Journal: Cell reports

Article Title: SARS-CoV-2 3CL pro (main protease) regulates caspase activation of gasdermin-D/E pores leading to secretion and extracellular activity of 3CL pro .

doi: 10.1016/j.celrep.2024.115080

Figure Lengend Snippet: Figure 1. SARS-CoV-2 3CLpro is released from infected cells by cell lysis and secreted by an unconventional protein-secretion mechanism (A) Immunoblots of 3CLpro precipitated by 33% trichloroacetic acid (TCA) from the conditioned media or in lysates from mock-infected or A549-ACE2 cells 48 hpi with SARS-CoV-2 (MOI 4, n = 5, N = 2). stds, 3CLpro standards. Densitometry of 3CLpro relative to 10 ng 3CLpro standard. (B) 3CLpro, b-tubulin, and GAPDH immunoblots and densitometry of 3CLpro in 33% TCA-precipitated conditioned media or cell lysate from mock-infected or SARS-CoV-2-infected A549-ACE2 cells 48 hpi with MOI 0.1 or 1.0. Dotted line, intracellular 3CLpro/tubulin ratio in MOI 1.0 cell lysate (n = 3/group). (C) Immunoblots and densitometry of 3CLpro in 33% TCA-precipitated conditioned media compared to matched lysates from SARS-CoV-2-infected Vero76 cells 48 hpi (MOI 1) or mock-infected cells (n = 3/group). 3CLpro ppt, 10 ng of 3CLpro precipitated from cell-naive media as a control; std, 10 ng of 3CLpro standard. (D) Immunoblots of 3CLpro in conditioned media concentrated by ultrafiltration and cell lysates from HEK293 cells transfected with 3CLpro expression plasmid for 48 h (n = 8, N = 2). (E) LDH activity in conditioned medium collected from 3CLpro transfected HEK293 cells (48 h) compared with the maximum amount of LDH released from lysed HEK293 cells (n = 6, N = 2). Absorbance was measured at 490 nm with 680 nm correction. (F) Immunoblots and densitometry of 3CLpro in concentrated conditioned medium and cell lysates from HEK293 cells transiently transfected with plasmids encoding 3CLpro or catalytic inactive 3CLpro Cys145Ala for 48 h (n = 5, N = 2). (G) Immunoblots and densitometry of 3CLpro or IFN-l1 after 24 h culture ± Golgi inhibitor 2 mM monensin (n = 6, N = 2). (H) 3CLpro, GSDMD, and b-actin loading control immunoblots and GSDMD densitometry of lysates from mock-infected or SARS-CoV-2-infected A549-ACE2 cells 48 hpi, MOI 0.1 or 1.0 (n = 3/group). (I) Schematic of candidate mechanisms of 3CLpro release from SARS-CoV-2-infected cells. Antibodies: anti-3CLpro (1:1,000, in-house), anti-b-tubulin (1:2,000, clone: BT7R), anti-b-actin (1:2,000, ab8226), anti-GAPDH (1:2,000, clone: GA1R), anti- GSDMD N-terminal antibody (1:500, clone: W18029B), and anti-FLAG M2 (IFN-l1 in G) (1:1,000, F3165). Data are shown as mean ± SD; statistical analysis of two groups was performed by two-tailed unpaired t test, three or more groups by one-way ANOVA with Tukey’s post hoc test, with a significance threshold of p < 0.05.

Article Snippet: SARS-CoV-2 3CLpro expression and purification Recombinant proteins were expressed from DNA expression vectors for active 3CLpro and inactive 3CLpro Cys145Ala control with C-terminal 3xFLAG-Myc-6xHis tags (Addgene 177334, 177335) or C-terminal 6xHis tag, purified as described5 and stored at 70 C in 3CLpro assay buffer (150 mM NaCl, 2 mM dithiothreitol (DTT), 1 mM ethylenediaminetetraacetic acid (EDTA), 50 mM Tris-HCl, pH 7.2).

Techniques: Infection, Lysis, Western Blot, Control, Transfection, Expressing, Plasmid Preparation, Activity Assay, Two Tailed Test

Journal: Cell reports

Article Title: SARS-CoV-2 3CL pro (main protease) regulates caspase activation of gasdermin-D/E pores leading to secretion and extracellular activity of 3CL pro .

doi: 10.1016/j.celrep.2024.115080

Figure Lengend Snippet: Figure 2. SARS-CoV-2 3CLpro cleaves GSDMD at LQ29Y30SS to block GSDMD pore formation, whereas caspase cleavage generates func- tional GSDMD pores that directly secrete 3CLpro and nucleocapsid protein (A) Schematic of 3CLpro cleavage sites (non-prime side [P] in blue, prime side [P0] in red) and the caspase cleavage site (violet) in human GSDMD. Y, scissile bond.

Article Snippet: SARS-CoV-2 3CLpro expression and purification Recombinant proteins were expressed from DNA expression vectors for active 3CLpro and inactive 3CLpro Cys145Ala control with C-terminal 3xFLAG-Myc-6xHis tags (Addgene 177334, 177335) or C-terminal 6xHis tag, purified as described5 and stored at 70 C in 3CLpro assay buffer (150 mM NaCl, 2 mM dithiothreitol (DTT), 1 mM ethylenediaminetetraacetic acid (EDTA), 50 mM Tris-HCl, pH 7.2).

Techniques: Blocking Assay

Journal: Cell reports

Article Title: SARS-CoV-2 3CL pro (main protease) regulates caspase activation of gasdermin-D/E pores leading to secretion and extracellular activity of 3CL pro .

doi: 10.1016/j.celrep.2024.115080

Figure Lengend Snippet: Figure 3. SARS-CoV-2 3CLpro is secreted through caspase-activated GSDMD and GSDME pores and retains activity in serum to dampen platelet activation (A) Recombinant 3CLpro (2 mM) was incubated with or without recombinant GSDME (3 mg) for 2 h at 37C and resolved on 4%–12% reducing SDS-PAGE stained with Coomassie (N = 2). 3CLpro with C-terminal 33FLAG-Myc-63His tag was used. (legend continued on next page)

Article Snippet: SARS-CoV-2 3CLpro expression and purification Recombinant proteins were expressed from DNA expression vectors for active 3CLpro and inactive 3CLpro Cys145Ala control with C-terminal 3xFLAG-Myc-6xHis tags (Addgene 177334, 177335) or C-terminal 6xHis tag, purified as described5 and stored at 70 C in 3CLpro assay buffer (150 mM NaCl, 2 mM dithiothreitol (DTT), 1 mM ethylenediaminetetraacetic acid (EDTA), 50 mM Tris-HCl, pH 7.2).

Techniques: Activity Assay, Activation Assay, Recombinant, Incubation, SDS Page, Staining

Journal: Cell reports

Article Title: SARS-CoV-2 3CL pro (main protease) regulates caspase activation of gasdermin-D/E pores leading to secretion and extracellular activity of 3CL pro .

doi: 10.1016/j.celrep.2024.115080

Figure Lengend Snippet: Figure 4. SARS-CoV-2 3CLpro cleaves and inactivates IFN-l1 (A) Coomassie-stained 4%–12% SDS-PAGE gel of recombinant human IFN-l1 (2 mg) incubated alone, with active 3CLpro, or with inactive Cys145Ala mutant (2.5 mM) for 20 h showing an 15-kDa cleavage fragment (band 5) (N = 12). Bands 1–4 were identified by Edman sequencing to be the mature N terminus of intact IFN-l1, thus indicating multiple post-translational modified proteoforms of IFN-l1. The 3CLpro-cleavage fragment also commenced with the mature N-terminal sequence (band 5), indicating C-terminal truncation. (B) Amino-terminal oriented mass spectrometry (ATOMS) identification of 3CLpro cleavage sites in IFN-l1 by MS/MS peak analysis of isotopically heavy [C13H3]2- dimethylated(*) neo-N-terminal peptides 130*ACIQPQPTAGPRPR and 155*EAPKKESAGCLEASVTFNLFR, which were identified only in 3CLpro-treated samples. (C) Time course of 3CLpro cleavage of peptides spanning the P6–P60 ILSQLQ129Y130ACIQPQ(YR) sequence of IFN-l1 (top) and a non-cleavable P1-Q129A peptide (bottom) incubated over 4 h at 37C (n = 2, N = 2). (D) Schematic of the human IFN-l1 sequence highlighting the mature N terminus (black), the non-prime P5–P1 sequence (blue), and the prime P10–P50 sequence (red) of both cleavage sites. (E) IFN-l1 ribbon diagram structural model color coded according to B-factor analysis (PDB: 3O6G).68 Helices A to E are labeled as are the first and last resolved N-terminal (S41) and C-terminal (L189) residues, respectively, and both cleavage sites at the P1-Q and P10-A/E.

Article Snippet: SARS-CoV-2 3CLpro expression and purification Recombinant proteins were expressed from DNA expression vectors for active 3CLpro and inactive 3CLpro Cys145Ala control with C-terminal 3xFLAG-Myc-6xHis tags (Addgene 177334, 177335) or C-terminal 6xHis tag, purified as described5 and stored at 70 C in 3CLpro assay buffer (150 mM NaCl, 2 mM dithiothreitol (DTT), 1 mM ethylenediaminetetraacetic acid (EDTA), 50 mM Tris-HCl, pH 7.2).

Techniques: Staining, SDS Page, Recombinant, Incubation, Mutagenesis, Sequencing, Mass Spectrometry, Tandem Mass Spectroscopy, Labeling

Journal: Journal of Biomolecular Structure & Dynamics

Article Title: Emerging role of artificial intelligence in therapeutics for COVID-19: a systematic review

doi: 10.1080/07391102.2020.1855250

Figure Lengend Snippet: Summary of artificial intelligence-based studies for therapeutics of COVID-19.

Article Snippet: Likewise, DFCNN was also applied to screen three other Targetmol compound libraries. shows top predictions among the approved drugs natural compounds and bioactive compounds that can inhibit 3CLpro of SARS-CoV-2.

Techniques: Binding Assay, Expressing, Activity Assay, Infection, Sequencing, In Silico, In Vitro, Stability Assay, High Throughput Screening Assay

Journal: Journal of Biomolecular Structure & Dynamics

Article Title: Emerging role of artificial intelligence in therapeutics for COVID-19: a systematic review

doi: 10.1080/07391102.2020.1855250

Figure Lengend Snippet: Target-wise categorization of identified drugs through AI for COVID-19 therapeutics.

Article Snippet: Likewise, DFCNN was also applied to screen three other Targetmol compound libraries. shows top predictions among the approved drugs natural compounds and bioactive compounds that can inhibit 3CLpro of SARS-CoV-2.

Techniques:

Journal: Analytical Chemistry

Article Title: Simple Nano-Luciferase-Based Assay for the Rapid and High-Throughput Detection of SARS-CoV-2 3C-Like Protease

doi: 10.1021/acs.analchem.2c02590

Figure Lengend Snippet: Principle of this assay for 3CLpro detection. (A) MBs-peptide-nLuc was added into PBS containing 3CLpro standards (or virus lysate). (B) Peptide on the surface of MBs-peptide-nLuc was cleaved by 3CLpro, and nLuc was released into the supernatant. (C) Supernatant was transferred into the 96-well plate pre-added with the luciferase substrate, and (D) the signal of each well was detected using the microplate reader.

Article Snippet: 3CLpro-His recombinant protein was purchased from Sino Biological Inc. HRP conjugated goat anti-mouse IgG (H+L), matrix metalloprotein 9 (MMP 9), and caspase 9 were purchased from Proteintech Group, Inc. Ultrasensitive ECL substrate was purchased from Thermo.

Techniques: Luciferase

Journal: Analytical Chemistry

Article Title: Simple Nano-Luciferase-Based Assay for the Rapid and High-Throughput Detection of SARS-CoV-2 3C-Like Protease

doi: 10.1021/acs.analchem.2c02590

Figure Lengend Snippet: Selection and optimization of the peptide substrate sequence of 3CLpro. (A) SARS-CoV-2 genome and 3CLpro potential restriction sites. (B) Bioinformation analysis revealed 11 potential cleavage sites of 3CLpro. (C) Prediction of the most likely amino acid sequence of the 3CLpro hydrolysis site.

Article Snippet: 3CLpro-His recombinant protein was purchased from Sino Biological Inc. HRP conjugated goat anti-mouse IgG (H+L), matrix metalloprotein 9 (MMP 9), and caspase 9 were purchased from Proteintech Group, Inc. Ultrasensitive ECL substrate was purchased from Thermo.

Techniques: Selection, Sequencing

Journal: Analytical Chemistry

Article Title: Simple Nano-Luciferase-Based Assay for the Rapid and High-Throughput Detection of SARS-CoV-2 3C-Like Protease

doi: 10.1021/acs.analchem.2c02590

Figure Lengend Snippet: Optimization of experimental parameters. (A) Feasibility of this assay for 3CLpro detection. The incubation time of GST-peptide-nLuc with 3CLpro was 7.5 min, 15 min, 30 min, 1 h, 2 h, 3 h, 4 h, 5 h, and 6 h, respectively. 3CLpro: 200 ng/mL. (B) Maximum binding quantity of GST-peptide-nLuc to the surface of 1 μg of GSH-MBs. (C) Effect of reaction buffer, and (D) the different incubation times of 3CLpro with GST-peptide-nLuc on signal intensity. 3CLpro: 200 ng/mL. The error bars represent the standard deviation of three independent measurements.

Article Snippet: 3CLpro-His recombinant protein was purchased from Sino Biological Inc. HRP conjugated goat anti-mouse IgG (H+L), matrix metalloprotein 9 (MMP 9), and caspase 9 were purchased from Proteintech Group, Inc. Ultrasensitive ECL substrate was purchased from Thermo.

Techniques: Incubation, Binding Assay, Standard Deviation

Journal: Analytical Chemistry

Article Title: Simple Nano-Luciferase-Based Assay for the Rapid and High-Throughput Detection of SARS-CoV-2 3C-Like Protease

doi: 10.1021/acs.analchem.2c02590

Figure Lengend Snippet: (A) Sensitivity of this assay for 3CLpro detection. Statistical analysis was performed using Student’s t -test. ** P ≤ 0.01. (B) Plots of signal vs the log values of different concentrations of 3CLpro. (C) Specificity of this assay for 3CLpro detection. 3CLpro: 20 ng/mL, MMP 9: 100 ng/mL, Caspase 9: 100 ng/mL, Trypsin: 10 μg/mL. (D) Inhibition efficiencies of different concentrations of Ebselen on the activity of 3CLpro. 3CLpro: 200 ng/mL. The error bars represent the standard deviation of three independent measurements.

Article Snippet: 3CLpro-His recombinant protein was purchased from Sino Biological Inc. HRP conjugated goat anti-mouse IgG (H+L), matrix metalloprotein 9 (MMP 9), and caspase 9 were purchased from Proteintech Group, Inc. Ultrasensitive ECL substrate was purchased from Thermo.

Techniques: Inhibition, Activity Assay, Standard Deviation