Journal: npj Viruses

Article Title: K5 polysaccharides inhibit SARS-CoV-2 infection by preventing spike-proteolytic priming

doi: 10.1038/s44298-025-00163-4

Figure Lengend Snippet: a Predominant composition of the disaccharide unit present in sulfated K5 derivatives is shown (see Table – for more details). Disaccharides are represented in their ionic form (sodium salt is the common counterion). b Fraction of spike bound to GAG at increasing concentrations of the indicated GAGs as measured by MST. c Inhibition of the interaction of immobilized heparin with spike by increasing concentrations of the indicated GAGs as measured by SPR. d Inhibition of the binding of spike RBD to immobilizedACE2 at increasing concentrations of the indicated GAGs as measured by SPR. For panel C and D, the responses are plotted as a percentage of the binding of spike in the absence of GAG. e Inhibition of cleavage of a peptide fragment containing the S /S 2 basic domain of spike by increasing concentrations of the indicated GAGs. The peptide was left untreated (- furin) or exposed to furin (25 ng/well) ( + furin) (red points and lanes) and after incubation in the absence and the presence of the GAG, spike cleavage was evaluated by optical density (O.D.) measurements as described in Experimental Procedures. –furin vs + furin: P value < 0.00001. Each point is the mean ± standard deviation (sd) of three to ten separate determinations (see Table for more details).

Article Snippet: Reagents and materials were used as received, unless otherwise mentioned, and were purchased from the following: Human recombinant SARS-CoV-2 Wuhan-Hu-1 spike His-Tag protein and RBD from Sino Biological (#40592-V08B); ACE2 from Acrobiosystem (#AC2-H52H8); Bovine Serum Albumin (BSA) from Merck (#810037); Human recombinant furin from OriGene Technologies Inc. (#TP304279M); Conventional heparin (13.6 kDa - purity ≥95%) from a commercial batch of unfractionated sodium heparin from Laboratori Derivati Organici S.p.A. (#9041-08-1).

Techniques: Inhibition, Binding Assay, Incubation, Standard Deviation

Journal: Cell Death Discovery

Article Title: The chaperone GRP94 interacts with the proprotein convertase furin and regulates TGF-beta maturation in human primary M2 macrophages

doi: 10.1038/s41420-025-02866-2

Figure Lengend Snippet: A Analysis by microscale thermophoresis of the specific binding between Nano-RED-labelled recombinant GRP94 (100 nM) and either recombinant furin (3.58 μM to 0.1 nM) or the negative control recombinant Hsp110 (2.33 μM to 0.07 nM). Changes in thermophoresis depending on ligand (furin or Hsp110) concentration from n = 3 experiments were plotted and expressed as ∆Fnorm (‰). B Staining and quantification of proximity ligation assays (Duolink™ PLA) of GRP94 and furin or the negative control Hsp110 in human PBMC-derived M2 macrophages ( n = 3 donors). Scale bars = 50 µm. The bar plot represents the mean number of spots for each cell analyzed +/− SEM. (**** p < 0.0001). C GRP94-furin co-immunoprecipitation in M2 macrophages. GRP94 was immunoprecipitated in M2 macrophages cell lysates using an anti-GRP94 antibody (IP:GRP94) or a non-relevant antibody (IgCT). Samples were analyzed by western-blot (representative images, n = 7).

Article Snippet: Specific binding between recombinant human furin (1503-SE, R&D Systems®, USA) and recombinant human GRP94 (RD172379100, Biovendor®, Czech Republic) was measured by MST assays.

Techniques: Microscale Thermophoresis, Binding Assay, Recombinant, Negative Control, Concentration Assay, Staining, Ligation, Derivative Assay, Immunoprecipitation, Western Blot

Journal: Cell Death Discovery

Article Title: The chaperone GRP94 interacts with the proprotein convertase furin and regulates TGF-beta maturation in human primary M2 macrophages

doi: 10.1038/s41420-025-02866-2

Figure Lengend Snippet: A Furin enzymatic activity analysis in cell lysates of MDA-MB-231 treated or not 24 h with GRP94 inhibitors, PU-WS13 12.5 or 25 µM or GRP94 inhibitor-1 (GRP94inh1) 2.5 or 5 µM ( n = 6). Fluorescence signal (expressed in relative fluorescence units, RFU) due to cleavage of the furin fluorogenic substrate, pERTKR-AMC, in cell lysates was recorded during 30 min. (**** p < 0.0001). Western- blot analysis of ( B ) active TGFβ secretion in supernatants (representative images, n = 7) and ( C ) pro-TGFβ expression in cell lysates (representative images, n = 5) from MDA-MB-231 treated or not 24 h with GRP94 inhibitors, PU-WS13 12.5 or 25 µM or GRP94 inhibitor-1 (GRP94inh1) 2.5 or 5 µM. (* p < 0.05; ** p < 0.01). D GRP94-furin co-immunoprecipitation in MDA-MB-231 cells. GRP94 was immunoprecipitated in MDA-MB-231 cell lysates using an anti-GRP94 antibody (IP:GRP94) or a non-relevant antibody (IgCT). Samples were analyzed by western-blot (representative images, n = 4). E Staining and quantification of proximity ligation assays (Duolink™ PLA) of GRP94 and furin or the negative control Hsp110 in MDA-MB-231 cells treated or not 24 h with GRP94 inhibitor-1 5 µM ( n = 3 experiments). Scale bars = 20 µm. The bar plot represents the mean number of spots for each cell analyzed +/− SEM. (**** p < 0.0001).

Article Snippet: Specific binding between recombinant human furin (1503-SE, R&D Systems®, USA) and recombinant human GRP94 (RD172379100, Biovendor®, Czech Republic) was measured by MST assays.

Techniques: Activity Assay, Fluorescence, Western Blot, Expressing, Immunoprecipitation, Staining, Ligation, Negative Control

Journal: bioRxiv

Article Title: Cathepsin K as a Key SARS-CoV-2 Cell Entry Protease and Dual-Inhibition Target

doi: 10.1101/2025.10.03.680348

Figure Lengend Snippet: (A) Schematic representation of the Spike (S) protein, highlighting the Furin and TMPRSS2 cleavage sites. The amino acid sequences of S proteins from various SARS-related coronaviruses are aligned for comparison. (B) Table compares the kinetic parameters of selected cysteine cathepsins toward FCS-Wu and FCS-Omic substrates. (C) Table compare the kinetic parameters of Furin and TMPRSS2 using two fluorogenic substrates derived from the S1/S2 site: FCS-Wu (Ac- AE (Edans) QTNSPRRARSVK (Dabcyl)) and FCS-Omic (Ac- AE (Edans) QTKSHRRARSVK (Dabcyl)). (D) Table compare the kinetic parameters of Furin, TMPRSS2, and selected cysteine cathepsins using the synthetic fluorogenic substrate TCS (Ac- AE (Edans) LPDPSKPSKRSFIK (Dabcyl). The kinetic data were obtained from two independent experiments, each performed in triplicate. Values are reported as mean ± standard deviation (SD).

Article Snippet: Recombinant human Furin and human liver cathepsin L were obtained from Fisher Scientific, Canada.

Techniques: Comparison, Derivative Assay, Standard Deviation

Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

Article Title: Development of ketobenzothiazole-based peptidomimetic TMPRSS13 inhibitors with low nanomolar potency

doi: 10.1080/14756366.2025.2466841

Figure Lengend Snippet: Selectivity heatmap of peptide inhibitors against fives targets. The heatmap displays the selectivity profiles of three compounds (N-0430, N-0130, and N-0388) tested against TMPRSS13, matriptase, Factor Xa, thrombin, and furin. The colour scale on the right ranges from red (log( K i ) = -5) to green (log( K i ) = -10), indicating the logarithmic inhibitory constant ( K i ) values for each compound-enzyme pair. Higher log( K i ) values (red) represent weaker inhibition, while lower log( K i ) values (green) indicate stronger inhibition. Data are available in Table S2 .

Article Snippet: Recombinant human furin, Factor Xa (R&D Systems) and thrombin (MilliporeSigma) were obtained from commercial sources.

Techniques: Inhibition