sars-cov-2 plpro protein  (Ubiquigent)

Journal: Frontiers in Chemistry

Article Title: Insights Into Drug Repurposing, as Well as Specificity and Compound Properties of Piperidine-Based SARS-CoV-2 PLpro Inhibitors

doi: 10.3389/fchem.2022.861209

Figure Lengend Snippet: High Throughput Screen (HTS) of the ReFRAME library for inhibitors of SARS-CoV-2 PLpro. (A) Schematic showing the screening cascade for the identification of selective PLpro inhibitors. 11,804 compounds were screened at 8.33, 4.17 or 0.83 µM through a primary screen against PLpro. Hits showing a median absolute deviation >2.5 (over the DMSO negative control) were selected for a 10-point titration assays against PLpro and a counter screen against USP21 (5 nM). Of the 53 total hits from the screen, 27 compounds retested showing activity against PLpro in the 10-point titration, and 31 showed activity against USP21. Two compounds, XL-999 and C1*, were found to be selective for PLpro over USP21. (B) 10-point titration for the top 12 compounds showing activity against SARS-CoV-2 PLpro. Compounds were assayed at a top concentration of 100 µM and titrated using 1:2 (PLpro) or 1:3 (USP21) serial dilutions. 100 µM rac5c was used as a positive control for the HTS. 10-point titration curves of rac5c and rac3k were performed in a separate control assay and are shown for comparison. (C) Two selective hits from the screening of the ReFRAME library were XL-999 and C1*, with an IC 50 of 48 and 51 μM, respectively. Both were found to be weak inhibitors of PLpro and were not further investigated. 10-point titration curves of rac5c and rac3k were performed in a separate control assay and are shown for comparison. IC 50 values were derived from one set of independent experiments ( n = 1).

Article Snippet: SARS-CoV-2 PLpro WT protein and compound 5c were supplied to Ubiquigent (Dundee, United Kingdom).

Techniques: High Throughput Screening Assay, Negative Control, Titration, Activity Assay, Concentration Assay, Positive Control, Control Assay, Derivative Assay

Journal: Frontiers in Chemistry

Article Title: Insights Into Drug Repurposing, as Well as Specificity and Compound Properties of Piperidine-Based SARS-CoV-2 PLpro Inhibitors

doi: 10.3389/fchem.2022.861209

Figure Lengend Snippet: Molecular basis for inhibition of SARS-CoV-2 PLpro by 3k . (A) Structure of SARS-CoV-2 PLpro bound to 3k in green, with inhibitor in wheat colour in ball-and stick representation representing the (R)-enantiomer. A superimposed structure of apo PLpro [pink, PDB 6WZU ] shows that the inhibitor does not induce global conformational changes. Catalytic residues are shown in ball and stick representation, and a bound zinc ion in apo PLpro is shown as a grey sphere. (B) Close-up view of the ligand binding site for 3k with key residues indicated. The chemical structure of 3k is also shown, with the stereocenter labelled in red. Hydrogen bonds are indicated by yellow dotted lines. (C) 2|Fo| – |Fc| electron density map contoured at 1 σ for 3k of molecule A (top) and molecule B (bottom) in the asymmetric unit. Also see . (D) Close-up view of the ligand binding site for inhibitor 3k overlaid with ubiquitin-bound PLpro in orange [PLpro ∼ Ub, PDB 6XAA ]. The catalytic Cys111 of PLpro was mutated to a Ser (C111S) in the compound complex. 3k binding inhibits PLpro catalytic activity by blocking the C-terminus of Ub or ISG15 entering the catalytic cleft.

Article Snippet: SARS-CoV-2 PLpro WT protein and compound 5c were supplied to Ubiquigent (Dundee, United Kingdom).

Techniques: Inhibition, Ligand Binding Assay, Binding Assay, Activity Assay, Blocking Assay

Journal: Frontiers in Chemistry

Article Title: Insights Into Drug Repurposing, as Well as Specificity and Compound Properties of Piperidine-Based SARS-CoV-2 PLpro Inhibitors

doi: 10.3389/fchem.2022.861209

Figure Lengend Snippet: Molecular basis for the inhibitor specificity towards PLpro variants. (A) Close-up view of the overlay between the ligand binding sites of 3k in complex with SARS-CoV-2 PLpro (green) and SARS-CoV PLpro in salmon (PDB 4OW0 ) or MERS-CoV PLpro in teal (PDB 4RNA ). Cross reactivity by 3k between species is a consequence from the conservation of key interacting residues as indicated. Crucial differences in interacting residues underpin compound specificity. (B) 5c was tested for specificity to inhibit SARS-CoV PLpro, MERS-CoV PLpro, SARS-CoV-2 PLpro, or SARS-CoV-2 PLpro with residues 267–272 of blocking loop 2 (BL2) substituted for those in MERS-CoV (SARS-CoV-2 BL ) in a UbRh assay. Inhibitor 5c is cross-reactive with SARS-CoV PLpro and SARS-CoV-2 PLpro but not with MERS-CoV PLpro. These data indicate that engaging the conserved BL2 is crucial for the inhibition of PLpro by 5c . Experiments were performed using the HTS assay as two independent experiments ( n = 2) each containing two technical replicates. Individual data points represent the mean replicate value for each experiment. (C) SPR assays for compound 5c against PLpro variants from (B) . The top panels show double referenced sensorgram data as a function of time and the bottom show steady-state dose response curves. Absence of inhibitor 5c cross-reactivity with MERS-CoV PLpro can be explained by a loss in direct binding. Minor binding can be detected when assayed against SARS-CoV-2 BL PLpro compared with MERS-CoV PLpro and confirms most free energy loss during binding is resulting from interactions with the conserved BL2. SPR data for 3k can be found in . All SPR experiments were performed in triplicate; a representative example is shown. See for all data and for the response curves of the remaining experiments.

Article Snippet: SARS-CoV-2 PLpro WT protein and compound 5c were supplied to Ubiquigent (Dundee, United Kingdom).

Techniques: Ligand Binding Assay, Blocking Assay, Inhibition, HTS Assay, Binding Assay

Journal: Frontiers in Chemistry

Article Title: Insights Into Drug Repurposing, as Well as Specificity and Compound Properties of Piperidine-Based SARS-CoV-2 PLpro Inhibitors

doi: 10.3389/fchem.2022.861209

Figure Lengend Snippet: Medicinal Chemistry elaborations to improve piperidine based PLpro inhibitors. Subset of tested piperidine based PLpro inhibitors molecules indicating how the 5c scaffold was altered. The blue box shows alterations to the naphthyl ring, the green box shows modifications of the amide linkage and the orange box shows a subset of the most potent alterations to the benzylic group. These data exposed key insights for improving inhibitor 5c (i) tight SAR is evident around the naphthyl position, with only minor modifications able to achieve <1 µM activity; (ii) the amide and its positioning is important to compound activity; (iii) modifications at the benzylic group gave the most potent compounds. This position appears the most malleable to achieve improved potency against SARS-CoV-2 PLpro. The calculated IC 50 (µM) is noted below each compound and is the average of two or four ( 9 only) independent experiments (n = 2) each containing two technical replicates. Dose response curves can be found in .

Article Snippet: SARS-CoV-2 PLpro WT protein and compound 5c were supplied to Ubiquigent (Dundee, United Kingdom).

Techniques: Activity Assay

Journal: Frontiers in Chemistry

Article Title: Insights Into Drug Repurposing, as Well as Specificity and Compound Properties of Piperidine-Based SARS-CoV-2 PLpro Inhibitors

doi: 10.3389/fchem.2022.861209

Figure Lengend Snippet: TCID50 Assays for compound 5c , 3k and 9 . (A) Schematic and time-course of TCID50 determination. Vero cells were infected with SARS-CoV-2 containing supernatant obtained from infected Calu-3 cells, and treated as shown in the cartoon (see Methods). (B) TCID50 data, mean and standard deviation for one of two representative experiments with six technical replicates each. Compound 9 retained antiviral activity in preventing viral replication, and in stabilising the naphthyl ring appears to correlate with a modest increase in antiviral activity. Remdesivir (RDV) was used for comparison and assayed at 12.5 µM. Experiments were performed as three independent experiments ( n = 3) each containing six technical replicates. Values shown are the mean of the three independent experiments, error bars indicate the standard error of the mean. LOD = limit of detection.

Article Snippet: SARS-CoV-2 PLpro WT protein and compound 5c were supplied to Ubiquigent (Dundee, United Kingdom).

Techniques: Infection, Standard Deviation, Activity Assay