DataSheet1_Novel inhibitors and activity-based probes targeting serine proteases.DOCX

Serine proteases play varied and manifold roles in important biological, physiological, and pathological processes. These include viral, bacterial, and parasitic infection, allergic sensitization, tumor invasion, and metastasis. The use of activity-based profiling has been foundational in pinpointing the precise roles of serine proteases across this myriad of processes. A broad range of serine protease-targeted activity-based probe (ABP) chemotypes have been developed and we have recently introduced biotinylated and “clickable” peptides containing P1 N-alkyl glycine arginine N-hydroxy succinimidyl (NHS) carbamates as ABPs for detection/profiling of trypsin-like serine proteases. This present study provides synthetic details for the preparation of additional examples of this ABP chemotype, which function as potent irreversible inhibitors of their respective target serine protease. We describe their use for the activity-based profiling of a broad range of serine proteases including trypsin, the trypsin-like protease plasmin, chymotrypsin, cathepsin G, and neutrophil elastase (NE), including the profiling of the latter protease in clinical samples obtained from patients with cystic fibrosis.</p

A Selective Irreversible Inhibitor of Furin Does Not Prevent <i>Pseudomonas Aeruginosa</i> Exotoxin A-Induced Airway Epithelial Cytotoxicity

<div><p>Many bacterial and viral pathogens (or their toxins), including <i>Pseudomonas aeruginosa</i> exotoxin A, require processing by host pro-protein convertases such as furin to cause disease. We report the development of a novel irreversible inhibitor of furin (QUB-F1) consisting of a diphenyl phosphonate electrophilic warhead coupled with a substrate-like peptide (RVKR), that also includes a biotin tag, to facilitate activity-based profiling/visualisation. QUB-F1 displays greater selectivity for furin, in comparison to a widely used exemplar compound (furin I) which has a chloromethylketone warhead coupled to RVKR, when tested against the serine trypsin-like proteases (trypsin, prostasin and matriptase), factor Xa and the cysteine protease cathepsin B. We demonstrate QUB-F1 does not prevent <i>P</i>. <i>aeruginosa</i> exotoxin A-induced airway epithelial cell toxicity; in contrast to furin I, despite inhibiting cell surface furin-like activity to a similar degree. This finding indicates additional proteases, which are sensitive to the more broad-spectrum furin I compound, may be involved in this process.</p></div

Evaluation of the selectivity of action of QUB-F1 and furin I versus a selection of serine proteases.

<p>Trypsin (A), matriptase (B), prostasin (C), factor Xa (D), neutrophil elastase (E), proteinase 3 (F), cathepsin G (G) and chymotrypsin (H) activity was assessed using their cognate fluorogenic substrates in the presence or absence of 10 μM inhibitor (QUB-F1 or furin I) (N = 3). Data represent mean ± s.e.m.; ***P<0.001.</p

Irreversible inhibition and visualization of recombinant human furin by QUB-F1.

<p>Western blot analysis was performed under reducing and denaturing conditions demonstrating QUB-F1 (100 μM) is irreversibly bound to furin (0.4 μg). The inhibitor-proteases complex can be visualized using streptavidin-HRP, which detects the biotin reporter group on the compound.</p

Additional file 1: of MAFb protein confers intrinsic resistance to proteasome inhibitors in multiple myeloma

Supplementary data. Figure S1. described Exposure to LiCl, an inhibitor of GSB3beta led to a stabilization of MAFb protein in all 5 MM cell lines within 120 min. The half-maximum inhibitory concentration (IC50) of proteasome inhibitors, Bzb and CFZ in each tested human MM cell lines are summarized at Table S1. (DOCX 812 kb

Cortisol stimulates angiogenesis in equine vessels.

<p>(a) Light microscopy images of new vessel outgrowths [i], which stained strongly (brown) for CD31 [ii], indicating they are likely to be predominantly endothelial in nature (Scale 0.2mm). (b) Light microscopy images of equine laminar vessel sections after incubation (5 days) with DMEM [i], foetal bovine serum [ii], cortisol [iii], or FBS with cortisol [iv]. These demonstrate the stimulatory effect of cortisol on new vessel growth from equine vessels. (c) New vessel outgrowths from laminar (n = 10) [i] and facial skin vessels (n = 10) [ii] of healthy horses were quantified in the presence of DMEM, foetal bovine serum (FBS), cortisol, FBS+ cortisol, cortisol + RU486, or cortisol + spironolactone. Data are mean ± SEM for (n) horses) and were analysed by one-way ANOVA and Dunnett’s post-hoc test at each time point. * P<0.05 in comparison to DMEM.</p

GO term enrichment analysis comparing murine samples incubated with FBS (control) with those incubated with cortisol.

<p>The top 8 up-regulated and down-regulated pathways are shown.</p

KEGG enrichment analysis of pathways up- or down-regulated in equine laminar vessels in response to cortisol.

<p>Thirty six KEGG pathways were identified as significantly enriched. Eight were up-regulated (red) and 28 were down-regulated (blue).</p

Genes identified by next-generation sequencing that were down-regulated in the horse by cortisol and up-regulated in the mouse.

<p>Genes identified by next-generation sequencing that were down-regulated in the horse by cortisol and up-regulated in the mouse.</p

Genes identified by next-generation sequencing that were up-regulated in the horse by cortisol and down-regulated in the mouse.

<p>Genes identified by next-generation sequencing that were up-regulated in the horse by cortisol and down-regulated in the mouse.</p