Development of small molecule non-covalent coronavirus 3CL protease inhibitors from DNA-encoded chemical library screening

Abstract

Variants of SARS-CoV-2 have continued to emerge across the world and cause hundreds of deaths each week. Due to the limited efficacy of vaccines against SARS-CoV-2 and resistance to current therapies, additional anti-viral therapeutics with pan-coronavirus activity are of high interest. Here, we screen 2.8 billion compounds from a DNA-encoded chemical library and identify small molecules that are non-covalent inhibitors targeting the conserved 3CL protease of SARS-CoV-2 and other coronaviruses. We perform structure-based optimization, leading to the creation of a series of potent, non-covalent SARS-CoV-2 3CL protease inhibitors, for coronavirus infections. To characterize their binding mechanism to the 3CL protease, we determine 16 co-crystal structures and find that optimized inhibitors specifically interact with both protomers of the native homodimer of 3CL protease. Since 3CL protease is catalytically competent only in the dimeric state, these data provide insight into the design of drug-like inhibitors targeting the native homodimer state. With a binding mode different from the covalent 3CL inhibitor nirmatrelvir, the protease inhibitor in the COVID drug Paxlovid, these compounds may overcome resistance reported for nirmatrelvir and complement its clinical utility.

Fig. 1. Screening of a DNA-encoded library (DELopen by WuxiAppTec) against SARS-CoV-2 3CL protease and its complex with known inhibitors.

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.

Fig. 2. DEL1 - DEL8 inhibit 3CL protease in a biochemical assay and suppress SARS-CoV-2 viral replication in a cellular assay.

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.

Fig. 3. Non-covalent binding of DEL2 and DEL7 to SARS-CoV-2 3CL protease.

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 APBS 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).

Fig. 4. Structure-activity relationship (SAR) study of DEL7.

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.

Fig. 5. Characterization of DEL7 analogs 1 and 2.

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.

Fig. 6. Analogs with modifications in the R1 group and their co-crystal structures with 3CL protease.

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 motif 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 interaction. 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.

Fig. 7. Structure-activity relationship (SAR) study on top of analog 14.

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.

Fig. 8. Evaluation of DEL7 analogs with neutral modifications in the R1 group: 25 and 26.

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.

Fig. 9. Analogs of DEL7 with neutral modifications.

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 APBS and rendered in PyMOL.