fluorogenic adam10 substrate peptide  (R&D Systems)

Journal: bioRxiv

Article Title: Discovery, characterisation and optimisation of bicyclic peptide inhibitors that disarm Staphylococcus aureus α-hemolysin

doi: 10.64898/2026.03.09.710508

Figure Lengend Snippet: a) Alexa Fluor 647-labeled AhlyH35A (7.5nM) was incubated with A549 cells in the presence of increasing concentrations of Peptide 88 or a control bicyclic peptide. Cell-associated fluorescence was quantified by flow cytometry and shown as histogram overlays. Negative control (cells only) shown in black; positive control (AhlyH35A without peptide) shown in red. b) Quantification of median fluorescence intensity plotted against peptide concentration. Data are normalized to the negative and positive controls. c) ADAM10 protease activation by Ahly (6µM) was measured using a whole-cell FRET peptide cleavage assay in the presence of Peptide 88 or a control bicyclic peptide (900µM). Mean of two biological replicates; error bars indicate standard deviation. Data were analysed using one-way ANOVA with Dunnett’s test: ns = not significant; ** = P < 0.01.

Article Snippet: Following incubation, cells were washed once with 25mM Tris buffer, pH 8.0 and a fluorogenic ADAM10 substrate peptide (Mca-PLAQAV-Dpa-RSSSR-NH 2 ; R&D Systems) was added at a final concentration of 10μM.

Techniques: Labeling, Incubation, Control, Fluorescence, Flow Cytometry, Negative Control, Positive Control, Concentration Assay, Activation Assay, Cleavage Assay, Standard Deviation

Journal: bioRxiv

Article Title: Repurposed Drugs Block Toxin-Driven Platelet Clearance by the Hepatic Ashwell-Morell Receptor to Clear Staphylococcus aureus Bacteremia

doi: 10.1101/2020.07.06.190322

Figure Lengend Snippet: (A) Effect of 10 μM aspirin (ASA) and 10 μM ticagrelor (TICA) (15 min pretreatment ex vivo ) on human platelet killing of MRSA for 2 h (n = 9). Experiments performed in triplicate and repeated three times. (B) Representative transmission electron microscopy image of platelets pre-treated with or without 10 μM TICA and exposed to MRSA at MOI: 0.1 for 2 h. (C) P2Y12 inhibitor (TICA) pretreatment blocks human platelet cytotoxicity by 5 μg/ml purified α-toxin as measured by LDH release (n = 3) in a (D) dose-dependent manner. Inhibitors: P2Y12 (ticagrelor), GPIIb/IIIa (eptifibatide), COX-1 (SC560), PAR-1 (vorapaxar), and PAR-4 (ML-354). (E) TICA treatment of human platelets reduces proteolytic cleavage of an ADAM10-specific fluorogenic substrate. Data representative of three independent experiments and statistical significance determined by least squares ordinary fit, *P < 0.5. (F) Measurement of intracellular calcium in human platelets loaded with 2 μM Fluo-3 dye and stimulated with 5 μg/mL recombinant α-toxin; calcium influx is measured every 30 sec by fluorescence and normalized to baseline (non-stimulated platelet control). ( G ) TICA treatment of human platelets did not alter surface ADAM-10 expression as determined by flow cytometry. Surface ADAM-10 expression on human platelets treated with TICA or control was measured by flow cytometry. ( H ) Human platelets with or without TICA treatment were infected with MRSA at MOI = 0.1 for 90 min. Surface glycoprotein-6 (GP6) was measured by flow cytometry and % decreased expression (GP6 shedding) calculated. ( I ) Human platelet P-selectin expression indicating platelet activation measured by flow cytometry with or without MRSA challenge (MOI = 0.1) and with or without TICA aor 90 min. All data represented as mean ± SEM and are representative of at least 3 independent experiments. Statistical significance was determined by One way ANOVA with Bonferroni’s multiple comparisons test (A,C,G), unpaired two-tailed Student’s T-test (H) and two-way analysis of variance (ANOVA) with Bonferroni’s multiple comparisons posttest (I). *P< 0.05, **P < 0.005. PBS, phosphate buffered saline; ns, not significant.

Article Snippet: Human platelets (2 × 10 7 ) were pre-treated with 10μM ticagrelor (Sigma) or vehicle control and incubated at 37°C with rotation for 20 min, at which time platelets were exposed to 5 mg/mL recombinant α-toxin and a fluorogenic ADAM10 specific substrate (PEPMCA001, Biozyme) at 37°C + 5% CO2 without shaking.

Techniques: Ex Vivo, Transmission Assay, Electron Microscopy, Purification, Recombinant, Fluorescence, Expressing, Flow Cytometry, Infection, Activation Assay, Two Tailed Test