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. 2023 Aug 17;14(10):2068–2078. doi: 10.1039/d3md00306j

Identification of novel 1,2,3-triazole isatin derivatives as potent SARS-CoV-2 3CLpro inhibitors via click-chemistry-based rapid screening†

Xiangyi Jiang a, Jing Li a, Antonio Viayna b,c, F Javier Luque b,c,d, Molly Woodson e,f, Lanlan Jing a, Shenghua Gao a, Fabao Zhao a, Minghui Xie a, Karoly Toth e,f, John Tavis e,f, Ann E Tollefson e,f,✉, Xinyong Liu a,✉, Peng Zhan a,✉
PMCID: PMC10583828  PMID: 37859715

Abstract

SARS-CoV-2 3-chymotrypsin-like protease (3CLpro) is considered an attractive target for the development of anti-COVID-19 agents due to its vital function. The N-substituted isatin derivative L-26 is a potential SARS-CoV-2 3CLpro inhibitor, but it has poor cell-based antiviral activity and high cytotoxicity. With L-26 as the lead compound, 58 isatin derivatives were prepared using click-chemistry-based miniaturized synthesis and their 3CLpro inhibitory activities were determined by a fluorescence resonance energy transfer-based enzymatic assay. Compounds D1N8 (IC50 = 0.44 ± 0.12 μM) and D1N52 (IC50 = 0.53 ± 0.21 μM) displayed excellent inhibitory potency against SARS-CoV-2 3CLpro, being equivalent to that of L-26 (IC50 = 0.30 ± 0.14 μM). In addition, the cytotoxicity of D1N8 (CC50 >20 μM) and D1N52 (CC50 >20 μM) decreased significantly compared with L-26 (CC50 <2.6 μM). Further molecular dynamics simulations revealed the potential binding interactions between D1N52 and SARS-CoV-2 3CLpro. These efforts lay a solid foundation for the research of novel anti-SARS-CoV-2 agents targeting 3CLpro.

SARS-CoV-2 3-chymotrypsin-like protease (3CLpro) is considered an attractive target for the development of anti-COVID-19 agents due to its vital function.graphic file with name d3md00306j-ga.jpg

1. Introduction

The coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), remains the most serious global infectious disease at this stage.1 As a single-stranded RNA virus, the extreme mutagenicity of SARS-CoV-2 results in dramatically reduced preventive efficacy of existing vaccines.2–4 Worse yet, the effective antiviral agents used to treat COVID-19 are fairly limited.5–7 Therefore, there is an urgent need to develop potent anti-SARS-CoV-2 drugs to alleviate patient symptoms and mortality. 3-Chymotrypsin-like protease (3CLpro) is a main protease (Mpro) that cleaves viral polyproteins into crucial non-structural proteins in cooperation with papain-like protease (PLpro).8 3CLpro provides an indispensable role in viral replication, and it is considered as one of the most preferred targets for the development of broad-spectrum anti-SARS-CoV-2 agents due to its high conservation and lack of human homologs.9,10

To date, a variety of chemotypes have been identified as SARS-CoV-2 3CLpro inhibitors and can be structurally divided into peptide-like and non-peptide molecules (Fig. 1).11 Peptidomimetics consist of a peptide-based scaffold mimicking natural substrates and a covalent warhead interacting with Cys145, such as nirmatrelvir (1), FB2001 (2), PF-00835231 (3), PF-07304814 (4) and GC-376 (5).9,12,13 However, the undesirable pharmacokinetic (PK) properties caused by the peptide-based scaffold severely limit their clinical applications. For example, nirmatrelvir, approved by the U.S. FDA, needs to be used in combination with ritonavir (cytochrome P450 enzymatic inhibitor) to improve metabolic stability in vivo.7 Consequently, some potential non-peptide 3CLpro inhibitors have attracted much attention, such as L-26 (6), S-217622 (7), AL-19 (8), GC-14 (9) and CCF981 (10).8,14–17 Among these, the N-substituted isatin derivative L-26 is a promising non-covalent 3CLpro inhibitor with an IC50 value of 0.045 μM.14 However, the relatively high cytotoxicity interfered with the quantitative determination of the antiviral activity against SARS-CoV-2, indicating that further structural optimization of L-26 is necessary.

Fig. 1. Representative peptide-like and non-peptide SARS-CoV-2 3CLpro inhibitors.

Fig. 1

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Unlike previous studies, the click-chemistry-based rapid screening technique was first applied to the identification of potent SARS-CoV-2 3CLpro inhibitors in this work. “Click chemistry” is an efficacious fragment-based assembly reaction for the construction of high-quality compound libraries owing to its high yields, remarkable selectivity, gentle reaction conditions and exceptional functional group compatibility.18 In particular, copper(i)-catalyzed azide–alkyne cycloaddition (CuAAC), the most generic click chemical reaction, can rapidly prepare 1,2,3-triazole-derived compound libraries.19 It is widely known that 1,2,3-triazole is a preferred fragment in drug design with unique properties such as hydrogen bonding capability, positive solubility, rigidity and stability in vivo.20 The previous structure–activity relationships (SARs) indicated that the amide group at the 5-position of isatin is the preferred pharmacophore for maintaining activity against SARS-CoV-2 3CLpro, while the N-substituted part could well accommodate various groups.14 Based on this, methylene alkynyl was introduced into the 1-position of 2,3-dioxoindoline-5-carboxamide to prepare the privileged alkynyl fragment, which was reacted with azide substituents in 96-well plates using CuAAC click chemistry (Fig. 2). A total of 58 triazole-containing isatin derivatives were obtained by miniaturized synthesis, and then were directly tested for inhibitory potency against SARS-CoV-2 3CLpro without purification. Finally, compounds D1N8 and D1N52 were identified as potent SARS-CoV-2 3CLpro inhibitors by further biological evaluation in vitro and molecular dynamics (MD) simulation.

Fig. 2. Design of novel 1,2,3-triazole isatin SARS-CoV-2 3CLpro inhibitors via click-chemistry-based rapid screening.

Fig. 2

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2. Results and discussion

2.1. Generation of combinatorial library by click chemistry

First, we synthesized the alkynyl fragment and a total of 58 azide substituents (Fig. 3). As shown in Scheme 1, the commercially available 2,3-dioxoindoline-5-carboxamide (11) was treated with 3-bromopropyne in the presence of cesium carbonate to prepare the privileged alkynyl fragment (D1).21 The synthesis of azide substituents N1–N58 is described at length in our previous work.22

Fig. 3. Structures of various azide substituents used in this study.

Fig. 3

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Scheme 1. Synthetic route of D1.

Scheme 1

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A 58-member isatin derivative library was built in microtiter plates via click-chemistry-based miniaturized synthesis. The various azide substituents N1–N58 (1.4 eq) were dissolved in dimethyl sulfoxide, and alkynyl fragment D1 (1.0 eq) was added, followed by tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA, 0.2 eq), CuSO4·5H2O (0.2 eq) and sodium ascorbate (5.0 eq). The minute reaction conditions are shown in Table 1. The reactions were stirred on a constant temperature oscillation incubator at room temperature for 24 h, and then were monitored on thin-layer chromatography (TLC) using iodine vapor and UV light (λ = 254, 365 nm).

The detailed reaction conditions.

Reagents Concentration Volume Final concentration
Azide-unit 35 mM DMSO 20 μL 7 mM
Alkyne-unit 25 mM DMSO 20 μL 5 mM
TBTA 10 mM DMSO 10 μL 20 mol%
CuSO4·5H2O 4 mM MilliQ 25 μL 20 mol%
Sodium ascorbate 20 mM MilliQ 25 μL 100 mol%
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2.2. In situ screening against SARS-CoV-2 3CLpro

The prepared compounds in the library were tested for their inhibitory potency against SARS-CoV-2 3CLpro using a fluorescence resonance energy transfer (FRET)-based enzymatic assay in order to identify potent 3CLpro inhibitors quickly, along with lead compound L-26 as the positive control. The biological results of tested compounds were expressed as inhibition percentage at a concentration of 1 μM. As shown in Fig. 4, the target compounds showed weak to excellent inhibitory potency against SARS-CoV-2 3CLpro, indicating that the various azide substituents have an obvious impact on the activity. Notably, compounds D1N8 (76.41%), D1N18 (73.34%) and D1N52 (78.12%) turned out to be the most active inhibitors, which were equivalent to lead compound L-26 (87.30%). Subsequently, they were synthesized (Scheme 2) on a milligram scale to quantitatively measure the 3CLpro inhibitory activity.

Fig. 4. SARS-CoV-2 3CLpro inhibitory activity in the presence of 1.0 μM D1NX. The blank is a reaction buffer without the azide substituent and alkynyl fragment.

Fig. 4

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Scheme 2. Synthesis of D1N8, D1N18 and D1N52.

Scheme 2

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2.3. Biological evaluation

Inhibitory activity towards SARS-CoV-2 3CLpro under gradient concentrations (0.1 μM, 0.5 μM, 1.0 μM, 5.0 μM) was determined for the newly prepared compounds D1N8, D1N18, and D1N52 and the lead compound L-26. The 50% inhibition concentrations (IC50) of the compounds are outlined in Table 2. Compounds D1N8 bearing 3-chlorobenzyl and D1N52 bearing 2-fluoro-4-(trifluoromethyl)benzyl showed outstanding anti-3CLpro potency with IC50 values of 0.44 ± 0.12 and 0.53 ± 0.21 μM, respectively, which are similar to that of L-26 (IC50 = 0.30 ± 0.14 μM), while D1N18 (IC50 = 1.12 ± 0.54 μM) containing 4-sulfamoylbenzyl is slightly weaker than the other compounds.

Inhibitory activity against SARS-CoV-2 3CLpro.

Compounds D1N8 D1N18 D1N52 L-26
IC50a (μM) 0.44 ± 0.12 1.12 ± 0.54 0.53 ± 0.21 0.30 ± 0.14
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a

IC50: 50% concentration required to inhibit SARS-CoV-2 3CLpro. The numbers in the table are the average of three independent tests.

Subsequently, the three compounds and L-26 were further evaluated for their anti-SARS-CoV-2 activity and cytotoxicity in Vero E6 cells. As shown in Table S1,† the tested compounds did not display significant anti-SARS-CoV-2 activity under the concentration of 20 μM, as well as lead L-26. Still, the three compounds (CC50 >20 μM) displayed remarkable reduction in cytotoxicity compared with L-26 (CC50 <2.2 μM). This study also confirms the conclusion that the drug discovery process is full of challenges and uncertainties, therefore a new round of iterative optimization is necessary.23

2.4. Molecular dynamics (MD) simulation studies

Docking of compound D1N52 led to similar poses in the binding pocket of both 7EN8 and 7V1T, where the isatin ring of D1N52 partially overlaps the 2-(methylcarbamoyl)-4-bromanyl-6-nitro-phenyl moiety of the ligand bound to 3CLpro in PDB ID 7EN8, and the 2-fluoro-4-trifluromethyl-benzene moiety is close to the hydrophobic area shaped by the methyl group of Thr25 and the side chain of Leu27. Compared with the docking performed using 7M8P as the template, the main difference is the orientation of the 2-fluoro-4-trifluromethyl-benzene unit, which lies close to Thr190 and Ala191 due to the enlarged cavity generated by the shift of the Asp187-Ala193 region in 7M8P (Fig. S1†).

Molecular dynamics was used to examine the stability of the ligand (D1N52) pose (Fig. 5A). The root-mean square deviation of the protein backbone was stable along the trajectory, as noted in an average value of 1.9 Å. Regarding the ligand, a structural change occurred after release of the positional restraints used to avoid artefactual changes in the binding pose during the equilibration of the system (at around 25 ns; see section 4.5). Then, D1N52 adopted a stable arrangement with a slight adjustment at around 130 ns, leading to a stable pose till the end of the MD simulation (Fig. 5B).

Fig. 5. A) Time (ns) evolution of the positional root-mean square deviation (RMSD; Å) of the protein backbone (black) and ligand (D1N52; red) bound to the catalytic pocket of 3CLpro. The profile for the protein backbone was determined by excluding the atoms pertaining to the N-termini (residues 1–8) and loops 188–208 and 280–290 (numbering in the PDB structure 7EN8). The RMSD was determined relative to the X-ray energy-minimized structure. B) Representation of the binding mode of D1N52 in the catalytic pocket of 3CLpro (shown as gray surface). The ligands sampled every 5 ns along the last 50 ns of the unrestrained MD simulation are shown as sticks (carbon atoms colored in green). Selected residues in the binding pocket are highlighted as sticks, and Cys44 and Cys144 are shown as spheres (carbon atoms colored in yellow).

Fig. 5

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The binding mode of D1N52 involved polar contacts between the isatin-bound amido group and the C Created by potrace 1.16, written by Peter Selinger 2001-2019 O and NH groups of Gln192, and between the amido NH2 unit of Asn119 and the trifluoromethyl group of the terminal benzyl unit, as well as hydrophobic contacts between the isatin ring and the side chains of Met165 and Leu167, the benzyl ring and the side chains of Thr25 and Leu27, and the carbon atoms of the triazole ring, which stacks against the imidazole ring of His41, are located close to the side chain of Met49. It can be noticed that the fluorine atom bound to the 4-trifluoromethylbenzene ring is oriented toward Cys44 or Cys144. The adoption of the two orientations is presumably facilitated by the lack of direct, specific interactions formed by the nitrogen atoms of the triazole ring in the binding pocket. In this regard, the presence of the 2-fluoro-4-(trifluoromethyl)benzyl unit is relevant to counterbalance the polarity of the triazole ring, as noted in a log P value of 2.5 estimated from QM (IEF-PCM/MST) continuum solvation calculations for the corresponding benzylated triazole fragment obtained upon exclusion of the isatin ring from D1N52. A similar log P value was estimated for the triazole fragment with chlorobenzyl (log P = 2.4), which may explain the similar inhibitory activity observed for D1N52 and D1N8 (IC50 = 0.52 μM and 0.44 μM, respectively; Table 2), whereas the presence of the sulfonamidobenzyl unit in D1N18 led to a more polar triazole fragment (log P = −1.7), which can be expected to favor the solvation in water, thus contributing to the 2-fold reduction in inhibitory potency measured for D1N18 (IC50 = 1.12 μM; Table 2).

2.5. DTT-dependent assay

To verify the target specificity of these isatin derivatives, the anti-3CLpro potency of compound D1N8 was measured using the FRET assay with or without 4 mM DL-dithiothreitol (DTT).24 The non-specific cysteine protease inhibitor ebselen and lead L-26 were selected as controls. At a concentration of 5 μM, all the compounds exhibited significant inhibitory activity against 3CLpro protease in the absence of DTT (Fig. 6). However, ebselen almost completely lost anti-3CLpro activity in the presence of 4 mM DTT. In contrast, isatin derivatives L-26 and D1N8 maintained high inhibitory activity against 3CLpro protease despite being slightly affected by 4 mM DTT (the reason is that DTT could directly react with isatin derivatives25). These results indicated that these isatin derivatives are likely to belong to specific SARS-CoV-2 3CLpro protease inhibitors.

Fig. 6. SARS-CoV-2 3CLpro inhibitory activity of compounds (5 μM) in the absence or presence of 4 mM DTT.

Fig. 6

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3. Conclusion

Herein, taking the promising SARS-CoV-2 3CLpro inhibitor L-26 as the lead compound, a 58-membered isatin derivative library was constructed via click-chemistry-based miniaturized synthesis, and the bioactivity against SARS-CoV-2 3CLpro was determined by rapid screening. Among them, compounds D1N8 (IC50 = 0.44 ± 0.12 μM) and D1N52 (IC50 = 0.53 ± 0.21 μM) were identified as the most potent inhibitors towards SARS-CoV-2 3CLpro, which is equivalent to the lead L-26 (IC50 = 0.30 ± 0.14 μM). In addition, the cytotoxicity of the tested compounds decreased significantly compared with L-26, but they did not have significant anti-SARS-CoV-2 activity under the concentration of 20 μM, as well as lead L-26. The MD simulation studies allowed us to propose a putative binding mode that balances hydrogen-bond and hydrophobic interactions, providing clues to understand the binding contribution of the different structural fragments present in the isatin derivatives. The DTT-dependent assay revealed that these isatin derivatives are likely to belong to specific 3CLpro protease inhibitors, although slightly affected by reactions with DTT. In our future study, target-specific cellular assays such as the FlipGFP assay26 will be conducted to further validate this hypothesis. All in all, these efforts provided valuable insights in seeking novel anti-SARS-CoV-2 agents targeting 3CLpro.

4. Experimental section

4.1. Chemistry

The melting point data of the prepared compounds were obtained on a micro melting point apparatus and were uncorrected. 1H NMR and 13C NMR spectra were recorded on a Bruker Avance-400 NMR spectrometer or a Bruker Avance-600 NMR spectrometer in DMSO-d6 with TMS as an internal reference. Chemical shifts and J values were expressed in δ units (ppm) and in hertz (Hz), respectively. Mass spectra were measured via electrospray ionization on an LC Autosampler Device: Standard G1313A instrument. The reactions were monitored on thin-layer chromatography (TLC) using iodine vapor and UV light (λ = 254, 365 nm). The mixtures were separated on a column packed with silica gel 60 (200–300 mesh) by flash column chromatography. Reagent-grade solvents were applied and purified via standard methods if necessary. A rotary evaporator (EYELA N-1300D) was used to concentrate the reaction solutions under reduced pressure condition.

2,3-Dioxo-1-(prop-2-yn-1-yl)indoline-5-carboxamide (D1)

A solution of 2,3-dioxoindoline-5-carboxamide (11, 0.1 g, 0.53 mmol) in 5 mL N,N-dimethylformamide was added to cesium carbonate (0.43 g, 1.33 mmol) and 3-bromopropyne (0.08 g, 0.64 mmol), and the mixture stirred at room temperature for 0.5 h. After being monitored by TLC, the resulting mixture was treated with 20 mL water, and extracted with ethyl acetate (3 × 10 mL). Then, the organic phase was washed with saturated sodium bicarbonate (3 × 20 mL), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and finally was purified by flash column chromatography (ethyl acetate: petroleum ether = 1 : 2) to afford intermediate D1 as a yellow solid with a yield of 82.0%. 1H NMR (400 MHz, DMSO-d6) δ 8.25 (dd, J = 8.3, 1.8 Hz, 1H, Ph-H), 8.11 (d, J = 1.6 Hz, 1H, Ph-H), 8.09 (s, 1H, 1/2 NH2), 7.44 (s, 1H, 1/2 NH2), 7.32 (d, J = 8.3 Hz, 1H, Ph-H), 4.59 (s, 2H, CH2), 3.37 (d, J = 2.4 Hz, 1H, alkynyl-H). ESI-MS: m/z 229.09 [M + H]+, C12H8N2O3 [228.05].

General procedure for the synthesis of target compounds D1N8, D1N18 and D1N52: the prepared compound D1 (1.0 eq), azide substituents (N8, N18 or N52, 1.2 eq), and ascorbic acid sodium (0.6 eq) and CuSO4·5H2O (0.3 eq) were dissolved in the solution of tetrahydrofuran/water (8 mL, v : v = 1 : 1). The mixture was stirred at room temperature for 8 h, and then monitored by TLC. The reaction mixture was treated with 20 mL water, and extracted with ethyl acetate (3 × 10 mL). The combined organic phase was washed with saturated salt water (3 × 15 mL), dried over anhydrous sodium sulphate, filtered, and concentrated under reduced pressure to give crude products, which were purified by flash column chromatography and recrystallized using ethyl acetate and petroleum ether to afford the corresponding target compounds D1N8, D1N18 and D1N52.

1-((1-(3-Chlorobenzyl)-1H-1,2,3-triazol-4-yl)methyl)-2,3-dioxoindoline-5-carboxamide (D1N8)

Yellow solid, yield: 14.5%, mp: 218–220 °C. 1H NMR (600 MHz, DMSO-d6) δ 8.24 (s, 1H, triazoyl-H), 8.15 (dd, J = 8.3, 1.8 Hz, 1H, Ph-H), 8.08 (d, J = 1.6 Hz, 1H, Ph-H), 8.04 (s, 1H, 1/2 NH2), 7.42 (s, 1H, 1/2 NH2), 7.40–7.37 (m, 2H, Ph-H), 7.34 (s, 1H, Ph-H), 7.25–7.20 (m, 2H, Ph-H), 5.59 (s, 2H, CH2), 5.00 (s, 2H, CH2). 13C NMR (100 MHz, DMSO-d6) δ 183.01, 166.72, 158.71, 152.61, 142.22, 138.75, 137.84, 133.76, 131.15, 129.63, 128.62, 128.25, 127.09, 124.45, 123.75, 117.92, 111.27, 52.55, 35.74. ESI-MS: m/z 396.20 [M + H]+, C19H14ClN5O3 [395.80].

2,3-Dioxo-1-((1-(4-sulfamoylbenzyl)-1H-1,2,3-triazol-4-yl)methyl)indoline-5-carboxamide (D1N18)

Yellow solid, yield: 12.0%, mp: 208–210 °C. 1H NMR (600 MHz, DMSO-d6) δ 8.25 (s, 1H, triazoyl-H), 8.16 (d, J = 7.8 Hz, 1H, Ph-H), 8.08 (s, 1H, Ph-H), 8.05 (s, 1H, 1/2 NH2), 7.80 (d, J = 7.7 Hz, 2H, Ph-H), 7.44 (d, J = 7.9 Hz, 3H, 1/2 NH2 + 2 Ph-H), 7.36 (s, 2H, NH2), 7.25 (d, J = 8.1 Hz, 1H), 5.66 (s, 2H, CH2), 5.00 (s, 2H, CH2). 13C NMR (100 MHz, DMSO-d6) δ 183.01, 166.73, 158.72, 152.61, 144.33, 142.25, 140.09, 137.84, 129.63, 128.89, 126.58, 124.43, 123.74, 117.94, 111.27, 52.73, 35.74. ESI-MS: m/z 441.4[M + H]+, 463.3 [M + Na]+, C19H16N6O5S [440.43].

1-((1-(2-Fluoro-4-(trifluoromethyl)benzyl)-1H-1,2,3-triazol-4-yl)methyl)-2,3-dioxoindoline-5-carboxamide (D1N52)

Yellow solid, yield: 12.0%, mp: 320–322 °C. 1H NMR (600 MHz, DMSO-d6) δ 8.25 (s, 1H, triazoyl-H), 8.15 (dd, J = 8.3, 1.9 Hz, 1H, Ph-H), 8.08 (d, J = 1.9 Hz, 1H, Ph-H), 8.03 (s, 1H, 1/2 NH2), 7.74 (d, J = 9.7 Hz, 1H), 7.61 (d, J = 7.9 Hz, 1H, Ph-H), 7.49 (t, J = 7.6 Hz, 1H, Ph-H), 7.41 (s, 1H, 1/2 NH2), 7.23 (d, J = 8.3 Hz, 1H, Ph-H), 5.73 (s, 2H, CH2), 5.00 (s, 2H, CH2). 13C NMR (100 MHz, DMSO) δ 183.01, 166.70, 160.28 (d, JCF = 249.8 Hz), 158.71, 152.59, 142.18, 137.82, 132.23 (d, JCF = 3.9 Hz), 131.46 (q, JCF = 10.3 Hz), 129.64, 128.02 (d, JCF = 14.4 Hz), 124.67, 123.74, 122.45 (q, JCF = 167.2 Hz), 122.27 (q, JCF = 4.0 Hz), 117.93, 113.71 (dq, JCF = 25.1, 3.7 Hz), 111.28, 47.12 (d, JCF = 3.4 Hz), 35.70. ESI-MS: m/z 448.4 [M + H]+, C20H13F4N5O3 [447.35].

4.2. 3CLpro enzymatic assay

The anti-3CLpro activity of the prepared compounds was evaluated using the fluorescence resonance energy transfer (FRET) method with the fluorescent substrate MCAAVLQSGFR-Lys(Dnp)-Lys-NH2 (Beyotime Biotechnology, Shanghai, China).15 The solution of 3CLpro and fluorescent substrate were respectively diluted to a concentration of 1.5 μM and 500 μM with the assay buffer (50 mM Tris-HCl, 150 mM NaCl, 20% glycerol, pH = 7.3) and stored at −20 °C. The tested compounds were dissolved in DMSO to prepare 5 mM stock solutions, and diluted to desired concentrations with assay buffer. The black 96-well plate was added with 50 μL of assay buffer, 20 μL of diluted enzyme solution, and 20 μL inhibitor solution, incubated for 10 minutes at 37 °C, and then 10 μL of substrate solution was added to each well. The fluorescence signal, with an excitation wavelength of 320 nm and emission wavelength of 405 nm, was detected using a SpectraMax iD5 multimode plate reader (Molecular Devices) every 10 seconds, lasting for 10 minutes. The control wells were blank wells with no enzyme or inhibitor and wells with no inhibitor. Inhibition (i%) in each well can be calculated using the following formula: i% = 1 − Vi/V0 × 100% (Vi for test well, and V0 for control well). All the tested compounds were measured for the inhibition rate at a concentration of 1 μM in preliminary screening, and the hit compounds were measured at 4 different concentrations (0.1 μM, 0.5 μM, 1 μM, and 5 μM, three independent experiments in each concentration) to determine the IC50 values (three independent experiments were performed).

4.3. Cellular anti-SARS-CoV-2 activity assay

Vero E6 cells (from Prof. Ann E. Tollefson's laboratory, Department of Molecular Microbiology and Immunology, Saint Louis University School of Medicine, United States) were plated at 1.7 × 104 cells per well into 96-well plates about 24 hours prior to infection. Test compounds were prepared to a 10 mM stock solution in DMSO. The test compounds were diluted to 80 μM in Dulbecco's MEM/5% FBS (fetal bovine serum) and then 1 : 3 dilutions were done on a dilution plate (each compound was done in duplicate), to form a dilution series. The medium was removed from the cells and 50 μL of the compound dilutions were added to each well of the cell plate. The plates were then transferred to the BSL-3 facility for infection with SARS-CoV-2. The virus was diluted to approximately 100 focus forming units (infectious particles) per 50 μL (the volume of diluted virus added per well). After the addition of virus to the wells, the plates were incubated at 37 °C, 5% CO2 for 1 hour. After the incubation, the medium containing methyl cellulose was added to each well (100 μL). The total final volume in each well was 200 μL. The final compound concentration range was 20 μM to 0.0274 μM. After the plates were incubated for 25–26 hours, the methyl cellulose overlay was removed and the plates were washed twice with PBS. 5% paraformaldehyde in PBS was then added to each well and the entire plates were subsequently submerged in 5% formalin for 15 minutes. The plates were then submerged in PBS for 15 minutes and then fresh PBS was added to each well and the plates were transferred out of the BSL-3 laboratory. The plates carrying immobilized Vero E6 cells were rinsed with focus forming assay buffer and incubated with a guinea pig anti-SARS-CoV antibody generated against virions that cross-reacts with SARS-CoV-2. The secondary antibody was goat anti-guinea pig IgG (horseradish peroxidase conjugate). The substrate was TrueBlue and the infected cells/foci appeared as individual or clusters of blue cells. After staining, the plates were read on an EliSpot reader. The relative surface area that was stained was used to calculate the inhibition activity of the concentration gradient of the drug, and to generate the EC50 curves. EC50 values were generated by curve fitting using Graphpad Prism 8.0.

4.4. Cellular toxicity assay

Vero E6 cells (from Prof. Ann E. Tollefson's laboratory, Department of Molecular Microbiology and Immunology, Saint Louis University School of Medicine, United States.) were plated on 96-well plates (Greiner) at 1.7 × 104 cells per well in DMEM (Dulbecco's modified Eagle medium) high glucose with 10% FBS. Cells were incubated for 24 h in 37 °C and 5% CO2 at saturating humidity. Cells were treated with compound in DMEM high glucose with 5% FBS at a final concentration of 1% DMSO for 26 h. Cellular viability was measured using the CellTiter 96 aqueous nonradioactive cell proliferation assay (Promega). Three or more replicate assays were done to calculate the average values. The 50% cytotoxic concentration (CC50) values were determined with GraphPad Prism 8.0 using the four-parameter variable slope algorithm with the bottom set to zero.

4.5. Molecular dynamics simulation

A combined strategy involving molecular docking and molecular dynamics simulations was used to investigate the binding mode of isatin derivatives to SARS-CoV-2 3CLpro. For the docking stage calculations were performed considering X-ray structures deposited in the Protein Data Bank27 with PDB codes 7EN8, 7M8P and 7V1T28–30 to take into account i) the non-covalent inhibitory binding mode of the enzyme-bound ligands, ii) the distinct arrangement of the side chains of residues Met49 and Gln189, and iii) the different conformation adopted by the flexible loop defined by residues Asp187-Ala193. It is worth noting that the binding mode of the ligands bound to these structures fills the distinct subpockets that can be identified in the binding site (see ESI† Fig. S2). Docking was accomplished using the XP score function of Glide31–33 in conjunction with a defined inner/outer box of 40/76 Å centered on the catalytic pocket of 3CLpro.

Molecular dynamics (MD) simulations were performed using the Amber20 package34 to explore the structural stability of D1N52 bound to 3CLpro. The ff19SB protein force-field35 was used for the enzyme, while the isatin derivative was parametrized using the gaff2 force field36,37 considering the RESP atomic charges38 determined by fitting the HF/6-31G(d) electrostatic potential. The enzyme–ligand complex was solvated with the OPC water model,39 and counterions were used to maintain the system neutrality and the physiological ionic atmosphere. Finally, production calculations were performed using the SHAKE algorithm40 and by treating electrostatic interactions with particle mesh Ewald (PME)41 with a cutoff of 10 Å for non-bonded interactions. Due to the flexibility of the 3CLpro binding pocket, the restraints were maintained along the first 25 ns, then reduced to 2 kcal mol−1 A−2 in the next 5 ns, and finally an unrestrained MD simulation (200 ns) was run under constant volume and temperature.

Quantum mechanical (QM) continuum solvation calculations were performed to estimate the hydrophobicity of the isatin derivatives. To this end, the n-octanol/water partition coefficient (log P) was calculated using the IEF-PCM/MST method42–44 as implemented in a local version of Gaussian 16.45 Molecular geometries were optimized at the B3LYP/6-31G(d) level of theory46–48 considering the solvation effects in both water and n-octanol. Finally, the log P was estimated by considering the solvation free energies in water and n-octanol.

4.6. DTT-dependent assay

The method is similar to the 3CLpro enzymatic assay in section 4.2. In the experimental group 4 mM DTT was added to the assay buffer, while in the control group DTT was not added to the assay buffer.

Conflicts of interest

The authors declare no conflict of interest.

Supplementary Material

MD-014-D3MD00306J-s001

Acknowledgments

The authors gratefully acknowledge financial support from Major Basic Research Project of Shandong Provincial Natural Science Foundation (no. ZR2021ZD17), Guangdong Basic and Applied Basic Research Foundation (no. 2021A1515110740), China Postdoctoral Science Foundation (no. 2021M702003), Science Foundation for Outstanding Young Scholars of Shandong Province (no. ZR2020JQ31), and Foreign Cultural and Educational Experts Project (no. GXL20200015001). A. V. and F. J. L. acknowledge financial support from the Spanish Ministerio de Ciencia e Innovación (AEI/10.13039/501100011033; grants PID2020-117646RB-I00 and CEX2021-001202-M), the Generalitat de Catalunya (grant 2021SGR00671). The Consorci de Serveis Universitaris de Catalunya (CSUC) is acknowledged for computational resources (Molecular Recognition project).

†

Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d3md00306j

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