Journal: Microorganisms

Article Title: Natural Flavonoid Derivatives Have Pan-Coronavirus Antiviral Activity.

doi: 10.3390/microorganisms11020314

Figure Lengend Snippet: Figure 1. Cell viability analysis. Different concentrations (100–0.4 µg/mL) of the selected 3CLpro inhibitors (A–F) were tested in order to assess their toxicity on Vero E6. Absorbance was reported as mean values ± SD, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: In order to examine the compounds with structural analogies to active flavonoids 7,8-Hydroxyflavone and Luteolin for their inhibitory activity against SARS-CoV-2, a preliminary screening using an enzymatic assay [3CLpro, Untagged SARS-CoV-2 Kit, AMSBio (Abingdon, UK) was performed.

Techniques:

Journal: Microorganisms

Article Title: Natural Flavonoid Derivatives Have Pan-Coronavirus Antiviral Activity.

doi: 10.3390/microorganisms11020314

Figure Lengend Snippet: Figure 2. (A–F) Antiviral activity of selected 3CLpro inhibitors. The inhibition of Omicron BA.5 infection was tested using different concentrations of the selected compounds and was assessed at 72 hpi. Mean values are reported for all the experimental replicates. Standard deviations were less than 10% of the mean values. An ordinary one-way ANOVA with a Tukey’s multiple comparisons test, with a single pooled variance, was performed. * p < 0.05 and ** p < 0.01.

Article Snippet: In order to examine the compounds with structural analogies to active flavonoids 7,8-Hydroxyflavone and Luteolin for their inhibitory activity against SARS-CoV-2, a preliminary screening using an enzymatic assay [3CLpro, Untagged SARS-CoV-2 Kit, AMSBio (Abingdon, UK) was performed.

Techniques: Activity Assay, Inhibition, Infection

Journal: Microorganisms

Article Title: Natural Flavonoid Derivatives Have Pan-Coronavirus Antiviral Activity.

doi: 10.3390/microorganisms11020314

Figure Lengend Snippet: Figure 3. Putative binding modes of the bioactive natural products investigated in this work within the catalytic site of SARS-CoV-2 3CLpro, as predicted by molecular docking simulations. (A) Baicalein; (B) Morin; (C) Hispidulin; (D) Isokaempferide; (E) Luteolin; and (F) 7,8-dihydroxyflavone. The protein is shown as a green cartoon and lines (only residues within 6 Å from the ligands are shown, the others are omitted). The ligand polar contacts are highlighted by black dashed lines. The residues contacted by the ligands are labeled. Natural products are shown as yellow sticks, and non-polar H atoms are omitted.

Article Snippet: In order to examine the compounds with structural analogies to active flavonoids 7,8-Hydroxyflavone and Luteolin for their inhibitory activity against SARS-CoV-2, a preliminary screening using an enzymatic assay [3CLpro, Untagged SARS-CoV-2 Kit, AMSBio (Abingdon, UK) was performed.

Techniques: Binding Assay, Labeling

Journal: International Journal of Molecular Sciences

Article Title: Human Superantibodies to 3CL pro Inhibit Replication of SARS-CoV-2 across Variants

doi: 10.3390/ijms23126587

Figure Lengend Snippet: Preparation of recombinant 3CL pro of SARS-CoV-2. ( A ) Amplicons of 3cl pro amplified from 3cl pro -pTriEx1.1-transformed DH5α E. coli clones. Lane M 1 kb DNA ladder; lane N, negative control (no DNA template); lanes 1, 2, 7, 9 and 10 show 3cl pro amplicons at ~1300 base pairs (bp) (arrowhead) that were amplified from DH5α E. coli clones 1, 2, 7, 9 and 10, respectively. Transformed DH5α E . coli clones 3–6 and 8 were negative for the recombinant vector. ( B ) Amplicons of 3cl pro amplified by using colonies of NiCo21 (DE3) E. coli as the direct DNA templates. Lane M, 1 kb DNA ladder; lane N, negative control (no DNA template); lane P, positive control; lanes 1–10, transformed NiCo21 (DE3) E. coli clones 1–10, respectively. Numbers at the left of ( A , B ) are DNA sizes in base pairs. ( C ) Recombinant 3CL pro produced by transformed NiCo21 (DE3) E. coli clones that contained recombinant 3cl pro -pTriEx1.1 vector. Lane M, pre-stained protein ladder; lanes 1–10, rCL pro (~34 kD, arrowhead) of 10 NiCo21 (DE3) E. coli clones. ( D ) Purified recombinant 3CL pro after SDS-PAGE and CBB staining. Lane M, pre-stained protein ladder; lane 1, crude lysate of transformed NiCo21 (DE3) E. coli ; lane 2, flow through fraction; lanes 3–12, 150 mM imidazole eluted fractions 1–10, respectively; lane 13, 1 M imidazole eluted fraction. Numbers at the left of ( C , D ) are protein masses in kDa.

Article Snippet: PEN-HuscFvs, 3CL pro inhibitor [GC376 positive control from 3CL protease, untagged (SARS-CoV-2) assay kit (BPS Bioscience, San Diego, CA, USA)], and buffer alone (negative inhibition control) were mixed separately with 20 µg of enzymatically active r3CL pro .

Techniques: Recombinant, Amplification, Transformation Assay, Clone Assay, Negative Control, Plasmid Preparation, Positive Control, Produced, Staining, Purification, SDS Page

Journal: International Journal of Molecular Sciences

Article Title: Human Superantibodies to 3CL pro Inhibit Replication of SARS-CoV-2 across Variants

doi: 10.3390/ijms23126587

Figure Lengend Snippet: Enzymatic activity of the r3CL pro tested by FRET assay using E . coli -derived recombinant CFP-TSAVLQ↓SGFRKM-YFP fusion protein as the fluorogenic substrate. ( A ) The r3CL pro (80 μg) was mixed with 5 μg of the fluorogenic substrate and kept at 25 °C for 60 min; the emitted relative fluorescence units (RFU) at 528 nm were measured at 5 min-intervals with the CFP excitation wavelength. The active 3CL pro cleaved the fluorogenic substrate resulted in a decrease of the emitted RFU at 528 nm ( y -axis) in a time-dependent manner ( x -axis). ( B ) Various amounts of r3CL pro (0.625–80 μg) were mixed with 5 μg of the fluorogenic substrate and incubated at 25 °C for 4 h. The r3CL pro cleaved the substrate in a dose-dependent manner. Inhibitor, r3CL Pro mixed with protease inhibitor of Untagged SARS-CoV-2 3CL Protease assay kit (BPS Bioscience) before adding the substrate.

Article Snippet: PEN-HuscFvs, 3CL pro inhibitor [GC376 positive control from 3CL protease, untagged (SARS-CoV-2) assay kit (BPS Bioscience, San Diego, CA, USA)], and buffer alone (negative inhibition control) were mixed separately with 20 µg of enzymatically active r3CL pro .

Techniques: Activity Assay, Derivative Assay, Recombinant, Fluorescence, Incubation, Protease Inhibitor, Protease Assay

Journal: International Journal of Molecular Sciences

Article Title: Human Superantibodies to 3CL pro Inhibit Replication of SARS-CoV-2 across Variants

doi: 10.3390/ijms23126587

Figure Lengend Snippet: HuscFvs that bound to r3CL pro by indirect ELISA. ( A ) Amplicons of HuscFv genes ( huscfvs ;) amplified from representative phage transformed-HB2151 E. coli colonies by direct colony PCR. Lane M, standard DNA marker; lanes 25–28, 30, and 33 show huscfv amplicons (1000 bp, arrowhead). Numbers at the left are DNA sizes in bp. ( B ) Western blot patterns of E-tagged-HuscFvs in lysates of representative huscfv -positive HB2151 E . coli clones (the expressed HuscFvs from some E. coli clones appeared as protein doublets at about 34–36 kDa; upper band, immature HuscFv with signal peptide; lower band, mature HuscFvs without signal peptide). ( C ) HuscFvs in lysates of 35 HB2151 E. coli clones (clones 1, 5, 7, 12, 19, 20, 25, 26, 27, 28, 30, 33, 34, 35, 36, 39, 44, 45, 51, 52, 54, 55, 57, 59, 66, 68, 72, 73, 75, 79, 80, 83, 84, 85 and 87) bound to r3CL pro and gave significant ELISA signals [optical density (OD) 405 nm to r3CL pro at least two times higher than the same lysate to the control antigen, BSA]. Broken line, arbitrarily cut-off OD 405 nm of the indirect ELISA. HB2151, lysate of original HB2151 E. coli used as negative (no antibody) binding control. Mouse anti-His was used as positive binding control (bound to 6× His 3CL pro ).

Article Snippet: PEN-HuscFvs, 3CL pro inhibitor [GC376 positive control from 3CL protease, untagged (SARS-CoV-2) assay kit (BPS Bioscience, San Diego, CA, USA)], and buffer alone (negative inhibition control) were mixed separately with 20 µg of enzymatically active r3CL pro .

Techniques: Indirect ELISA, Amplification, Transformation Assay, Marker, Western Blot, Clone Assay, Enzyme-linked Immunosorbent Assay, Binding Assay

Journal: International Journal of Molecular Sciences

Article Title: Human Superantibodies to 3CL pro Inhibit Replication of SARS-CoV-2 across Variants

doi: 10.3390/ijms23126587

Figure Lengend Snippet: Computerized homology modeling and intermolecular docking of HuscFvs and 3CL pro . ( A ) Left panel is three-dimensional structure of monomeric 3CL pro (ivory) showing important residues for homodimerization. The amino acids are shown in CINEMA color scheme: polar negative Asp and Glu are red; polar positive His, Lys, and Arg are blue; polar neutral Ser, Thr, Asn and Gln are green; non-polar aromatic Phe and Tyr are magenta; non-polar aliphatic Ala, Val, Leu, Ile, and Met are white; Pro and Gly are brown); middle panel of ( A ) shows contact interface between 3CL pro and HuscFv27 (green); right panel of ( A ) shows important interacting residues of 3CL pro and HuscFv27. ( B ) Highlight the substrate-binding sites, i.e., S1 (blue), S1′ (violet), S2 (orange) and S4 (red) of the 3CL pro (ivory). The S3 (E166) cannot be seen as it is located under the S1. ( C ) Left panel shows surface of 3CL pro (blue, red, orange and pink) that formed contact interface with HuscFv33; middle panel shows contact interface between 3CL pro and HuscFv33 (green); right panel shows important interacting residues of 3CL pro and HuscFv33. ( D ) Left panel shows surface of 3CL pro (blue, red, orange and pink) that forms contact interface with HuscFv34; middle panel shows contact interface between 3CL pro and HuscFv34 (green); right panel shows important interacting residues of 3CL pro and HuscFv34.

Article Snippet: PEN-HuscFvs, 3CL pro inhibitor [GC376 positive control from 3CL protease, untagged (SARS-CoV-2) assay kit (BPS Bioscience, San Diego, CA, USA)], and buffer alone (negative inhibition control) were mixed separately with 20 µg of enzymatically active r3CL pro .

Techniques: Binding Assay

Journal: International Journal of Molecular Sciences

Article Title: Human Superantibodies to 3CL pro Inhibit Replication of SARS-CoV-2 across Variants

doi: 10.3390/ijms23126587

Figure Lengend Snippet: SARS-CoV-2 3CL pro domains and residues that formed contact interface with the HuscFv27, HuscFv33 and HuscFv34, and the interactive bonds.

Article Snippet: PEN-HuscFvs, 3CL pro inhibitor [GC376 positive control from 3CL protease, untagged (SARS-CoV-2) assay kit (BPS Bioscience, San Diego, CA, USA)], and buffer alone (negative inhibition control) were mixed separately with 20 µg of enzymatically active r3CL pro .

Techniques: Binding Assay

Journal: International Journal of Molecular Sciences

Article Title: Human Superantibodies to 3CL pro Inhibit Replication of SARS-CoV-2 across Variants

doi: 10.3390/ijms23126587

Figure Lengend Snippet: Inhibition of 3CL pro activity by the PEN-HuscFvs as determined by FRET assay. In the assay, 20 μg of recombinant 3CL pro was mixed with buffer (negative inhibition control), inhibitor (positive inhibition control), or various amounts of PEN-HuscFvs (2.5, 5, 10 and 20 μg). Individual mixtures were added to appropriate wells of a 96-well flat and clear bottom black plate and the plate was kept on an orbital shaker at 25 °C for 30 min. The r3CL pro fluorescent substrate (5 μg) was added to each well and the plate was kept shaking for 4 h. Fluorescent signals were recorded at the excitation wavelength 436 nm and emission wavelength 528 nm using Synergy H1 Microplate reader. ( A – C ) are relative 3CL pro activities in the presence of PEN-HuscFv27, PEN-HuscFv33, and PEN-HuscFv34, respectively. ns, not significantly different; * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001.

Article Snippet: PEN-HuscFvs, 3CL pro inhibitor [GC376 positive control from 3CL protease, untagged (SARS-CoV-2) assay kit (BPS Bioscience, San Diego, CA, USA)], and buffer alone (negative inhibition control) were mixed separately with 20 µg of enzymatically active r3CL pro .

Techniques: Inhibition, Activity Assay, Recombinant

Journal: International Journal of Molecular Sciences

Article Title: Human Superantibodies to 3CL pro Inhibit Replication of SARS-CoV-2 across Variants

doi: 10.3390/ijms23126587

Figure Lengend Snippet: Biocompatibility of the PEN-HuscFvs to mammalian cells, mammalian cells expressing 3CL pro , and ability of the PEN-HuscFvs to enter mammalian cells. ( A ) Percent viability of human lung epithelial (A549) cells after exposure to 0.5–3.0 µM of PEN-HuscFv27, PEN-HuscFv33, and PEN-HuscFv34, compared to cells incubated with 100 mM DTT (cytotoxic control) and cells in culture medium alone (non-cytotoxic control; Normal). ( B ) Flow cytometric analysis of cells that expressed 3CL pro (3CL pro -HEK293T cells; dark magenta) compared to cells without 3CL pro (blue). ( C ) The 3CL pro -HEK293T cells containing intracellular 3CL pro were incubated with PEN-HuscFv27, PEN-HuscFv33, and PEN-HuscFv34. The PEN-HuscFvs (green) were found to co-localize with the intracellular 3CL pro (magenta) which then appear light pink or white upon merging. Nuclei stained blue by DAPI.

Article Snippet: PEN-HuscFvs, 3CL pro inhibitor [GC376 positive control from 3CL protease, untagged (SARS-CoV-2) assay kit (BPS Bioscience, San Diego, CA, USA)], and buffer alone (negative inhibition control) were mixed separately with 20 µg of enzymatically active r3CL pro .

Techniques: Expressing, Incubation, Staining

Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

Article Title: Design, synthesis, and biological activity evaluation of dihydromyricetin derivatives against SARS-CoV-2-Omicron virus

doi: 10.1080/14756366.2024.2390909

Figure Lengend Snippet: Inhibitory effect of compounds 1 – 10 against 3CL pro at 1.0 μM. Note: n = 3, mean ± SD. Compared with the DHM: * p < .05 and ** p < .01.

Article Snippet: The inhibitory activity of DHM derivatives against SARS-CoV-2 3CL pro was detected by using the SARS-CoV-2 3CL pro Inhibitor Screening Kit (Enhanced) #P0315M (Beyotime, China) .

Techniques:

Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

Article Title: Design, synthesis, and biological activity evaluation of dihydromyricetin derivatives against SARS-CoV-2-Omicron virus

doi: 10.1080/14756366.2024.2390909

Figure Lengend Snippet: Molecular docking score and IC 50 of compounds 1 – 10 ^a with 3CL pro .

Article Snippet: The inhibitory activity of DHM derivatives against SARS-CoV-2 3CL pro was detected by using the SARS-CoV-2 3CL pro Inhibitor Screening Kit (Enhanced) #P0315M (Beyotime, China) .

Techniques: Binding Assay

Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

Article Title: Design, synthesis, and biological activity evaluation of dihydromyricetin derivatives against SARS-CoV-2-Omicron virus

doi: 10.1080/14756366.2024.2390909

Figure Lengend Snippet: SAR of dihydromyricetin derivatives inhibition SARS-CoV-2 3CL pro .

Article Snippet: The inhibitory activity of DHM derivatives against SARS-CoV-2 3CL pro was detected by using the SARS-CoV-2 3CL pro Inhibitor Screening Kit (Enhanced) #P0315M (Beyotime, China) .

Techniques: Inhibition

Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

Article Title: Design, synthesis, and biological activity evaluation of dihydromyricetin derivatives against SARS-CoV-2-Omicron virus

doi: 10.1080/14756366.2024.2390909

Figure Lengend Snippet: Molecular docking of compounds 1 ( DHM ), 3 , 5, and 10 with 3CL pro . (A) Schematic diagram, (B) 3D enlargement, and (C) 2D schematic of the hydrogen-bond interaction between compounds 1, 3, 5, 10, and 3CL pro , respectively.

Article Snippet: The inhibitory activity of DHM derivatives against SARS-CoV-2 3CL pro was detected by using the SARS-CoV-2 3CL pro Inhibitor Screening Kit (Enhanced) #P0315M (Beyotime, China) .

Techniques:

Journal: American Journal of Physiology - Endocrinology and Metabolism

Article Title: Vitamin D and lumisterol novel metabolites can inhibit SARS-CoV-2 replication machinery enzymes

doi: 10.1152/ajpendo.00174.2021

Figure Lengend Snippet: The binding pattern of identified compounds with SARS-CoV-2 M pro . A : structural representation of the protein in complex with selected sterols and secosteroids. B : selected compounds blocking the binding pocket and making significant interactions with the functionally important residues of SARS-CoV-2 M pro . C : surface representation of conserved substrate-binding pocket of SARS-CoV-2 M pro in complex with selected compounds. D : zoomed view of the substrate-binding pocket of SARS-CoV-2 M pro in complex with selected compounds.

Article Snippet: The SARS-CoV-2 3CL Pro (M pro ), MBP-tagged Assay Kit (BPS Biosciences, San Diego, CA) was used to measure 3CL Protease (M pro ) activity for screening and profiling applications.

Techniques: Binding Assay, Blocking Assay

Journal: American Journal of Physiology - Endocrinology and Metabolism

Article Title: Vitamin D and lumisterol novel metabolites can inhibit SARS-CoV-2 replication machinery enzymes

doi: 10.1152/ajpendo.00174.2021

Figure Lengend Snippet: The binding pattern of identified sterols and secosteroids with SARS-CoV-2 RdRP. A : structural representation of the protein in complex with selected compounds. B : active site residues of the RdRP-binding pocket making significant interactions with each of the identified compounds. C surface view of the RdRP active site with the electrostatic potential from red (negative) to blue (positive) in complex with selected compounds. RdRP, RNA-dependent RNA polymerase.

Article Snippet: The SARS-CoV-2 3CL Pro (M pro ), MBP-tagged Assay Kit (BPS Biosciences, San Diego, CA) was used to measure 3CL Protease (M pro ) activity for screening and profiling applications.

Techniques: Binding Assay

Journal: Biochemical and Biophysical Research Communications

Article Title: Highly polymerized proanthocyanidins (PAC) components from blueberry leaf and stem significantly inhibit SARS-CoV-2 infection via inhibition of ACE2 and viral 3CLpro enzymes

doi: 10.1016/j.bbrc.2022.04.072

Figure Lengend Snippet: Inhibitory activity of ACE2, BSS, and virus-derived 3CL protease enzymes by BB-PAC. A. ACE2 enzyme activity by BB-PAC fractions of Fr1(dotted line) as a control and Fr7 (solid line) at several different concentrations was measured by the ACE2 activity assay kit (BPS Bioscience). The right figure shows the results using the blueberry leaf fraction and the left figure shows the results using the blueberry stem fraction. B. TMPRSS2 enzyme activity by BB-PAC fractions of Fr1(dotted line) as a control and Fr7 (solid line) at several different concentrations was measured by the TMPRSS2 activity assay kit (BPS Bioscience). C. 3CLpro enzyme activity by BB-PAC fractions of Fr1(dotted line) as a control and Fr7 (solid line) at several different concentrations was measured by the 3CLpro activity assay kit (BPS Bioscience).

Article Snippet: C. 3CLpro enzyme activity by BB-PAC fractions of Fr1(dotted line) as a control and Fr7 (solid line) at several different concentrations was measured by the 3CLpro activity assay kit (BPS Bioscience).

Techniques: Activity Assay, Derivative Assay