Identification of SARS-CoV-2 3CL protease inhibitors from DNA-encoded chemical library screening

Aiming to screen 2.8 billion compounds in a DNA-encoded chemical library (DELopen by WuXi AppTec) for SARS-CoV-2 3CL protease inhibitors, we captured His-tagged 3CL protease on Ni-NTA magnetic beads for binding affinity screening (Supplementary Fig. 1). After incubation with the DELopen library, 3CL protease beads or control empty beads were magnetically pulled down and washed before being heated to 95 °C to release bound library molecules.

In the first screen, four compounds (DEL1 through DEL4) emerged as top screening hits with consistently high enrichment scores in three replicates for binding to 3CL protease (Fig. 1a). We designed a counterscreen to distinguish between compounds binding at the same binding site as known inhibitors or different binding sites. In this second screen, four copies of the DELopen library were separately screened against beads capturing 3CL alone, the 3CL-GC376 complex, the 3CL-Compound 4 complex, or no protein as a negative control. GC376 and Compound 4 are known 3CL inhibitors described in previous studies^4,13,14. This counterscreen enables identification of protein binders with the same binding site as the known inhibitors, because they are expected to bind less efficiently to the protein-inhibitor complex than to the protein alone. Meanwhile, small molecules with binding sites different from the known inhibitors are expected to bind to the protein-inhibitor complex and to the protein alone in an indistinguishable manner.

A Enrichment scores of top screening hits (DEL1 through DEL4) on three replicates of DELopen for binding to 3CL protease. B Enrichment scores of top screening hits (DEL5 through DEL8) in a counterscreen against beads capturing 3CL alone, the 3CL-GC376 complex, and the 3CL-Compound 4 complex, to distinguish between compounds that share binding sites with known inhibitors or target different binding sites. C Structures of top screening hits DEL1 through DEL8. For close analogs of DEL7, structure differences from DEL7 are shown in red color.

Four compounds (DEL5 through DEL8) emerged as top screening hits with enrichment for 3CL alone compared to the 3CL-GC376 complex, the 3CL-Compound 4 complex, and the negative control (Fig. 1b, c). Since both GC376 and Compound 4 are known to bind to the active site of the 3CL protease, DEL5 through DEL8 are likely to interact with the active site as well. No compound was found to be enriched for the protein-inhibitor complex or the protein alone, suggesting the known active site might be the most suitable binding site for small molecules on the surface of 3CL protease, and that additional allosteric binding sites are not likely to exist or to be easy to target.

Our further evaluation of these screening hits started from measuring their enzymatic inhibition potency using purified 3CL protease in vitro. We found that DEL2, DEL7, DEL1, DEL3, and DEL8 inhibited the activity of the 3CL protease on a specific fluorogenic substrate, with IC₅₀ values of 0.3 µM, 0.3 µM, 4 µM, 4 µM, and 6 µM, respectively (Fig. 2a). Indeed, these five compounds are close structural analogs of each other, providing a 3CL protease inhibitor scaffold and structure-activity relationships for further optimization (Fig. 1c).

A The dose-dependent effects of DEL1 through DEL8 on the activity of 20 nM SARS-CoV-2 3CL protease were tested. The fluorogenic peptide MCA-AVLQSGFR-Lys(DNP)-Lys-NH2, corresponding to the nsp4/nsp5 cleavage site in the virus was applied as the substrate. Mean of 2 biological replicates is plotted. Source data are provided as a Source Data file. B Ability of DEL1 through DEL8 to inhibit SARS-CoV-2 viral infection in human Huh-7 cells, which were transduced to overexpress ACE2 receptor (Huh-7^ACE2) before infection with SARS-CoV-2 virus (2019-nCoV/USA-WA1/2020). Data are plotted as the mean ± s.d., n = 3 biological replicates. Source data are provided as a Source Data file.

We evaluated these five compounds for inhibition of SARS-CoV-2 viral replication in human Huh-7 cells, which were transduced to stably overexpress the ACE2 receptor (Huh-7^ACE2) before infection with a reporter SARS-CoV-2 virus (USA-WA1/2020 strain). We found that DEL2, DEL7, and DEL1 blocked viral infection, with EC₅₀ values of 2 μM, 3 μM, and 4 μM, respectively (Fig. 2b). DEL3 and DEL8 also suppressed viral replication but exhibited EC₅₀ values above 10 µM.

Co-crystal structures of DEL2 and DEL7 with SARS-CoV-2 3CL protease suggest non-covalent binding to the active site

Aiming to directly visualize the binding mechanism of top screening hits to 3CL protease, as well as to guide structure-based optimization efforts, we determined crystal structures of 3CL protease with DEL2 and DEL7 at resolutions of 1.85 Å and 1.74 Å, respectively (Fig. 3a, b, Supplementary Fig. 2a, Supplementary Table 1, 2). The X-ray crystal structures revealed that both compounds bind non-covalently to the substrate-binding pocket of 3CL protease. Since DEL7 is a close analog of DEL2, differing only by a fluoro substitution at the para position of the phenyl group, the two compounds share similar binding modes to the 3CL protease. Primary amines on both compounds strongly interact with the catalytic C145 residue. In both structures, the benzonitrile group occupies the S1 site, anchored by a hydrogen bond with the side chains of H163 (Fig. 3a, c, Supplementary Fig. 2b).

A The co-crystal structures of 3CL-DEL7 (yellow) complex. The inhibitor-interacting residues of 3CL protease subunit A (cyan) and subunit B (green, the other protomer of the 3CL homodimer) are shown with stick models. The Fo-Fc electron density omit map at 3σ is shown with blue mesh and hydrogen bonds are represented by red dash lines. C145 appeared to be oxidized to sulfenic acid during crystal growth and interacted with the primary amine of DEL-7. B Summary of atomic distances of hydrogen bonds between 3CL protease and DEL7 or DEL7 analogs in the co-crystal structures. C The structural overlay of 3CL protease bound to DEL7 with 3CL protease bound to GC376 (PDB code: 7JSU), compound 4 (PDB code: 7JT7), and nirmatrelvir (PDB code: 7U28). Substrate binding sites are labeled. The black arrow highlights how the peptidic backbones of GC376, compound 4, and nirmatrelvir overlaps well with each other, while the peptidic backbone of DEL7 and its analogs is flipped by almost 140° as compared with those of g-lactam-containing inhibitors. The depicted electrostatic surface (K_bT/e = ±5.0) of 3CL protease alone (PDB code: 7JST) was generated using the program APBS⁴² and rendered in PyMOL. Clashes of inhibitors with the surfaces demonstrated how inhibitors re-organized the substrate-binding site and its plasticity. D MALDI-MS spectra of 3CL only (gray), 3CL treated with 10 equivalent (eq) DEL7 for 1 h at 4 °C (blue), 3CL preincubated with DMSO for 1 h at 4 °C before treatment of 10 eq Compound 4 for 1 h at 4 °C (green), 3CL pre-incubated with 1 eq GC376 for 1 h at 4 °C before treatment of 10 eq Compound 4 for 1 h at 4 °C (red), and 3CL preincubated with 1 eq DEL7 for 1 h at 4 °C before treatment of 10 eq Compound 4 for 1 h at 4 °C (purple).

The dichlorophenyl group of both compounds is favorably embedded in the hydrophobic S2 site, surrounded by the alkyl portion of the side chains of H41, M49, M165, D187, and Q189. More importantly, the dichlorobenzyl group forms methionine-aromatic motifs¹⁵ (sulfur-π interactions) with both M49 and M165, which were found to yield additional stabilization as compared with purely hydrophobic interaction via isobutyl groups commonly seen in the S2 site with previously reported 3CL inhibitors^7,13 (Fig. 3c, Supplementary Fig. 2c).

The p-Cl group establishes either a hydrogen bond or hydrophobic interactions with the side chain of H41. Extending into the S3 and S4 sites, the NH of the terminal amide bond of DEL2 and DEL7 is stabilized by a hydrogen bond with the side chain of E166. However, we observed stronger electron densities, which typically indicate a more specific recognition and tighter binding, in the S3 and S4 sites for DEL7, primarily due to the formation of a hydrogen bond between the p-F group and the NH backbone of A191.

The crystal structure of the 3CL protease with DEL7 revealed a strong, continuous electron density map between the primary amine and the thiol of the catalytic C145 residue. Considering the implausibility of a covalent interaction between these two groups, this suggested that Cys145 was oxidized to a sulfenic acid in the course of crystal growth. Such oxidation of cysteine thiols has been observed at acidic pH with neighboring histidine as a proton acceptor, which is in line with our crystallization conditions (pH = 5–6) and the presence of H41 in the active site. Indeed, the presence of an oxygen atom bridging a sulfur and nitrogen atom to form the S-O-N entity is a widespread occurrence¹⁶.

To complement this crystallographic study, we further investigated the binding mode of DEL7 by mass spectrometry (MS). In a Matrix-Assisted Laser Desorption Ionization (MALDI) MS analysis of the intact 3CL protease protein after incubating it with DEL7 or covalent inhibitor Compound 4, we observed covalent modification only by the control Compound 4, with a mass shift matching its molecular weight (Fig. 3d). No mass shift induced by DEL7 was observed, which was consistent with a non-covalent binding mechanism of DEL7 to the 3CL protease.

In prior studies, we found that the reversible covalent inhibitor GC376 would not shift the mass of 3CL in MALDI MS due to the sensitivity of its covalent bond to pH changes, but pre-incubation of the 3CL protease with one equivalent of GC376 would occupy the active site and block the subsequent binding of Compound 4 to 3CL protease. Accordingly, we pre-incubated 3CL protease with one equivalent of DEL7 or GC376 before the addition of Compound 4. While GC376 consistently blocked Compound 4 binding through a reversible covalent interaction, DEL7 exhibited no such effect (Fig. 3d). Together, these data support a non-covalent binding mechanism of DEL7 to the SARS-CoV-2 3CL protease, as expected based on its chemical structure lacking an electrophilic warhead and several other crystal structures that we determined with DEL7 analogs.

DEL7 and its analogs specifically inhibit coronavirus 3CL proteases

Selectivity is considered a major challenge for targeting SARS-CoV-2 proteases for the treatment of COVID-19, as reported 3CL protease inhibitors, including GC376, are also potent inhibitors of human cathepsin L and B¹⁷. Cathepsin L was shown to mediate SARS-CoV-2 viral entry, but the virus may adopt alternative entry pathways, such as TMPRSS2, a transmembrane serine protease^18,19. Therefore, dual inhibitors of 3CL protease and cathepsin L exhibit a partial loss of their antiviral activities in cells expressing TMPRSS2, which limits their clinical usefulness¹⁷. This selectivity limitation may be especially relevant for repurposing inhibitors of other proteases for 3CL protease or applying compounds with covalent warheads designed to target cysteine proteases.

By comparison, DEL7 and its analogs are non-covalent inhibitors identified from targeted screening specifically for the SARS-CoV-2 3CL protease. In line with this, we found that none of these hits inhibited human cathepsin L at concentrations up to 10 µM (Supplementary Fig. 3a), while GC376 exhibited an IC₅₀ value of 0.33 nM on cathepsin L.

We further evaluated DEL7 against a panel of additional human proteases of various classes (Supplementary Fig. 3b). The result confirmed that DEL7 is highly selective for 3CL inhibition, displaying IC₅₀ values of >10 μM against all tested human proteases. For therapeutic consideration, we measured the cellular toxicity of all DEL7 analogs in human cells, and no compound exhibited toxicity at concentrations up to 10 μM (Supplementary Fig. 4).

Considering that the 3CL protease substrate-binding pocket is highly similar across 12 different coronaviruses, we hypothesized that small-molecule inhibitors of the SARS-CoV-2 3CL protease have the potential to be broadly effective against other coronaviruses^5,6. To test this hypothesis, we transfected HEK293T cells with 3CL proteases from different coronaviruses to induce cytotoxicity so that inhibitors of the corresponding 3CL protease could protect cells²⁰. Accordingly, we found that DEL7 not only exhibited inhibitory effect on the SARS-CoV-2 3CL protease in cells, but also suppressed the 3CL protease activity of SARS-CoV and MERS-CoV in cells (Supplementary Fig. 3c). Moreover, this cell-based assay confirmed that DEL7 can effectively inhibit the tested viral proteases within a cellular context, in agreement with the expected mechanism of its antiviral activity.

Structural optimization of DEL7 led to the discovery of analogs with improved biochemical and biophysical potency for SARS-CoV-2 3CL protease

We focused on the creation of improved analogs of DEL7, based on its highly specific binding and strong electron density in the co-crystal structure. To optimize the structure of DEL7, we used a structure-based approach. We defined five modifiable groups in the chemical structure of DEL7, termed R1, R2, R3, R4, and R5 (Fig. 4). In the design of the DEL7 analogs, we modified one group at a time.

Structures of DEL7 analogs, along with their IC₅₀ values against SARS-CoV-2 3CL protease activity in a biochemical assay and EC₅₀ values against SARS-CoV-2 viral replication in a Huh7^ACE2 cellular viral assay. Structure differences from DEL7 are shown in red color.

Since the R1 group has weaker electron density than other groups in the co-crystal structure of DEL7 with the 3CL protease, more analogs with modifications on R1 were synthesized initially (Fig. 4). Compared with DEL7 in the biochemical assay, two analogs featured improved inhibition potency on SARS-CoV-2 3CL protease: 1 and 2, with IC₅₀ values of 70 nM and 20 nM, respectively. Structurally, both analogs have an elongated alkyl moiety with a terminal dimethyl amino group, where 2 features one additional methylene group in the alkyl chain compared with 1. Other analogs (3–9) with modifications on R1 exhibited similar or lower potency in comparison with DEL7.

DEL2 (Fig. 4) is a close analog of DEL7, differing solely by the fluorine on the R2 phenyl group. It exhibited comparable biochemical potency to DEL7, prompting the testing of fluorine removal on additional optimized DEL7 analogs.

For R3, we substituted both chlorines in DEL7 with fluorines and generated 10, which exhibited a poorer biochemical potency than DEL7, with an IC₅₀ value of 2 µM. This may be due to the loss of hydrophobic interactions or a hydrogen bond interaction between the p-Cl in the R3 group of DEL7 and the side chain of H41. For the R4 group, which resides next to the unoccupied S1’ site in the co-crystal structure of DEL7 with 3CL protease, we added acetyl or tert-butyloxycarbonyl (Boc) groups onto the amine, aiming to occupy the S1’ site to establish more favorable interactions. The resulting analogs 11 and 12 lost inhibitory potency in the biochemical assay, suggesting the importance of interactions between the primary amine in the R4 group of DEL7 and C145.

DEL1 (Fig. 4) is also a close analog of DEL7, the only difference being the substitution of the nitrile moiety in R5 with a nitro group. Additionally, the R5 group occupies the S1 site, where a γ-lactam group has commonly been designed in previously reported 3CL protease inhibitors, including GC376¹³, compound 4¹⁴, and nirmatrelvir⁷. We therefore substituted the R5 group of DEL7 with a γ-lactam and generated 13. However, both DEL1 and 13 were less active than DEL7 in the biochemical assay, suggesting that benzonitrile is preferred in the R5 group in the context of the DEL7 scaffold.

The structural overlay of 3CL protease bound to DEL7 with 3CL protease bound to GC376, compound 4, or nirmatrelvir provides a tantalizing clue to why the lactam is not a preferred moiety for DEL7: the peptidic backbone of DEL7 and its analogs is flipped by almost 140° as compared with those of γ-lactam-containing inhibitors (Fig. 3c, Supplementary Fig. 2c).

We further evaluated the binding of DEL7 and its improved analogs 1 and 2 to the 3CL protease in a surface plasmon resonance (SPR) assay and observed K_D (dissociation constant) values of 434 nM, 196 nM, and 63 nM, respectively (Fig. 5a). The improved biophysical binding affinities observed for 1 and 2 are in line with their improved biochemical potencies in the 3CL protease assay.

A Surface plasmon resonance (SPR) analysis of the in vitro binding of DEL7, 1, and 2 to SARS-CoV-2 3CL protease. Different concentrations of inhibitors were serially injected onto a sensor chip with immobilized 3CL protease. The equilibrium dissociation constant (Kd) of the inhibitors was obtained by fitting binding response units to the Hill equation. B, C The co-crystal structures of 3CL-1 (purple) and 3CL-2 (orange) complex. The inhibitor-interacting residues of 3CL protease subunit A (cyan) and subunit B (green, the other protomer of the 3CL homodimer) are shown with stick models. The Fo-Fc electron density omit map at 3σ is shown with blue mesh. Hydrogen bonds and hydrophobic interactions are represented by red and blue dash lines, respectively. In panel (C), C145 appeared to be oxidized to sulfenic acid during crystal growth, but it didn’t form a covalent bond with the primary amine of DEL7-5. D Metabolic stability of DEL7 and 2 in human and mouse (CD-1) liver microsomes.

Aiming to decipher the basis of improvement over DEL7 as well as to guide further optimization efforts, we solved the X-ray crystal structures of 3CL protease with 1 and 2 at resolutions of 1.93 Å and 1.56 Å, respectively (Fig. 5b, c, Supplementary Table 3, 4). While both compounds inherited the interactions that DEL7 featured with the protease, the elongated alkyl chains in the R1 group further extended beyond the S3 site, reaching toward the other protomer (B) of the physiological homodimer of 3CL protease. Specifically, the tertiary amine in the R1 group of 2 was anchored by additional hydrophobic interactions with S1 and N214 of protomer B, which may explain its improvement over DEL7. These interactions were absent in previously reported γ-lactam-containing 3CL inhibitors (Fig. 3c). Moreover, we found that the C145 of the 3CL protease co-crystalized with 1 had been oxidized to sulfenic acid (Fig. 5b). This observation confirmed that the continuous electron density map between the DEL7 primary amine group and the thiol group of C145 is an oxygen atom bridging a sulfur and nitrogen atom to form the S-O-N entity due to crystallographic artifacts introduced during crystal growth at pH 5-6¹⁶.

For consideration of therapeutic applications, we evaluated the metabolic stability of DEL7 and 2 in human and mouse liver microsomes and observed a significant improvement of 2 (T_{1/2, human} = 7.6 min, T_{1/2, mouse} = 41 min) over DEL7 (T_{1/2, human} = 1 min, T_{1/2, mouse} = 0.9 min), indicating potential suitability for further animal studies and an approach to enhance the metabolic stability of DEL7 via modifications of the R1 group (Fig. 5d).

Cell permeability limits antiviral potencies of DEL7 analogs but can be overcome by reducing net charge

Based on the improved biochemical potency observed for 2 over 1 and DEL7, we continued modifying the R1 group in DEL7. We elongated the alkyl chain in 2 by adding one or two methylene groups and generated 14 and 15, respectively (Fig. 6a). We also elongated the R1 group of 2 by incorporating the nitrogen-containing heterocycles triazole or piperazine and generated 16 and 17, respectively (Fig. 6a).

A Structures of DEL7 analog 14, 15, 16, and 17, along with their IC₅₀ values against SARS-CoV-2 3CL protease activity in a biochemical assay and EC₅₀ values against SARS-CoV-2 viral replication in a Huh7^ACE2 cellular viral assay. B–E The co-crystal structure of 3CL-14 (brown), 3CL-15 (magenta), 3CL-16 (pink), and 3CL-17 (violet) complex. The inhibitor-interacting residues of 3CL protease subunit A (cyan), subunit B (green, the other protomer of the 3CL homodimer), and subunit C (pink, a neighboring protomer in the crystal packing, not part of the 3CL homodimer formed by subunit A and B) are shown with stick models. The Fo-Fc electron density omit map at 3σ is shown with blue mesh. Hydrogen bonds, hydrophobic interactions, and methionine-aromatic motif¹⁵ are represented by red, blue, and green dash lines, respectively. The methionine-aromatic motif (Sulfur-π interactions) was found to yield additional stabilization as compared with purely hydrophobic interaction¹⁵. In panel E, C145 appeared to be oxidized to a sulfenic acid during crystal growth, but it didn’t form a covalent bond with the primary amine of 17.

When tested against the SARS-CoV-2 3CL protease, 14 and 15 exhibited further improvements over 2. Both 14 and 15 featured IC₅₀ values of 10 nM, the highest possible potency in the biochemical assay, which uses 20 nM protease to cleave enough fluorogenic substrates to produce a fluorescence signal detectable by our instrument. Analogs 16 and 17 were also able to efficiently inhibit 3CL protease, with IC₅₀ values of 0.5 µM and 30 nM, respectively. This validated the strategy of modifying the R1 group for further optimization.

Aiming to decipher the basis for these improvements, as well as to guide further optimization efforts, we determined the X-ray crystal structures of the 3CL protease with 14, 15, 16, and 17, at resolutions of 1.55 Å, 1.67 Å, 1.51 Å, and 1.72 Å, respectively (Fig. 6b–e, Supplementary Table 5, 6, 7, 8). Compared with the co-crystal structure of 2, these four compounds retained interactions that 2 featured with the active site of the protease, including the hydrogen bonds with H163, E166, and A191 (Fig. 6b). Furthermore, the R1 groups of these compounds were engaged in and stabilized by additional interactions with the active site and the interacting protomer B of the native 3CL protease homodimer. Specifically, the R1 group of 15 recruited the penultimate residue F305 of protomer B into hydrophobic interactions (Fig. 6c). The R1 group of 16 was held tightly by two additional hydrogen bonds engaging the side chain of H64 from a neighboring protease protomer (C) and the backbone carbonyl of P168, in addition to a π-π interaction between the triazole ring of 16 and F305 of protomer B (Fig. 6d). In contrast, the piperazine nitrogen of 17 forms a hydrogen bond with the backbone carbonyl of N214 of protomer B (Fig. 6e). These additional interactions stabilize the R1 group of these compounds in the substrate binding site and on the surface of the other protomer of 3CL homodimer, as evidenced by strong and well-defined electron densities in the crystal structures. This is in line with their high potency in biochemical assays.

However, when tested in cells infected with SARS-CoV-2, 2 did not show improvement over DEL7. Indeed, its EC₅₀ value (4.2 µM) was poorer than that of DEL7 (3.3 µM). Analogs 1, 14, 15, and 17 only exhibited slight improvements over DEL7 in this cellular viral assay, with EC₅₀ values of 1.2 µM, 1.3 µM, 1.7 µM, and 0.8 µM, respectively. However, their EC₅₀/IC₅₀ ratios (1.2/0.07 = 17, 1.3/0.01 = 130, 1.7/0.01 = 170, 0.8/0.03 = 27, respectively) were markedly higher than for DEL7 (3.3/0.3 = 11). We speculated that this poor cellular potency might be due to stability or permeability issues. To investigate how we could resolve this issue by optimizing the structure, we synthesized seven additional analogs of the most biochemically potent compound, 14, with modifications on R1 through R5 (Fig. 7).

Structures of DEL7 analogs designed on top of analog 14, along with their IC₅₀ values against SARS-CoV-2 3CL protease activity in a biochemical assay and EC₅₀ values against SARS-CoV-2 viral replication in a Huh7^ACE2 cellular viral assay. Structure differences from DEL7 are shown in red color.

We first shortened the linker to R1 by one methylene and generated 18 (Fig. 7); this caused a complete loss of potency. This is likely due to the loss of a critical hydrogen bond between the R1 amide NH and the E166 side chain. We then replaced the fluorine in the R2 group with hydrogen and prepared the DEL2-like compound 19, as DEL-2 featured a better EC₅₀/IC₅₀ ratio (2/0.3 = 7) than DEL7. However, this trend was not reproduced in the case of 19, as it showed an EC₅₀/IC₅₀ ratio of 103 (3.1/0.03). For R3, we removed either of the chloro groups from the dichlorobenzene to decrease the molecular weight of the compounds. However, the resulting analogs 20 and 21 exhibited much lower potency than 14 in both biochemical and cellular assays, indicating that both chlorines are important for potency. Moreover, all crystal structures complexed with DEL compounds confirmed this finding, as p-Cl either forms polar or non-polar interactions with H41 and D87, while o-Cl interacts with the F-benzyl group of each inhibitor.

We then modified the chirality of the (R)-amino group in R4 by generating a diastereomer 22 with (S)-NH₂. We observed a substantial loss of inhibition potency, suggesting a specific stereochemistry requirement for the primary amine.

We found that removal of the R4 amino group was tolerated, as the corresponding analog 23 (R4 = H) exhibited an IC₅₀ value of 0.07 µM and an EC₅₀ value of 2.3 µM. Interestingly, a better EC₅₀/IC₅₀ ratio (2.3/0.07 = 33) was observed for 23. Compared with 14, which carries two positive charges at physiological pH due to the primary amine in R4 and the tertiary amine in R1, 23 only carries one positive charge. Highly charged small molecules are typically less cell permeable, so removal of charge from DEL7 analogs appeared to be a feasible approach to improve cellular permeability, as well as cellular antiviral potency. This is evidenced by the undesirably high EC₅₀/IC₅₀ ratio observed on highly charged DEL7 analogs, including 2 (4.2/0.02 = 210), 15 (1.7/0.01 = 170), and 19 (3.1/0.03 = 103).

We also replaced the nitrile group in R5 of 14 with an amide group, but it caused a substantial decrease of inhibition potency (24, IC₅₀ = 0.3 µM).

Aiming to validate the strategy of restricting net charge in DEL7 structural optimization, we subsequently modified R1 with neutral groups, as modification of R1 was shown to be more efficient in improving DEL7 as compared to other sites. We, therefore, synthesized 25 and 26 (Fig. 8a), elongating the alkyl chain of DEL7 R1 group by three methylene groups in 26, and incorporating an alkyne in the R1 group of 25. Compared with DEL7 (IC₅₀ = 0.3 µM, EC₅₀ = 3.3 µM) in the biochemical and cellular assay, 25 (IC₅₀ = 0.1 µM, EC₅₀ = 0.9 µM) and 26 (IC₅₀ = 0.2 µM, EC₅₀ = 0.8 µM) exhibited moderate improvements.

A Structures of DEL7 analog 25 and 26, along with their IC₅₀ values against SARS-CoV-2 3CL protease activity in a biochemical assay and EC₅₀ values against SARS-CoV-2 viral replication in a Huh7^ACE2 cellular viral assay. Structure differences from DEL7 are shown in red color. B The co-crystal structure of 25 (dark purple) complex. The inhibitor-interacting residues of 3CL protease subunit A (cyan) and subunit B (green, the other protomer of the 3CL homodimer) are shown with stick models. The Fo-Fc electron density omit map at 3σ is shown with blue mesh. Hydrogen bonds and hydrophobic interactions are represented by red and blue dash lines, respectively. C Cytosolic concentrations of DEL7, 14, and 25 in HEK293T cells treated with 10 µM of each compound. Data are plotted as the mean ± s.d., n = 3 biological replicates. Source data are provided as a Source Data file.

When we further evaluated 25 for inhibition of HCoV-Alpha-229E and HCoV-Beta-OC43 viral infection in Huh7 or RD cells, we found that 25 effectively blocked viral infection of HCoV-Alpha-229E and HCoV-Beta-OC43 with EC₅₀ values of 2.5 µM and 3.3 µM, respectively (Supplementary Table 9). The consistent activity of 25 on human coronaviruses in addition to SARS-CoV-2 (EC₅₀ = 0.9 µM) suggested the potential of developing DEL7 scaffold into a pan-coronavirus inhibitor.

We determined a crystal structure of the 3CL protease with 25, at resolution 1.81 Å (Fig. 8b, Supplementary Table 10). The electron density corresponding to 25 is not the highest among the DEL7 analogs that we determined; its terminal methyl group does not show well-defined electron density, despite forming a hydrophobic interaction with Cα of G170. This is in line with its moderate potency in inhibiting the purified protease.

However, to test our hypothesis that cell permeability is the limiting factor for cellular potency, we treated 293 T cells with 10 µM DEL7, 14, or 25, and quantified each compound’s concentration in cells using LC-MS. The results suggested that 14, which is doubly charged, is much less permeable than DEL7 and 25, which carry only one positive charge at the primary amine (Fig. 8c). Thus, the alkyne in R1 on 25 allows high cell accumulation. This supports our hypothesis and confirms the strategy to optimize analogs with less net charge.

Structural optimization of DEL7 with restrictions on net charge led to discovery of analogs with improved antiviral potency for SARS-CoV-2

Aiming to optimize the DEL7 structure into a potent antiviral inhibitor in cellular context, we prioritized synthesizing DEL7 analogs with less net charge than 14. This strategy included modification of R1 with neutral groups and substitution of unnecessary charged groups on compounds exhibiting high potency. We therefore synthesized a panel of 25 additional analogs of DEL7 (Fig. 9a and Supplementary Figs. 5, 6).

A Structures of DEL7 analogs 27, 28, 29, 30, 31, 32, and 46, along with their IC₅₀ values against SARS-CoV-2 3CL protease activity in a biochemical assay and EC₅₀ values against SARS-CoV-2 viral replication in a Huh7^ACE2 cellular viral assay. Structure differences from DEL7 are shown in red color. B–H, The co-crystal structures of 3CL-27 (navy), 3CL-28 (dark pink), 3CL-29 (dark green), 3CL-30 (chocolate), 3CL-31 (light green), 3CL-32 (salmon), and 3CL-46 (olive) complex. The inhibitor-interacting residues of 3CL protease subunit A (cyan), subunit B (green, the other protomer of the 3CL homodimer), and subunit C (pink, a neighboring protomer in the crystal packing, not part of the 3CL homodimer) are shown with stick models. The Fo-Fc electron density omit map at 3σ is shown with blue mesh. Hydrogen bonds and hydrophobic interactions are represented by red and blue dash lines, respectively. I, The structural overlay of 3CL protease bound to DEL7 with 3CL protease bound to 15 DEL7 analogs. The shown electrostatic surface (K_bT/e = ±5.0) of 3CL-DEL7 complex was generated using program APBS⁴² and rendered in PyMOL.

Compared with DEL7 (IC₅₀ = 0.3 µM, EC₅₀ = 3.3 µM) in the biochemical and cellular assay, 27 (IC₅₀ = 0.1 µM, EC₅₀ = 0.6 µM), 28 (IC₅₀ = 0.02 µM, EC₅₀ = 0.7 µM), 29 (IC₅₀ = 0.04 µM, EC₅₀ = 0.3 µM), and 30 (IC₅₀ = 0.05 µM, EC₅₀ = 1.0 µM) exhibited substantial improvements (Fig. 9a). All these compounds feature modifications to their R1 groups.

While the alkyl chain of the 25 R1 group is elongated by two more methylene groups in 27, the tertiary amine of 14 was replaced by pyridine to generate 29 and 30, as pyridine is also nitrogen-containing but neutral at physiological pH (pKa = 5.2). Alternatively, we attempted to alter the charge of 14 by substituting its tertiary amine with pyrrolidine, a cyclic amine. The resulting analog 28 was still a potent 3CL inhibitor (IC₅₀ = 0.02 µM) but its permeability was only slightly improved, as it exhibited an EC₅₀/IC₅₀ ratio of 35 (0.7/0.02), which is the highest among these four compounds. In contrast, when we modified R1 with neutral groups containing phenyl or ether moieties to generate 31 or 32, they exhibited the lowest EC₅₀/IC₅₀ ratios of all DEL7 analogs (0.3/0.5 = 0.6 and 0.4/0.3 = 1.3). Although 31 and 32 only feature moderate potency in inhibiting the purified protease (IC₅₀ = 0.5 µM and 0.3 µM, respectively), they exhibited substantial improvements over DEL7 in the cellular antiviral assay. The correlations between net charge and EC₅₀/IC₅₀ ratio were also reflected in DEL7 analogs with other modifications of the R1 group, including imidazole (33), morpholine (34), nitrile (35 and 36), carboxylic acid (37), and sulfonyl (38 and 39), which further validated this strategy.

In addition to modifying the R1 group, we also attempted to remove the charged primary amine in the R4 site of 7, initially through masking it with acyl or sulfonyl groups. We observed a substantial loss of inhibition potency in the corresponding analogs 40, 41, 42, and 43. We tested replacement of the primary NH₂ with neutral groups OH or Cl. Analogs with hydroxyl in place of amine, 44 and 45, were much less potent than 7 (IC₅₀ = 0.01 µM) in inhibiting the purified protease (IC₅₀ = 0.5 µM and 0.6 µM). However, we found that substitution of the amino group by chloro was tolerated, as the corresponding analog 46 exhibited an IC₅₀ value of 0.01 µM; but it still exhibited a high EC₅₀/IC₅₀ ratio, suggesting modification of R1 is more efficient to develop analogs with improved cellular antiviral potency.

We determined crystal structures of 3CL protease with 27, 28, 29, 30, 31, 32, and 46, at resolution 1.90 Å, 1.71 Å, 1.52 Å, 1.71 Å, 1.55 Å, 1.57 Å, and 1.55 Å, respectively (Fig. 9b–i, Supplementary Table 11, 12, 13, 14, 15, 16, 17). Compared with the co-crystal structure with DEL7, we found that these compounds retained interactions that DEL7 featured with the active site of the protease, including the hydrogen bond or polar interactions with H41, H163, E166, and A191. Similar to what we observed with 14 and 15, the R1 groups of these compounds were engaged and stabilized by additional interactions with the active site and the interacting protomer B of the 3CL protease homodimer. Specifically, the alkyl chain in the R1 group of 46 was anchored by multiple hydrophobic interactions with G170 and surrounding side chains of residues from neighboring protomers. The pyridine in the R1 group of 29 engaged in π-π interactions with F305 of protomer B and H64 from a neighboring protease protomer (C). These additional interactions stabilized the R1 group of these compounds in the substrate binding site. The electron densities corresponding to 29 and 30 are among the highest of all DEL7 analogs that we determined. This is in line with their high potency in biochemical assays.

For consideration of therapeutic applications, we evaluated the metabolic stability of DEL7, 27, 29, and 32 in mouse liver microsomes and observed improvements of 27, 29, and 32 (T_1/2 = 11 min, 2.5 min, and 14 min, respectively) over DEL7 (T_1/2 = 0.9 min), indicating modifications of the R1 group can enhance both antiviral potency and the metabolic stability of DEL7 (Supplementary Table 18). When we further evaluated 29 for inhibition of viral replication of the Omicron variant of SARS-CoV-2 (XBB lineage, hCov-19/USA/CA-Stanford-109_S21/22 strain) in human Caco-2 cells, we found that 29 fully blocked viral infection of the Omicron variant, at concentrations as low as 0.1 µM (Supplementary Table 19).

To summarize the structural optimization of DEL7, we found that elongation of the alkyl chain of the R1 group with incorporation of nitrogen-containing groups allows extension beyond the S3 site, reaching towards the other protomer of the native homodimer of 3CL protease, engaging additional interactions with the active site and the interacting protomer, and substantially improving potency for inhibiting the purified protease (Supplementary Fig. 7). In addition, net charges on the R1 group should be eliminated to enhance cell permeability and cellular antiviral potency.

Combining both strategies, analog 29 was found to be a potent 3CL protease inhibitor that can efficiently block SARS-CoV-2 replication in cells, so it may serve as a development candidate for treating COVID-19 and other coronavirus infections. We have termed this compound hermestat, after the Greek deity Hermes, who could rapidly move between worlds, reflecting the non-covalent nature of this compound.