A small molecule compound with an indole moiety inhibits the main protease of SARS-CoV-2 and blocks virus replication

The study addresses the urgent need for specific antivirals against SARS-CoV-2 beyond remdesivir. SARS-CoV-2 Mpro is essential for viral polyprotein processing and is a validated antiviral target; it is highly conserved and structurally similar to SARS-CoV Mpro (96% identity), suggesting potential cross-reactivity of prior SARS-CoV Mpro inhibitors. The authors hypothesize that two previously developed SARS-CoV Mpro inhibitors, GRL-1720 (indoline chloropyridinyl ester) and 5h (indole-based), will inhibit SARS-CoV-2 Mpro enzymatic activity and block viral replication in cells, with favorable cytotoxicity profiles. The purpose is to characterize their antiviral potency, mechanism of action, structural binding to Mpro, and potential synergy with remdesivir.

The paper builds on prior work demonstrating structural conservation of coronavirus main proteases and the development of SARS-CoV Mpro inhibitors (e.g., peptidomimetics and chloropyridyl esters). Reports have claimed activity of compounds such as shikonin and HIV-1 protease inhibitors (nelfinavir, atazanavir) against SARS-CoV-2, often based on docking or enzymatic assays, but cell-based antiviral evidence has been inconsistent. The authors reference structural studies of SARS-CoV-2 Mpro and remdesivir’s mechanism as an RdRp inhibitor, motivating combination therapy targeting distinct viral enzymes.

- Cell lines and virus: VeroE6 (JCRB), Calu-3, primary human PBMC and HBTEC; primary human airway epithelial cells in an air–liquid interface (ALI) model. SARS-CoV-2 JPN/TY/WK-521 strain used. - Antiviral assays: VeroE6 cells infected at MOI 0.05 for 1 h, virus washed, then cultured ± compounds for 3 days. Viral RNA in supernatants quantified by RT-qPCR (One Step PrimeScript III; primers/probe targeting envelope gene). Assays in duplicate. - Cytotoxicity: Continuous 3-day exposure to compounds; viability measured by Cell Counting Kit-8 in VeroE6, Calu-3, PBMC, HBTEC; CC50 determined. - Immunocytochemistry: Fixed cells stained with convalescent COVID-19 IgG (primary), anti-human IgG-Alexa Fluor 488 (secondary), phalloidin (F-actin), and DAPI; imaged on Cytation 5 to assess infected cells and cytoskeletal integrity; used to detect viral breakthrough under different drug conditions. - Combination studies: Remdesivir combined with 5h at multiple concentrations; viral RNA quantified; synergy assessed by Bliss additivism model; visualization by immunocytochemistry. - Enzymatic inhibition kinetics: SARS-CoV-2 Mpro with authentic N- and C-termini; continuous FRET-based fluorescence assay (substrate UIVT3) in HEPES buffer; for GRL-1720 (irreversible covalent) time-dependent inhibition parameters determined (kobs, Ki, kinact/Ki). Tight-binding IC50 values assessed (Morrison equation for 5h). - Structural biology: X-ray crystallography of Mpro-5h complex at 1.25 Å (asymmetric dimer), mapping binding interactions, covalent adduct with Cys145; PDB IDs 7JKV and 6XR3. - Mass spectrometry: nanoLC-ESI-QTOF-MS of Mpro after incubation with GRL-1720 or 5h (30 min and 3 h) to detect covalent mass shifts; observed +~145 Da adduct for GRL-1720; no mass shift for 5h consistent with reversible covalent binding. - Thermal stability (DSF): Differential scanning fluorimetry to measure Tm shifts of Mpro alone and with GRL-1720, 5h, or lopinavir; interpret stabilization/destabilization trends. - Molecular modeling: Docking and covalent reaction modeling of GRL-1720 with Mpro using Maestro/Glide and OPLS3 force field, to rationalize covalent binding and interactions. - Protein expression/purification and crystallization: GST-tagged Mpro expressed in E. coli, cleaved with thrombin, purified; 5h incubated with Mpro; crystals grown by hanging-drop vapor diffusion; data collected at SPring-8 and processed with DIALS/REFMAC5.

- Antiviral potency in cells: GRL-1720 and 5h blocked SARS-CoV-2WK-521 infection in VeroE6 cells with EC50 values of 15 ± 4 µM and 4.2 ± 0.7 µM, respectively. No detectable cytotoxicity for 5h up to 200 µM in Calu-3, PBMC, or HBTEC; remdesivir exhibited cytotoxicity in HBTEC with CC50 138 ± 9 µM. - Immunocytochemistry: At 100 µM, both GRL-1720 and 5h eliminated detectable infection; remdesivir reduced infection but allowed viral breakthrough at high concentrations (≥150–200 µM), whereas 5h showed no breakthrough from 20–200 µM. Shikonin was highly cytotoxic and inactive; nelfinavir and atazanavir showed no antiviral activity (nelfinavir toxic at 100 µM). - Enzymatic inhibition: GRL-1720 is an irreversible covalent Mpro inhibitor with kobs = 2.53 ± 0.27 min−1, Ki = 2.15 ± 0.49 µM, kinact/Ki = 19,610 M−1 s−1 (10-min incubation). GRL-1720 IC50 on Mpro = 0.52 ± 0.06 µM; reported tight-binding value 17.6 ± 3.2 nM. 5h acts as a tight-binding reversible covalent inhibitor by Morrison analysis. - Combination synergy: Remdesivir plus 5h showed synergy by Bliss analysis. Examples: 4 µM each yielded 24-fold suppression vs 0.53-fold (remdesivir) or 2.0-fold (5h) alone; 10 µM each yielded ~590,000-fold suppression vs 20-fold (remdesivir) and 210-fold (5h) alone; 20 µM each yielded ~1,600,000-fold suppression. Mean EC50 from this dataset: 5h 4.0 µM; remdesivir 9.0 µM. - Structural mechanism: X-ray structure (1.25 Å) shows 5h occupies all substrate pockets and forms six to eight direct hydrogen bonds (notably with Glu166, Gln189, His164, His163) and hydrophobic contacts (e.g., Leu27, Met49, Met165). The Cys145 sulfur forms a covalent adduct with 5h’s carbonyl (tetrahedral ketal), with interaction changes including loss of catalytic water and His41 repositioning. GRL-1720 covalently acylates Cys145 after chloropyridinyl group departure; MS confirmed a +~145 Da adduct corresponding to a bound indoline-4-carbonyl group. - Biophysical confirmation: DSF showed GRL-1720 decreases Mpro Tm (53.63 °C to 47.95 °C at 100 µM) consistent with covalent destabilization; 5h increases Tm (51.17 °C to 55.01 °C at 100 µM), consistent with strong binding; lopinavir decreased Tm, suggesting nonspecific destabilization. MS detected covalent adduct with GRL-1720 but not with 5h, indicating 5h forms a reversible covalent interaction under MS conditions.

Findings support the hypothesis that SARS-CoV-2 Mpro is a druggable target and that indole/indoline-based inhibitors can effectively block viral replication. Compound 5h demonstrated superior antiviral activity and a favorable cytotoxicity profile compared to GRL-1720 and remdesivir in VeroE6 assays. Importantly, detailed immunocytochemistry distinguished true antiviral effects from cytostatic/cytotoxic artifacts, refuting purported activities of shikonin and certain HIV-1 protease inhibitors under these conditions. Structural data reveal that 5h engages conserved main-chain interactions and forms a reversible covalent bond with Cys145, potentially minimizing the impact of active-site mutations and rationalizing its potency. The strong synergy between 5h (Mpro inhibitor) and remdesivir (RdRp inhibitor) indicates that combination therapy targeting distinct viral processes can dramatically enhance suppression and prevent viral breakthrough in vitro, echoing principles of combination antiviral therapy in HIV treatment.

The study identifies two SARS-CoV-2 Mpro-targeting small molecules, with 5h as a particularly promising lead showing potent antiviral activity, absence of detectable cytotoxicity up to high concentrations, and no viral breakthrough in vitro. Structural, enzymatic, and biophysical analyses provide a mechanistic basis for 5h’s efficacy, including reversible covalent engagement with Cys145 and extensive conserved interactions. Combining 5h with remdesivir yields strong synergy and complete protection from infection in vitro. Future work should optimize 5h’s pharmacological properties, assess efficacy and safety in primary human airway models and animal studies, evaluate resistance barriers, and explore clinical translation and rational combinations with polymerase inhibitors.

- The antiviral assessments are primarily in VeroE6 cells; primary human airway epithelial cells in ALI culture were poorly susceptible to SARS-CoV-2WK-521, limiting evaluation in a physiologically relevant respiratory model. - No in vivo efficacy, pharmacokinetics, or safety data are presented. - RT-qPCR antiviral assays were conducted in duplicate with limited replicates; broader statistical power and dose–response replication would strengthen conclusions. - Synergy analyses used Bliss additivity under specific conditions; additional models and time points could further validate combination effects. - The reversible covalent nature of 5h inferred from MS may depend on assay conditions; the covalent equilibrium in cellular contexts remains to be fully characterized.