P2Y(1) antagonists: Combining receptor-based modeling and QSAR for a quantitative prediction of the biological activity based on consensus scoring

P2Y(1) is an ADP-activated G protein-coupled receptor (GPCR). Its antagonists impede platelet aggregation in vivo and are potential antithrombotic agents. Combining ligand and structure-based modeling we generated a consensus model (LIST-CM) correlating antagonist structures with their potencies. We docked 45 antagonists into our rhodopsin-based human P2Y(1) homology model and calculated docking scores and free binding energies with the Linear Interaction Energy (LIE) method in continuum-solvent. The resulting alignment was also used to build QSAR based on CoMFA, CoMSIA, and molecular descriptors. To benefit from the strength of each technique and compensate for their limitations, we generated our LIST-CM with a PLS regression based on the predictions of each methodology. A test set featuring untested substituents was synthesized and assayed in inhibition of 2-MeSADP-stimulated PLC activity and in radioligand binding. LIST-CM outperformed internal and external predictivity of any individual model to predict accurately the potency of 75% of the test set.

Development of Novel Activity-Based Probes for the Detection of Serine Proteases

Cystic Fibrosis (CF) is a genetic disease featuring a chronic cycle of inflammation and infection in the airways of sufferers. Mutations lead to altered ion transport, which in turn causes dehydrated airways and reduced mucociliary clearance which predisposes the patient to infection, resulting in a severe immune response and tissue destruction (1). Airway dehydration is primarily caused by the hyperabsorption of sodium by the epithelial sodium channel (ENaC) (2). ENaC is activated by the action of a number of predominantly trypsin-like Channel Activating Proteases (CAPs) including prostasin, matriptase and furin (3). Additional proteases known to activate ENaC include human airway trypsin (3), plasmin, neutrophil elastase and chymotrypsin (4).

Activity profiling is a valuable technique which involves the use of small inhibitory molecules called Activity-Based Probes (ABPs) which can be used to covalently label the active site of proteases and provide a range of information regarding its structure, catalytic mechanism, location and function within biological systems. The development of novel ABPs for CAPs, would enhance understanding of the role of these proteases in CF airways disease and in particular their role in ENaC activation and airway dehydration. This project investigates the application of a range of novel broad-spectrum ABPs targeting the various subclasses of serine proteases, to include those proteases involved in ENaC activation. Additionally, the application of more selective ABPs in detecting specific serine proteases is investigated.

Compounds were synthesised by Solid-Phase Peptide Synthesis (SPPS) using a standard Fmoc/tBu strategy. Kinetic evaluation of synthesised ABPs against various serine proteases was determined by fluorogenic steady-state enzyme assays. Furthermore, application of ABPs and confirmation of irreversible nature of the compounds was carried out through SDS-PAGE and electroblotting techniques.

Synthesised compounds showed potent irreversible inhibition of serine proteases within their respective targeting class (NAP855 vs Trypsin k3/Ki = 2.60 x 106 M-1 min-1, NFP849 vs Chymotrypsin k3/Ki = 1.28 x 106 M-1 min-1 and NVP800 vs Neutrophil Elastase k3/Ki = 6.41 x 104 M-1 min-1). Furthermore ABPs showed little to no cross-reactivity between classes and so display selectivity between classes. The irreversible nature of compounds was further demonstrated through labelling of proteases, followed by separation and detection via SDS-PAGE and electroblotting techniques. Targeted labelling of active proteases only, was demonstrated by failure of ABPs to detect previously inactivated proteases. Extension of the substrate recognition site within probes resulted in an increased potency and selectivity in the detection of the target proteases. Successful detection of neutrophil elastase from CF sputum samples by NVP800, demonstrated the application of compounds within biological samples and their potential use in identifying further proteases involved in ENaC activation and airway dehydration in CF patients.

The application of a novel, cell permeable activity-based probe for the detection of cysteine cathepsins.

Cysteine cathepsins, such as cathepsin S (CTSS), are implicated in the pathology of a wide range of diseases and are of potential utility as diagnostic and prognostic biomarkers. In previous work, we demonstrated the potency and efficiency of a biotinylated diazomethylketone (DMK)-based activity-based probe (ABP), biotin-PEG-LVG-DMK, for disclosure of recombinant CTSS and CTSS in cell lysates. However, the limited cell permeability of both the biotin and spacer groups restricted detection of CTSS to cell lysates. The synthesis and characterisation of a cell permeable ABP to report on intracellular CTSS activity is reported. The ABP, Z-PraVG-DMK, a modified peptidyl diazomethylketone, was based on the N-terminus of human cystatin motif (Leu-Val-Gly). The leucine residue was substituted for the alkyne-bearing proparcylglycine to facilitate conjugation of an azide-tagged reporter group using click chemistry, following irreversible inhibition of CTSS. When incubated with viable Human Embryonic Kidney 293 cells, Z-PraVG-DMK permitted disclosure of CTSS activity following cell lysis and rhodamine azide conjugation, by employing standard click chemistry protocols. Furthermore, the fluorescent tag facilitated direct detection of CTSS using in-gel fluorescent scanning, obviating the necessity for downstream biotin-streptavidin conjugation and detection procedures.

MicroRNA-155 Promotes Resolution of Hypoxia-Inducible Factor 1{alpha} Activity

The hypoxia-inducible factor (HIF) is a key regulator of the transcriptional response to hypoxia. While the mechanism underpinning HIF activation is well understood, little is known about its resolution. Both the protein and the mRNA levels of HIF-1a (but not HIF-2a) were decreased in intestinal epithelial cells exposed to prolonged hypoxia. Coincident with this, microRNA (miRNA) array analysis revealed multiple hypoxia-inducible miRNAs. Among these was miRNA-155 (miR-155), which is predicted to target HIF-1a mRNA. We confirmed the hypoxic upregulation of miR-155 in cultured cells and intestinal tissue from mice exposed to hypoxia. Furthermore, a role for HIF-1a in the induction of miR-155 in hypoxia was suggested by the identification of hypoxia response elements in the miR-155 promoter and confirmed experimentally. Application of miR-155 decreased the HIF-1a mRNA, protein, and transcriptional activity in hypoxia, and neutralization of endogenous miR-155 reversed the resolution of HIF-1a stabilization and activity. Based on these data and a mathematical model of HIF-1a suppression by miR-155, we propose that miR-155 induction contributes to an isoform-specific negative-feedback loop for the resolution of HIF-1a activity in cells exposed to prolonged hypoxia, leading to oscillatory behavior of HIF-1a-dependent transcription.

The persistent activity of Jupiter-family comets at 3-7 AU

We present an analysis of comet activity based on the Spitzer Space Telescope component of the Survey of the Ensemble Physical Properties of Cometary Nuclei. We show that the survey is well suited to measuring the activity of Jupiter-family comets at 3-7 AU from the Sun. Dust was detected in 33 of 89 targets (37 ± 6%), and we conclude that 21 comets (24 ± 5%) have morphologies that suggest ongoing or recent cometary activity. Our dust detections are sensitivity limited, therefore our measured activity rate is necessarily a lower limit. All comets with small perihelion distances (q <1.8 AU) are inactive in our survey, and the active comets in our sample are strongly biased to post-perihelion epochs. We introduce the quantity ɛfρ, intended to be a thermal emission counterpart to the often reported Afρ, and find that the comets with large perihelion distances likely have greater dust production rates than other comets in our survey at 3-7 AU from the Sun, indicating a bias in the discovered Jupiter-family comet population. By examining the orbital history of our survey sample, we suggest that comets perturbed to smaller perihelion distances in the past 150 yr are more likely to be active, but more study on this effect is needed.

Strategies for detection and quantification of cysteine cathepsins-evolution from bench to bedside

The cysteine cathepsins are a family of closely related thiol proteases, normally found in the endosomal and lysosomal compartments of cells. A growing body of evidence has clearly linked the dysregulated activity of these proteases with many diseases and pathological conditions, offering therapeutic, prognostic and diagnostic potential. However, these proteases are synthesised as inactive precursors and once activated, are controlled by factors such as pH and presence of endogenous inhibitors, meaning that overall protein and activity levels do not necessarily correlate. In order to fully appreciate the role and potential of these proteases, tools are required that can detect and quantify overall cathepsin activity. Two main strategies have evolved; synthetic substrates and protease-labelling with affinity-binding probes (or activity-based probes). This review examines recent innovations in these approaches as the field moves towards developing tools that could ultimately be used in patients for diagnostic or prognostic applications.

Active site labeling of cysteine cathepsins by a straightforward diazomethylketone probe derived from the N-terminus of human cystatin C

We designed a straightforward biotinylated probe using the N-terminal substrate-like region of the inhibitory site of human cystatin C as a scaffold, linked to the thiol-specific reagent diazomethylketone group as a covalent warhead (i.e. Biot-(PEG)2-Ahx-LeuValGly-DMK). The irreversible activity-based probe bound readily to cysteine cathepsins B, L, S and K. Moreover affinity labeling is sensitive since active cathepsins were detected in the nM range using an ExtrAvidin®-peroxidase conjugate for disclosure. Biot-(PEG)2-Ahx-LeuValGly-DMK allowed a slightly more pronounced labeling for cathepsin S with a compelling second-order rate constant for association (kass = 2,320,000 M−1 s−1). Labeling of the active site is dose-dependent as observed using 6-cyclohexylamine-4-piperazinyl-1,3,5-triazine-2-carbonitrile, as competitive inhibitor of cathepsins. Finally we showed that Biot-(PEG)2-Ahx-LeuValGly-DMK may be a simple and convenient tool to label secreted and intracellular active cathepsins using a myelomonocytic cell line (THP-1 cells) as model.

Extracellular cathepsin S and intracellular caspase 1 activation are surrogate biomarkers of particulate-induced lysosomal disruption in macrophages

BACKGROUND: Particulate matter has been shown to stimulate the innate immune system and induce acute inflammation. Therefore, while nanotechnology has the potential to provide therapeutic formulations with improved efficacy, there are concerns such pharmaceutical preparations could induce unwanted inflammatory side effects. Accordingly, we aim to examine the utility of using the proteolytic activity signatures of cysteine proteases, caspase 1 and cathepsin S (CTSS), as biomarkers to assess particulate-induced inflammation.

METHODS: Primary peritoneal macrophages and bone marrow-derived macrophages from C57BL/6 mice and ctss(-/-) mice were exposed to micro- and nanoparticulates and also the lysosomotropic agent, L-leucyl-L-leucine methyl ester (LLOME). ELISA and immunoblot analyses were used to measure the IL-1β response in cells, generated by lysosomal rupture. Affinity-binding probes (ABPs), which irreversibly bind to the active site thiol of cysteine proteases, were then used to detect active caspase 1 and CTSS following lysosomal rupture. Reporter substrates were also used to quantify the proteolytic activity of these enzymes, as measured by substrate turnover.

RESULTS: We demonstrate that exposure to silica, alum and polystyrene particulates induces IL-1β release from macrophages, through lysosomal destabilization. IL-1β secretion positively correlated with an increase in the proteolytic activity signatures of intracellular caspase 1 and extracellular CTSS, which were detected using ABPs and reporter substrates. Interestingly IL-1β release was significantly reduced in primary macrophages from ctss(-/-) mice.

CONCLUSIONS: This study supports the emerging significance of CTSS as a regulator of the innate immune response, highlighting its role in regulating IL-1β release. Crucially, the results demonstrate the utility of intracellular caspase 1 and extracellular CTSS proteolytic activities as surrogate biomarkers of lysosomal rupture and acute inflammation. In the future, activity-based detection of these enzymes may prove useful for the real-time assessment of particle-induced inflammation and toxicity assessment during the development of nanotherapeutics.

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

Many bacterial and viral pathogens (or their toxins), including Pseudomonas aeruginosa exotoxin A, require processing by host pro-protein convertases such as furin to cause dis- ease. We report the development of a novel irreversible inhibitor of furin (QUB-F1) consist- ing 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 com- pound (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 P. aerugi- nosa 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.