Journal: bioRxiv

Article Title: ISGylation of the SARS-CoV-2 N protein by HERC5 impedes N oligomerization and thereby viral RNA synthesis

doi: 10.1101/2024.05.15.594393

Figure Lengend Snippet: ( A ) ISGylation of FLAG-tagged N, E, and M of SARS-CoV-2 in HEK293T cells that were transfected for 24 h with either empty vector or V5-tagged ISG15 along with plasmids encoding E1 (UBE1L), E2 (UBCH8), and E3 (HERC5), determined by FLAG pull-down (PD:FLAG) and immunoblotting (IB) with anti-V5 and anti-FLAG. Whole cell lysates (WCLs) were immunoblotted with the indicated antibodies. ( B ) ISGylation of FLAG-tagged NSP16 and N in HEK293T cells that were co-transfected for 24 h with empty vector or V5-tagged PLpro WT or C111A together with E1, E2, E3, and V5-ISG15, determined by PD:FLAG and IB with anti-V5 and anti-FLAG. ( C ) ISGylation of N protein in A549-hACE2 cells that were transfected for 24 h with either control non-targeting siRNA (si.C) or ISG15-specific siRNA (si.ISG15), followed by mock treatment or stimulation with 1000 U/mL IFN-α for 24 h and subsequent infection with SARS-CoV-2 (MOI 1) for 24 h in the absence or presence of the PLpro inhibitor GRL-0617 (25 µM), determined in the WCLs by IB with anti-N (SARS-CoV-2). WCLs were further immunoblotted with the indicated antibodies. ( D ) ISGylation of FLAG-tagged N in A549 cells transfected with the indicated siRNAs for 24 h, followed by mock treatment or stimulation with 1000 U/mL IFN-α for 24 h and subsequent transfection with FLAG-N for 24 h, determined by PD:FLAG and IB with the indicated antibodies. ( E ) Binding of Myc-tagged HERC5 to FLAG-tagged N in transiently transfected HEK293T cells, determined by PD:FLAG and IB with anti-Myc and anti-FLAG. si.C, nontargeting control siRNA. Asterisks (*) in ( A ), ( C ) and ( D ) indicate non-specific bands. Arrowheads in ( A ) and ( B ) indicate non-ISGylated form of transfected proteins. Data are representative of at least two independent experiments.

Article Snippet: GRL-0617 was purchased from AdooQ Bioscience.

Techniques: Transfection, Plasmid Preparation, Western Blot, Infection, Binding Assay

Journal: bioRxiv

Article Title: Discovery and characterization of highly potent and selective covalent inhibitors of SARS-CoV-2 PLpro

doi: 10.1101/2023.05.02.539082

Figure Lengend Snippet: Summary of identification of HUP0109 and its analogs. A. Identification of HUP0109 and its analogs through medicinal chemistry campaign starting from GRL0617 and its analogs. B. PLpro enzymatic inhibition plot of GRL0617, HUP0109 and DX-027. C. Antiviral activity plot of X-027 in A549:hACE2 cells infected by the Omicron B.1.1.529 strain. The EC 50 was determined by measuring the % Spike protein as a function of inhibitor concentration. X-027 is the racemic form of HUP0109, while DX-027 is the deuterated analog of the racemic form of HUP0109.

Article Snippet: To improve efficiency and accuracy in the design of analogs, we employed virtual screening using the ICM Molsoft docking method based on a 4-dimensional docking approach utilizing a crystal structure with non-covalent inhibitor GRL0617 (PDB ID: 7JRN) and two crystal structures with peptidomimetic covalent inhibitors, VIR251 and VIR250 (6WX4 and 6WUU).

Techniques: Inhibition, Activity Assay, Infection, Concentration Assay