A novel, covalent broad-spectrum inhibitor targeting human coronavirus Mpro

Abstract

Human coronaviruses (CoV) cause respiratory infections that range from mild to severe. CoVs are a large family of viruses with considerable genetic heterogeneity and a multitude of viral types, making preventing and treating these viruses difficult. Comprehensive treatments that inhibit CoV infections fulfill a pressing medical need and may be immensely valuable in managing emerging and endemic CoV infections. As the main protease (M^pro) is highly conserved across many CoVs, this protease has been identified as a route for broad CoV inhibition. We utilize the advanced generative chemistry platform Chemistry42 for de novo molecular design and obtained novel small-molecule, non-peptide-like inhibitors targeting the SARS-CoV-2 M^pro. ISM3312 is identified as an irreversible, covalent M^pro inhibitor from extensive virtual screening and structure-based optimization efforts. ISM3312 exhibits low off-target risk and outstanding antiviral activity against multiple human coronaviruses, including SARS-CoV-2, MERS-CoV, 229E, OC43, NL63, and HKU1 independent of P-glycoprotein (P-gp) inhibition. Furthermore, ISM3312 shows significant inhibitory effects against Nirmatrelvir-resistant M^pro mutants, suggesting ISM3312 may contribute to reduced viral escape in these settings. Incorporating ISM3312 and Nirmatrelvir into antiviral strategy could improve preparedness and reinforce defenses against future coronavirus threats.

Fig. 1. The generative AI-powered Chemistry 42 platform designs novel SARS-CoV-2 main proteinase inhibitors.

a Workflow of Chemistry 42 platform to generate SARS-CoV-2 main proteinase inhibitors. Performance of the design rounds is provided on the right side as bar charts, where SD- and SD+ are the number of inactives and actives in the single-dose experiments respectively, Tier I shows the number of compounds in the IC₅₀ range of >10 µM, Tier II: between 10 µM and 1 µM, Tier III: 1 µM–500 nM, Tier IV: 500 nM–100 nM, Tier V: below 100 nM. The IC50 values of the most active isomers were considered only, less active stereoisomers were not considered in these statistics. b The structures of AI based M^pro inhibitors and IC₅₀ (measurement after 90 min incubation, mean ± SD values shown, n = 3) and EC₅₀ values (mean values shown; n = 2) of each compound. c In vitro ADME and pharmacokinetics data of AI-based M^pro inhibitors. Source data are provided as a Source Data file.

Fig. 2. ISM3312 exhibits pan-coronavirus and irreversible inhibitory activity against different M^pros.

a Crystal structure of ISM3312 in complex with SARS-CoV-2 M^pro. Cartoon representation of dimeric M^pro bound with ISM3312. M^pro is in grey and ISM3312 is shown as magenta ball-and-stick models in the active sites of M^pro. b ISM3312 binds to the pocket of the SARS-CoV-2 M^pro active site and interacts with the amino acid residues surrounding it by forming hydrogen bonds (shown in dashed line). The residues involved are visualized as sticks. c Enzymatic dilution assay against SARS-CoV-2 M^pro highlights the irreversible binding mechanism of ISM3312. d The predicted binding model of ISM3312 with M^pro from human coronavirus. e ISM3312 showed broad and potent inhibition against M^pro proteins from the listed coronavirus types. Measurement after 90 min incubation, mean values are shown; n = 3. Source data are provided as a Source Data file.

Fig. 3. ISM3312 exhibits potent antiviral activity against coronaviruses, including SARS-CoV-2 variants, MERS-CoV, 229E, NL63, and OC43.

a. Compound inhibition and EC50 values are calculated according to SARS-CoV-2’s CPE rates. Three independent experiments were performed with eight concentration gradients, each with quadruplicate wells. b EC₅₀ values and fold-change relative to EC₅₀ of different drugs with Elacridar (in parentheses) of different SARS-CoV-2 variants by VeroE6 CPE-based assay. c The inhibition of compounds and the value of EC₅₀ is calculated according to MERS-CoV inhibition rate by indirect immunofluorescence assay (IFA). Inhibition Rate = [1 - (Infection Rate of the Test Compound - Cell Control) / Infection Rate of the Virus Control] × 100%. Following the analysis of inhibition rates, the EC₅₀ is determined using a four-parameter fitting process. Representative images are shown on the right at 0.19 μM. Scale bar=100 μm. n = 3 biological replicates. d EC₅₀ values of respective compounds against infection with 229E (Alphacoronavirus), NL63 (Alphacoronavirus), OC43 (Betacoronavirus) using IFA. Representative images are shown on the right at 0.19 μM (229E), 0.78 μM (NL63), and 0.19 μM (OC43), respectively. n = 3 biological replicates. In the bar plots, data are presented as the mean±s.e.m. Data were analyzed by using two-way ANOVA (a, c) or a one-way ANOVA (d) followed by Tukey’s multiple comparisons test. Exact P values are reported in the figure. Source data are provided as a Source Data file.

Fig. 4. ISM3312 exhibits potent antiviral activity against various coronaviruses in human proximal airway organoid model.

Comparison of the antiviral efficacy of ISM3312, Nimatrelvir, and Ensitrelvir against SARS-CoV-2 BA.2.3 was evaluated in 2D human proximal airway organoids. Viral RNA of supernatants from the apical and basolateral chambers of the 2D proximal airway organoids was quantified by real-time PCR. Representative data from independent experiments utilizing organoids from three different donors were displayed (a), n = 3 biological replicates. Organoids were stained with anti-NP (green) and DAPI (blue). Representative immunofluorescence images of SARS-CoV-2 infected organoids at 72 h post-infection (b), with a scale bar indicating 50 μm. Anti-MERS-CoV activity measurement of ISM3312 in 2D Airway Organoids. Viral RNA in the apical and basolateral chamber supernatants were quantified by qRT–PCR (c), n = 3 biological replicates. Representative immunofluorescence images of MERS-CoV infected organoids at 72 h post-infection (d), with a scale bar indicating 50 μm. e–g Viral RNA gene copy number were detected to evaluate antiviral efficacy of ISM3312 against 229E, NL63, OC43, and HKU1 in 2D airway organoids at two concentrations (20 nM, 200 nM). 229E, NL63, and HKU1 were performed with three biological replicates, while OC43 was performed with three biological samples and three technical replicates. Representative immunofluorescence images were shown in f and g. Scale bar = 50 μm. Data were analyzed by using two-way ANOVA followed by Tukey’s multiple comparisons test (a, c, e). Data in Fig.4a, c, e are presented as mean values ± SEM. Exact P values are reported in the figure. Source data are provided as a Source Data file.

Fig. 5. Therapeutic postexposure administration of ISM3312 against SARS-CoV-2 variants and various human coronaviruses in vivo.

a Weight loss and survival curve of Nirmatrelvir and ISM3312 against ancestral SARS-CoV-2 using H11-K18-hACE2 C57BL/6 mice (Mock, n = 4 mice; other groups, n = 8 mice). Virus titers in ancestral SARS-CoV-2 infected lungs (dpi 1) and brains (dpi 3) were quantified by FFA (Mock, n = 4 mice; other groups, n = 8 mice). b Weight loss and survival curve of Nirmatrelvir and ISM3312 against SARS-CoV-2 BA.2.3 using H11-K18-hACE2 C57BL/6 mice (n = 8 mice). Virus titers in the SARS-CoV-2 BA.2.3 infected lungs (dpi 1) and brains (dpi 4) were quantified by FFA (Mock, n = 4 mice; other groups, n = 8 mice). c Virus titers in the SARS-CoV-2 XBB.1 infected lungs (dpi 1) and brains (dpi 4) were quantified by FFA (n = 5 mice). d. Weight loss and survival curve of ISM3312 against MERS-CoV mouse adapted virus MA30 using hDPP4 knockin C57BL/6 mice (n = 6 mice). Viral titers in MERS-CoV MA30 infected lungs were quantified by FFA (n = 4–5 mice). e Viral gene copy number in 229E infected lungs from hAPN KI BALB/c mice were quantified by qRT–PCR (n = 5 mice). f Viral gene copy number in NL63 infected lungs from Ad5-hACE2 sensitized IFNARKO BALB/c mice were quantified by qRT–PCR (n = 4–5 mice). g. Weight loss and survival curve of ISM3312 against OC43 using WT C57BL/6 mice (n = 5–6 mice). Viral gene copy numbers in the OC43 infected brains and spinal cords at 5 dpi were quantified by qRT–PCR (n = 4 mice). Data were analyzed by using one-way ANOVA (a, c) or two-way ANOVA followed by Tukey’s multiple comparisons test (d, g). Two-sided unpaired t-tests were performed in e and f with Welch’s correction in e (data of Dpi 3). Data are presented as mean values ± SEM. Exact P values are reported in the figure. Source data are provided as a Source Data file.

Fig. 6. ISM3312 treatment reduces the risk of drug resistance.

SARS-CoV-2 BA.2.3 was serially passaged 18 times in VeroE6 cells with escalating concentrations of ISM3312 and Nirmatrelvir and P-gp inhibitor Elacridar (n = 3 biological replicates) (Method). Inhibition of passage eighteen viruses by ISM3312 (a) and Nirmatrelvir (b). c EC₅₀ of ISM3312 and Nirmatrelvir against passage 18 resistant SARS-CoV-2. Fold change of EC₅₀ mean values relative to inhibition of Ctrl strain from three biologically independent experiments. Color coding for variant frequency: white, no resistance ( < threefold); light blue, low-level resistance (3- to 10-fold); Medium blue, moderate-level resistance (10- to 50-fold); dark blue, high-level resistance ( > 50-fold). d Mutations in M^pro of ISM3312 and Nirmatrelvir resistance from the indicated passages. Dots indicate Ctrl at that residue. Mutations are shaded according to frequency. e IC₅₀ (Compounds are compared for IC₅₀ values under identical incubation time and conditions in each mutant assay) of Nirmatrelvir, Ensitrelvir, and ISM3312 against a panel of M^pro mutants induced by Omicron (P132H) and Nirmatrelvir (Y54A, N133H, F140A, E166A, E166V, L167F, H172Y) those previous reported and ISM3312 (T21I, L50L, P252L were bold labeled) using biochemical FRET assay. Colorimetric mapping of the dAffinity value (kcal/mol) for ISM3312-M^pro complex (f) and Nirmatrelvir-M^pro complex (g) by virtual alanine scanning. Residues around the binding sites are shown. Colors range from blue (negative values, indicating increased protein-ligand affinity) to red (positive values, indicating decreased protein-ligand affinity). ISM3312 is shown in pink, and Nirmatrelvir is shown in orange. h The affinity values for four mutations, including F140A, E166A, E166V and L167F. dAffinity values of E166A and E166V mutations in the Nirmatrelvir-bound system (bold labeled) were significantly higher than those in the ISM3312-bound system. Colorimetric mapping of the dStability value (kcal/mol) for ISM3312-M^pro comple x (i) and Nirmatrelvir-M^pro complex ( j) by virtual alanine scanning. Colors range from blue (negative values, indicating increased protein stability) to red (positive values, indicating decreased protein stability). k The dStability values for 4 mutations, including F140A, E166A, E166V and L167F. Data are presented as mean values ± SEM. Source data are provided as a Source Data file.