor concentrations on the product formation rate for 60 min, and the data were analyzed by nonlinear regression using the equation is the product concentration, v i is the initial rate, k obs is the observed first-order rate constant, and d is the offset. Subsequently, k obs was plotted versus the inhibitor concentration [I], and a nonlinear regression using the equation k obs = (k inac × [I])/([I] + K i × (1 + [S]/K m )) was performed. The deviation of each data point from the calculated nonlinear regression was less than 10%. Data were analyzed using GraphPad Prism 8.0.
Assay against Recombinant SARS-CoV-1 M pro and MERS-CoV M pro . Recombinant SARS-CoV-1 Mpro and MERS-CoV Mpro were purchased from R&D Systems. Proteolytic activity was determined using 10 μM MCA-AVLQSGFR-K(DNP)-K-NH 2 for SARS-CoV-1 Mpro and 10 μM Ac-Abu-Tle-Leu-Gln-ACC for MERS-CoV Mpro. Fluorescence was monitored in a Synergy HTX (Biotek) plate reader using excitation/emission wavelengths of 320/400 nm for MCA-AVLQSGFR-K(DNP)-K-NH 2 and 360/460 nm for Ac-Abu-Tle-Leu-Gln-ACC. All assays were performed in 384-well black plates with a 15 min preincubation of the compounds with the enzyme. The screen was performed with 10 μM of inhibitors. Compounds that inhibited enzyme activity by 50% or more in the initial screen had their dose−response curve determined. For each compound, two independent experiments were performed, each in triplicate and monitored for 2 h. The percentage of inhibition was calculated by comparison to a DMSO control. The protease inhibitor GC373 was used as a positive control. Assays were performed at 25°C in a final volume of 30 μL per well of 50 mM HEPES (pH 7.5), 150 mM sodium chloride, 1 mM EDTA, and 0.01% Tween 20 in the presence of 50 nM enzyme and 10 μM substrate. 51,52,65 The half-maximal inhibitory concentration (IC 50 ) was determined by nonlinear regression analysis of the velocity vs inhibitor concentration plot using GraphPad Prism 6. 66 Eleven inhibitor concentrations were used to generate each curve.
All cell lines were incubated at 37°C and 5% CO 2 in a humidified atmosphere.
Cell Vitality Assay. To determine cell vitality of Calu-3 cells treated with inhibitors, the CellTiter-Glo Luminescent Cell Viability Assay Kit (Promega) was used. Cells were grown in 96-well plates until reaching 50−60% confluency before they were incubated with DMSO (solvent control) or M pro inhibitors at a concentration of 10 μM for 24 h. Next, cell culture supernatants were removed, and 50 μL of the CellTiter-Glo substrate was added to each well and incubated for 30 min on a rocking platform. Finally, samples were transferred into white 96-well plates, and luminescence was measured using a Hidex Sense plate luminometer (Hidex). Antiviral Activity. All work with infectious SARS-CoV-2 was conducted under BSL-3 conditions at the German Primate Centre, Goẗtingen/Germany. Calu-3 cells were grown in 48-well plates until reaching approx. 70% confluency. Cells were incubated with 10-fold serial dilutions (10−0.001 μM) of M pro inhibitors for 1 h at 37°C prior infection. Next, the inhibitor containing the cell culture medium was removed, and cells were infected with SARS-CoV-2 isolate NK, Pango lineage B.1.513, at an MOI of 0.01 in an inoculum volume of 400 μL for 1 h at 37°C. At 1 h post infection (p.i.), the inoculum was removed, and cells were washed with PBS three times and further incubated in a cell culture medium containing the respective inhibitor for 24 h. Viruscontaining supernatants were harvested and stored at −80°C until further usage.
To determine viral titers, confluent grown Vero E6 cells were inoculated for 1 h at 37°C with 10-fold serial dilutions of viruscontaining supernatants. Next, the inoculum was removed, and cells were washed once with PBS before they were overlaid with 1% plaque agarose (Biozym) dissolved in Eagle's minimal essential medium without phenol red (Lonza) and further incubated. At 48 h p.i., virusinduced plaques were counted, and viral titers were determined as plaque forming units (PFU)/mL. Crystallography Procedure. The cDNA of SARS-CoV-2 M pro (GenBank: MN908947.3) with the N-terminal SUMO tag was cloned into the pET-15b vector. The plasmid was transformed into BL21 (DE3) cells for protein expression. The expressed protein was purified by a Ni-NTA column (GE Healthcare) and cleaved by the SUMO specific peptidase 2 (SENP2) to remove the SUMO tag. The resulting protein sample was further purified by Q-Sepharose followed by a sizeexclusion chromatography (GE Healthcare). The eluted protein samples were stored in a solution (10 mM Tris, pH 7.5).
The purified protease was concentrated to 7 mg/mL for crystallization. To obtain complex structures, the protein was incubated with 4 mM 3w and 3af for 1 h before crystallization condition screening. Crystals of the complexes were obtained under the condition of 10− 22% PEG6000, 100 mM MES, pH 5.75−6.25, and 3% DMSO. Crystals were flash frozen in liquid nitrogen in the presence of the reservoir solution supplemented with 20% glycerol. X-ray diffraction data were collected at beamline BL02U1 at the Shanghai Synchrotron Radiation Facility. 67 The data were processed with HKL3000 software packages. 68 The complex structures were solved by molecular replacement using the program PHASER 69 with a search model of PDB code 6M2N. 67 The model was built using Coot 68 and refined with XYZ (reciprocal-space), individual B factors, TLS parameters, and occupancies implemented in the program PHENIX. 69 The refined structures were deposited to the Protein Data Bank with accession codes listed in Table S2 in the Supporting Information. The complete statistics as well as the quality of the solved structures are also shown in Table S2.
Glutathione (GSH) Stability Assay. The GSH stability assay for compounds was performed according to the reported procedures. 70,71 A suitable HPLC/MS method was prepared, and the HPLC method was adjusted to inject a sample every 10 min. To achieve a final concentration of 10 μM inhibitor and 5 mM GSH in a final volume of 1 mL, 1 μL of the inhibitor stock solution (10 mM) was added to 949 μL of the phosphate buffer (pH 7.4) as well as 50 μL of the stock solution (100 mM) GSH. The solution was directly pipetted into a HPLC vial and vortexed thoroughly. The temperature of the autosampler was set to 30°C. Ten to 30 repeated measurements were performed. The reaction of the inhibitor with GSH was monitored by measuring the decreasing area under the curve (AUC) of each compound.