AI Promoted Virtual Screening, Structure-Based Hit Optimization, and Synthesis of Novel COVID-19 S-RBD Domain Inhibitors

Abstract

Coronavirus disease 2019 (COVID-19) is caused by a new, highly pathogenic severe-acute-respiratory syndrome coronavirus 2 (SARS-CoV-2) that infects human cells through its transmembrane spike (S) glycoprotein. The receptor-binding domain (RBD) of the S protein interacts with the angiotensin-converting enzyme II (ACE2) receptor of the host cells. Therefore, pharmacological targeting of this interaction might prevent infection or spread of the virus. Here, we performed a virtual screening to identify small molecules that block S-ACE2 interaction. Large compound libraries were filtered for drug-like properties, promiscuity and protein-protein interaction-targeting ability based on their ADME-Tox descriptors and also to exclude pan-assay interfering compounds. A properly designed AI-based virtual screening pipeline was applied to the remaining compounds, comprising approximately 10% of the starting data sets, searching for molecules that could bind to the RBD of the S protein. All molecules were sorted according to their screening score, grouped based on their structure and postfiltered for possible interaction patterns with the ACE2 receptor, yielding 31 hits. These hit molecules were further tested for their inhibitory effect on Spike RBD/ACE2 (19-615) interaction. Six compounds inhibited the S-ACE2 interaction in a dose-dependent manner while two of them also prevented infection of human cells from a pseudotyped virus whose entry is mediated by the S protein of SARS-CoV-2. Of the two compounds, the benzimidazole derivative CKP-22 protected Vero E6 cells from infection with SARS-CoV-2, as well. Subsequent, hit-to-lead optimization of CKP-22 was effected through the synthesis of 29 new derivatives of which compound CKP-25 suppressed the Spike RBD/ACE2 (19-615) interaction, reduced the cytopathic effect of SARS-CoV-2 in Vero E6 cells (IC₅₀ = 3.5 μM) and reduced the viral load in cell culture supernatants. Early in vitro ADME-Tox studies showed that CKP-25 does not possess biodegradation or liver metabolism issues, while isozyme-specific CYP450 experiments revealed that CKP-25 was a weak inhibitor of the CYP450 system. Moreover, CKP-25 does not elicit mutagenic effect on Escherichia coli WP2 uvrA strain. Thus, CKP-25 is considered a lead compound against COVID-19 infection.

Figure 1

S(RBD) illustration as brown cartoon with the three pillar residues shown as sticks and labeled.

Figure 2

Virtual screening. (A) Comparison between the adopted CNN-rescoring approach and Smina on the DUD-E benchmark data set. (B) Molecule ranking scores after applying our VS pipeline on the initially filtered library (∼60K molecules) and selection of the top-2% (1227).

Figure 3

(A) UMAP visualization of the finally selected hits (n = 31) compared to the top-2%, on a reduced feature space of shape descriptors. (B) Crystal structure of wt-Spike protein (PDB ID: 6M0J) colored in brown as surface representation for the RBD domain. (C) Docking solution of hit compound CKP-22 (Chembridge code: 5979349) shown as magenta-colored sticks residing in region A of the RBD domain.

Figure 4

Virtual screening workflow, structures of identified hit compounds inhibiting S(RBD)-ACE2 interaction in a dose-dependent manner and of inhibitor of SARS-CoV-2 entry hit 5979349 (CKP-22).

Figure 5

Design strategy of 5979349 (CKP-22) derivatives.

Figure 6

Overlay of COVID-19 line representation for S1 spike glycoprotein residues consisting described regions A and B for wt (PDB entry 6m0j), alpha (PDB entry 7ekf), beta (PDB entry 7ekg), gamma (PDB entry 7ekc), delta (PDB entry 7wbq), kappa (PDB entry 7tez), omicron BA.1 (PDB entry 7u0n), omicron BA.2 (PDB entry 7xo9), omicron BA.3 (PDB entry 7xb1) and omicron BA.4/5 (PDB entry 7zxu).

Scheme 1. Synthesis of 4-Nitro- and 4-Amino-N¹-Substituted Benzimidazoles

Reagents and conditions: (a) HNO₃/H₂SO₄, 0 °C, 3 h; (b) 2-(bromomethyl)naphthalene (for 3) or 4-biphenylmethyl bromide (for 4), K₂CO₃, DMF, 90 °C, 2 h; (c) SnCl₂·2H₂O, conc. HCl, gl. AcOH, rt, 1 h; (d) 2-(bromomethyl)naphthalene (for 8) or 4-biphenylmethyl bromide (for 9) or 7-(bromomethyl)quinoline hydrobromide salt (for 10), K₂CO₃, DMF, rt, 3–18 h; (e) trimethyl orthoformate, cat. PTSA, PhMe, 80 °C, 2–18 h or trimethyl orthoformate, BF₃·Et₂O, DCM, rt, 5 h.

Scheme 2. Synthesis of 4-Methoxy and 4-Hydroxy-N¹-Substituted Benzimidazoles

Reagents and conditions: (a) DPPA/TEA, t-BuOH, 100 °C, 4 h; (b) 2-(bromomethyl)naphthalene (for 19) or 4-biphenylmethyl bromide (for 20) or 7-(bromomethyl)quinoline hydrobromide salt (for 21), NaH, DMF, rt, 3 h; (c) TFA, DCM, 0 °C, 1 h; (d) SnCl₂, conc. HCl, MeOH, 50 °C, 18 h; (e) trimethyl orthoformate, cat. PTSA, PhMe, 80 °C, 2-18 h; (f) BF₃·S(CH₃)₂, DCM, rt, 24–36 h.

Scheme 3. Synthesis of Benzimidazolium Salts

Reagents and conditions: (a) 2-(bromomethyl)naphthalene (for CKP22) or 2-(2-bromoethyl)naphthalene (for 33) or 4-biphenylmethyl bromide (for 34) or 7-(bromomethyl)quinoline hydrobromide salt (for 35), 3,4-dimethoxybenzyl methanesulfonate (for 36), NaH, THF, rt, 1–3 h; (b) BF₃·S(CH₃)₂, DCM, rt, 24 h; (c) (E)-4-nitro-cinnamyl bromide (for 38, 40-43, 45) or (E)-4-chloro-cinnamyl bromide (for 39, 44, 46), 1,4-Dioxane, 100 °C, 24 h.

Figure 7

In vitro screening of computationally predicted compounds for Spike/ACE2 inhibitors. (A) Immunoblot analysis of HEK293T cells transiently transfected with expression clones encoding the PA-mCitrine-Spike RBD and cmyc-NL-ACE2 (19–615) proteins generated for the quantification of the Spike RBD/ACE2 (19–615) interaction. Immunoblots were detected with an anti-Spike antibody or an anti-ACE2 antibody. GAPDH was used as a loading control. (B) Effects of computationally predicted compounds on Spike RBD/ACE2 (19–615) interaction in LuTHy assay. The interacting proteins PA-mCitrine-Spike RBD and cmyc-NL-ACE2 (19–615) were co-produced in HEK293T cells in the presence of compounds (100 μM). The cBRET signal corresponding to Spike RBD/ACE2 (19–615) interaction was quantified 48 h post-treatment. Eleven compounds significantly reduced the interaction as compared to the cells treated with DMSO or the positive control Emodin (100 μΜ).

Figure 8

Concentration–response curves obtained for the inhibitory effect of compound 5979349 (CKP-22) in cell-based assays. Inhibition of (Α) Spike RDB/ACE2 (aa19–615) interaction in LuTHy assay and (Β) SARS-CoV-2 pseudovirus entry. Data are mean ± SD and were fitted with standard sigmoid curves for IC₅₀ determination.

Figure 9

Antiviral activity of 5979349 (CKP-22) against SARS-CoV-2 in Vero E6 cells. (A) Brightfield microscopy of Vero E6 cells. Vero E6 cells were infected with SARS-CoV-2 in the absence and presence of compound CKP-22 (50 μM). Two days post-infection, cells were monitored by microscopy. (B) CPE activity for CKP-22 in cells. Cells infected with SARS-CoV-2 were cultured in the absence or presence of CKP-22 (50 μM) for 48 h. After incubation, CPE was recorded using an inverted microscope with phase contrast. CKP-22 decreases the cytopathic effect of SARS-CoV-2 compared to cells treated only with DMSO. (C) Quantification of SARS-CoV-2 viral load in the supernatant of Vero E6 cells. Viral RNA measured by RT-qPCR in cell supernatant infected with SARS-CoV-2 (MOI of 0.01) and treated with CKP-22 or DMSO (positive control) for 48 h. Treatment with CKP-22 leads to significant reduction in viral load compared to cells treated only with DMSO.

Figure 10

Cytotoxicity and effects of the synthesized compounds on the Spike RBD/ACE2 (aa19–615) interaction. (A) Viability of HEK293T cells treated with the new compounds. Cell viability was measured 48 h later using a MTT assay. Cells treated with DMSO served as control. (B) Effect of the new compounds on Spike RBD/ACE2 (aa19–615) interaction using a cell-based quantification assay. Interaction of PA-mCitrine-Spike RBD and cmyc-NL-ACE2 (aa19–615) proteins was quantified in HEK293T cells in the presence of the new compounds used at the highest non-cytotoxic concentration. The cBRET signal corresponding to Spike RBD/ACE2 (aa19–615) interaction was quantified 48 h post treatment. Data are presented as a mean of triplicates ± SD.

Figure 11

Derivatives of 1-(2-naphthylmethyl)-1H-benzimidazole (CKP-22) protect from infection with SARS-CoV-2. (Α) Quantification (cBRET signal) of Spike-RBD/ACE2 (19–615) interaction in the presence of CKP-22 derivatives (CKP-25, CKP-27, CKP-40 and CKP-49, 25 μM final concentration) or the solvent DMSO. (Β) SARS-CoV-2 CPE in Vero E6 cells treated with the new compounds (12.5–25 μM final concentration) compared to the solvent DMSO. Τhe commercial SARS-CoV-2 inhibitor nirmatrelvir was used as a positive control. (C) SARS-CoV-2 load in the supernatant of infected Vero E6 cells treated with derivative compounds or the solvent DMSO. (D) TCID₅₀ of SARS-CoV-2 after treatment with compounds CKP-25, CKP-27, CKP-49 or the solvent DMSO. (E) Brightfield microscopy of SARS-CoV-2 infected Vero E6 cells treated with various varying concentrations (ranging from 0.01 to 25 μM) of compound CKP-25. (F) Concentration-dependent effect of compound CKP-25 inhibition in SARS-CoV-2 CPE (IC₅₀ = 3.5 ± 0.5 μΜ). Treatment with DMSO was arbitrarily set to 100%. Data in bar graphs are shown as mean ± SD (*p-value < 0.05, **p-value < 0.0.01). (G) Docking solution of compound CKP-25 shown as white colored sticks residing in region A of the RBD domain.

Figure 12

(A) Enzyme (catalytic) activity of the CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 isoenzymes upon the administration of CKP-25 at 1 μΜ (60 min). RFU, relative fluorescence units. Reaction Not Disturbed, reaction without test-compound. (B) CYP450 (%) inhibition of CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 isoenzymes upon the administration of CKP-25 at 1 μM (60 min). (C) CYP450 (%) metabolic activity of CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 isoenzymes upon the administration of CKP-25 at 1 μM (60 min).