Journal: Molecular Pharmaceutics

Article Title: A Positron Emission Tomography Tracer Targeting the S2 Subunit of SARS-CoV-2 in Extrapulmonary Infections

doi: 10.1021/acs.molpharmaceut.2c00584

Figure Lengend Snippet: Schematic illustration of the EK1 peptide probe targeting SARS-CoV-2 infections in live subjects. (A) SARS-CoV-2 uses spike (S) protein to invade host cells, including anchor ACE2 (with S1 subunit) and membrane fusion (with S2 subunit). Compared to the mutable S1 subunit, the S2 subunit is very conservative. Also, the probes that target the heptad repeat 1 (HR1) domain of the S2 subunit are more effective. (B) To accurately mimic the disease process of COVID-19 in the BSL-2 environment, we generated a HEK293T/ACE2 cells xenograft-bearing mice model infected with the SARS-CoV-2 pseudovirus to evaluate the imaging probes for tracking the S-protein. The probe’s effectiveness for detecting extrapulmonary infection was also tested in mouse hepatic virus 59 (MHV-A59) infected C57BL/6 mice using PET/CT imaging.

Article Snippet: The competitive binding result of NOTA-EK1 was curve-fitted to a three-parameter competitive binding model using GraphPad Prism 8.0 to derive the half-maximal inhibitory concentration (IC 50 ).

Techniques: Membrane, Generated, Infection, Imaging, Virus, Positron Emission Tomography-Computed Tomography

Journal: Molecular Pharmaceutics

Article Title: A Positron Emission Tomography Tracer Targeting the S2 Subunit of SARS-CoV-2 in Extrapulmonary Infections

doi: 10.1021/acs.molpharmaceut.2c00584

Figure Lengend Snippet: Preparation and characterization of [ 64 Cu]Cu-NOTA-EK1. (A) Diagram of the radiosynthesis procedure of [ 64 Cu]-NOTA-EK1 by [ 64 Cu]CuCl 2 and precursor NOTA-EK1. (B) Radio-HPLC profiles for final purified [ 64 Cu]Cu-NOTA-EK1 (molar activity: 10.5–15.0 GBq/μmol). (C) Stability of [ 64 Cu]Cu-NOTA-EK1 was measured by radio-HPLC after incubation in saline and human serum for up to 24 h. (D) Saturation binding experiments tested the binding dissociated constant ( K d ) of Biotin-EK1 to S-protein of SARS-CoV-2. (E) IC50 values of NOTA-EK1 binding to S-protein of SARS-CoV-2 were tested in a competitive binding assay, indicating that NOTA-EK1 bind to the S-protein of SARS-CoV-2 with nanomolar affinity. (F) Cell uptake of [ 64 Cu]Cu-NOTA-EK1 in 293 T/S + and HEK293 T/S – cell lines. Data are presented as means ± SD, n = 4.

Article Snippet: The competitive binding result of NOTA-EK1 was curve-fitted to a three-parameter competitive binding model using GraphPad Prism 8.0 to derive the half-maximal inhibitory concentration (IC 50 ).

Techniques: Purification, Activity Assay, Incubation, Saline, Binding Assay, Competitive Binding Assay

Journal: Molecular Pharmaceutics

Article Title: A Positron Emission Tomography Tracer Targeting the S2 Subunit of SARS-CoV-2 in Extrapulmonary Infections

doi: 10.1021/acs.molpharmaceut.2c00584

Figure Lengend Snippet: [ 64 Cu]Cu-NOTA-EK1 targeting engaged with the S-protein of SARS-CoV-2 in HEK293T/S + cell xenograft-bearing mice. (A) Flow chart for PET/CT imaging in the HEK293T/S + cell xenograft model. (B) PET/CT imaging of HEK293T/S + xenograft-bearing mice (top panel) and HEK293T/S – control mice (bottom panel). (C) Quantification of radioactivity (SUVmax) of xenografts at 1, 4, 8, 12, and 24 h p.i. of [ 64 Cu]Cu-NOTA-EK1. (D) Xenograft-to-normal ratios ( X / N ) were significantly different between HEK293T/S + and HEK293T/S – xenograft-bearing mice at each time point. (E and F) No significant difference in the time active curve (TAC) was found between the two groups in the kidney and heart. (G) Ex vivo biodistribution of [ 64 Cu]Cu-NOTA-EK1 at 24 h postinjection. (H) H&E and IHC staining for the anti-S-protein antibody for both HEK293T/S + and HEK293T/S – xenograft tissues (scale bar: 50 μm). (I) Anti-S-protein antibody (red) and DAPI (blue) from IF staining of the HEK293T/S + and HEK293T/S – xenograft (scale bar: 50 μm). Data are presented as mean ± SD, all n = 3 (* P < 0.05, ** P < 0.01).

Article Snippet: The competitive binding result of NOTA-EK1 was curve-fitted to a three-parameter competitive binding model using GraphPad Prism 8.0 to derive the half-maximal inhibitory concentration (IC 50 ).

Techniques: Positron Emission Tomography-Computed Tomography, Imaging, Control, Radioactivity, Ex Vivo, Immunohistochemistry, Staining

Journal: Molecular Pharmaceutics

Article Title: A Positron Emission Tomography Tracer Targeting the S2 Subunit of SARS-CoV-2 in Extrapulmonary Infections

doi: 10.1021/acs.molpharmaceut.2c00584

Figure Lengend Snippet: [ 64 Cu]Cu-NOTA-EK1 targeting engaged with the S2 subunit of SARS-CoV-2 in HEK293T/ACE2 infection xenograft-bearing mice. (A) Flow chart for PET/CT imaging in the spike-pseudotyped virus-infected HEK293T/ACE2 xenograft model. (B) PET/CT imaging of the HEK293T/ACE2 xenograft model treating with 50 μL of omicron PsV + 50 μL PBS (top panel) and 100 μL of PBS (bottom panel). (C) Quantification of radioactivity (SUVmax) of xenograft tumors and normal at 1, 4, 12, and 24 h postinjection of [ 64 Cu]Cu-NOTA-EK1. (D) Xenograft-to-normal ratios ( X / N ) of the HEK293T/ACE2 xenograft model among omicron and control groups at each time point ( n = 3). (E and F) No significant difference of the TAC was found between the two groups in the kidney and heart. (G) Ex vivo biodistribution of [ 64 Cu]Cu-NOTA-EK1 at 24 h postinjection. (H) Representative images for the anti S-protein antibody (red) and DAPI (blue) from IF staining of the HEK293T/ACE2 xenograft model with different treatments (scale bar: 100 μm). Data are presented as mean ± SD, n = 3. (** P < 0.01).

Article Snippet: The competitive binding result of NOTA-EK1 was curve-fitted to a three-parameter competitive binding model using GraphPad Prism 8.0 to derive the half-maximal inhibitory concentration (IC 50 ).

Techniques: Infection, Positron Emission Tomography-Computed Tomography, Imaging, Virus, Radioactivity, Control, Ex Vivo, Staining

Journal: Molecular Pharmaceutics

Article Title: A Positron Emission Tomography Tracer Targeting the S2 Subunit of SARS-CoV-2 in Extrapulmonary Infections

doi: 10.1021/acs.molpharmaceut.2c00584

Figure Lengend Snippet: PET/CT imaging indicated that [ 64 Cu]Cu-NOTA-EK1 targeting engaged with MHV-A59 in the infection C57BL/6 mice. (A) Flow chart for PET/CT imaging in the MHV-A59 infection model. (B) Representative MIP of PET images of C57BL/6 mice intranasal injected with PBS (upper) and MHV-A59 virus (bottom). (C) Quantification of radioactivity (SUVmax) at 1, 4, 12, and 24 h postinjection of [ 64 Cu]Cu-NOTA-EK1, which were significantly different in the MHV-A59(−) mice model and MHV-A59(+) mice model at 4, 12, and 24 h ( n = 3). (D) Liver-to-normal (L/N) ratios of MHV-A59(+) mice were significantly higher than the control group in 4, 12, and 24 h. (E and F) No significant difference of the TAC was found between the two groups in the kidney and heart. (G) Ex vivo biodistribution of [ 64 Cu]-NOTA-EK1 at 24 h postinjection. (H) Representative images from H&E of the liver tissue. There were plenty of lesions in the liver tissue. Data are presented as mean ± SD, n = 3. (* P < 0.05, ** P < 0.01).

Article Snippet: The competitive binding result of NOTA-EK1 was curve-fitted to a three-parameter competitive binding model using GraphPad Prism 8.0 to derive the half-maximal inhibitory concentration (IC 50 ).

Techniques: Positron Emission Tomography-Computed Tomography, Imaging, Infection, Injection, Virus, Radioactivity, Control, Ex Vivo

Journal: bioRxiv

Article Title: Drug Repurposing for the SARS-CoV-2 Papain-Like Protease

doi: 10.1101/2021.06.04.447160

Figure Lengend Snippet: (A) Initial screening of PLpro inhibition by 33 deubiquitinase inhibitors. (B) Initial screening of PLpro inhibition by 37 cysteine protease inhibitors. Reaction conditions: 20 nM PLpro, 50 μM Z-LRGG-AMC, 200 μM inhibitor, 20 mM Tris-HCl, 300 mM NaCl, pH 7.5.

Article Snippet: The normalized PLPro activity against drug concentration was analyzed with the inhibition curve (three parameters) analysis option in GraphPad 8.0 to determine the IC 50 values.

Techniques: Inhibition

Journal: bioRxiv

Article Title: Drug Repurposing for the SARS-CoV-2 Papain-Like Protease

doi: 10.1101/2021.06.04.447160

Figure Lengend Snippet: IC 50 characterizations for 16 small molecules on their inhibition of PLpro using 50 μM LRGG-AMC as a substrate. Experiments at different conditions were performed in triplicates.

Article Snippet: The normalized PLPro activity against drug concentration was analyzed with the inhibition curve (three parameters) analysis option in GraphPad 8.0 to determine the IC 50 values.

Techniques: Inhibition

Journal: bioRxiv

Article Title: Drug Repurposing for the SARS-CoV-2 Papain-Like Protease

doi: 10.1101/2021.06.04.447160

Figure Lengend Snippet: IC 50 assays for 6 deubiquitinase inhibitors on their inhibition of 20 nM PLpro using 5 μM Ub-AMC as substrate. Experiments at different conditions were performed in triplicates.

Article Snippet: The normalized PLPro activity against drug concentration was analyzed with the inhibition curve (three parameters) analysis option in GraphPad 8.0 to determine the IC 50 values.

Techniques: Inhibition