Journal: Science Advances

Article Title: Strategy to overcome a nirmatrelvir resistance mechanism in the SARS-CoV-2 nsp5 protease

doi: 10.1126/sciadv.adv8875

Figure Lengend Snippet: ( A ) Structures of nsp5 inhibitors: nirmatrelvir (NIR) and GC376. Both compounds share a lactam ring (green) and a warhead (yellow) to form a covalent bond with Cys 145 . ( B ) Nsp5 dimer structure and close-up of one of the nsp5 active sites with covalently bound NIR (PDB ID: 7RFW). Nsp5 is shown as a light gray surface, and the N terminus of the other subunit (S1′) is shown as a teal surface.

Article Snippet: For biochemical assays, SARS-CoV-2 nsp5 was cloned into the pGEX-6P-1 vector using BamHI and XhoI and then synthesized commercially (GenScript, Piscataway, NJ, USA).

Techniques:

Journal: Science Advances

Article Title: Strategy to overcome a nirmatrelvir resistance mechanism in the SARS-CoV-2 nsp5 protease

doi: 10.1126/sciadv.adv8875

Figure Lengend Snippet: The β-branched V166 sterically interacts with the tert -butyl of NIR forcing it to reposition in the E166V-nsp5 BA.1 active site. ( A ) Structure of E166V-nsp5 BA.1 (orange) in complex with NIR compared to WT-nsp5 BA.1 (blue; PDB ID: 7TLL). The alternate binding mode of NIR with a lower occupancy is shown in tan. ( B ) Rotated view of the catalytic C145 showing the increased distance between the C145 Sγ and the carbon of the nitrile warhead of the non-cross-linked binding mode (3.3 Å, which is incompatible with covalent binding). ( C ) Rotated view of the putative steric clash caused by the V166 Cγ 1 with the NIR tert -butyl group, forcing NIR to reposition.

Article Snippet: For biochemical assays, SARS-CoV-2 nsp5 was cloned into the pGEX-6P-1 vector using BamHI and XhoI and then synthesized commercially (GenScript, Piscataway, NJ, USA).

Techniques: Binding Assay

Journal: Science Advances

Article Title: Strategy to overcome a nirmatrelvir resistance mechanism in the SARS-CoV-2 nsp5 protease

doi: 10.1126/sciadv.adv8875

Figure Lengend Snippet: GC376 changes conformation in the E166V-nsp5 BA.1 and E166V-nsp5 WA1 active sites. ( A ) The benzyl group of GC376 adopts two different conformations ( IIa and IIb ; conformation IIb shown in light orange) when in complex with E166V-nsp5 BA.1 (orange), both of which point away from V166 and do not align with the GC376 conformation ( I ) seen in WT-nsp5 BA.1 (dark blue; PDB ID: 7TOB). ( B ) In E166V-nsp5 WA1 (yellow), GC376 adopts a conformation similar to IIa in E166V-nsp5 BA.1 (A). Aligned with WT-nsp5 WA1 (light blue; PDB ID: 7JSU).

Article Snippet: For biochemical assays, SARS-CoV-2 nsp5 was cloned into the pGEX-6P-1 vector using BamHI and XhoI and then synthesized commercially (GenScript, Piscataway, NJ, USA).

Techniques:

Journal: Science Advances

Article Title: Strategy to overcome a nirmatrelvir resistance mechanism in the SARS-CoV-2 nsp5 protease

doi: 10.1126/sciadv.adv8875

Figure Lengend Snippet: Covalent binding of NIR to WT-nsp5 BA.1 ( A ) but not to E166V-nsp5 BA.1 ( B ). Covalent binding of GC376 to WT-nsp5 BA.1 ( C ) and E166V-nsp5 BA.1 ( D ). Experiments were performed in triplicate at each of the three different inhibitor concentrations (50, 75, and 100 μM).

Article Snippet: For biochemical assays, SARS-CoV-2 nsp5 was cloned into the pGEX-6P-1 vector using BamHI and XhoI and then synthesized commercially (GenScript, Piscataway, NJ, USA).

Techniques: Binding Assay

Journal: Science Advances

Article Title: Strategy to overcome a nirmatrelvir resistance mechanism in the SARS-CoV-2 nsp5 protease

doi: 10.1126/sciadv.adv8875

Figure Lengend Snippet: E166V decreases the dimerization of nsp5. SEC-MALS with WT-nsp5 BA.1 and E166V-nsp5 BA.1 proteins at 60 μM ( A ) and 15 μM ( B ). ( C ) WT-nsp5 BA.1 (15 μM) in the presence of DMSO, NIR, or GC376. ( D ) E166V-nsp5 BA.1 (15 μM) in the presence of DMSO, NIR, or GC376. Points indicate the molecular weight (MW) calculated based on the light scattering and UV absorbance (normalized UV absorbance shown as a trace). The expected MWs are ~33.8 kDa for a pure monomeric population and ~67.6 kDa for a pure dimeric population.

Article Snippet: For biochemical assays, SARS-CoV-2 nsp5 was cloned into the pGEX-6P-1 vector using BamHI and XhoI and then synthesized commercially (GenScript, Piscataway, NJ, USA).

Techniques: Molecular Weight

Journal: Sensors and Actuators Reports

Article Title: A BRET-based Mpro Biosensor Containing a Nanobody and Tandem Cleavage Sites shows an Increased Cleavage Rate

doi: 10.1016/j.snr.2025.100315

Figure Lengend Snippet: Fig. 3. Characterization of Mpro biosensor constructs in living cells. (A-E) Bioluminescence spectra of the 1× (A), 2× (B), 4× (C), 8× (D) and 12× -syn (E) Mpro biosensor constructs in the absence (left panels) and in the presence (right panels) of Mpro. Data shown are from a representative of three independent experiments. (F,G) Graph showing BRET (F) and total bioluminescence (G) values obtained from cells expressing the indicated Mpro biosensor constructs in the absence and presence of Mpro. Data shown are mean ± s.d. from three in dependent experiments, with each experiment performed in triplicates. Numbers on top in (F) are percentage change in BRET (mean ± s.d.) from three independent experiments, with each experiment performed in triplicates. p values shown are adjusted p-values obtained from a two-way ANOVA (adjusted p-values; Sidak’s multiple comparisons test; in the absence and presence of Mpro). (H) Western blot analysis of the cell lysates collected from HEK 293T cells co-transfected with indicated Mpro biosensor constructs and either pLVX- EF1alpha-SARS-CoV-2-nsp5–2× Strep-IRES-Puro (Mpro WT) or FLAG-HA- pcDNA3.1 (No Mpro control) after 48 h. Top panel: anti-His tag blot showing the cleavage of 1×, 2×, 4×, 8×, and 12×-syn Mpro biosensors in the presence of SARS-CoV-2 Mpro. Bottom panel: anti-Strep-tag blot showing the expression of SARS-CoV-2 Mpro.

Article Snippet: The cells were co-transfected with either pmNG-Mpro-Nter-auto-NLuc (1×) or pmNG-Mpro-Nter-auto-2×-NLuc (2× WT) and pLVX-EF1alpha-SARSCoV-2-nsp5–2× Strep-IRES-Puro (Mpro WT) (Addgene plasmid # 141,370) at a ratio of 1:5.

Techniques: Construct, Expressing, Western Blot, Transfection, Control, Strep-tag