John Unitt

Pulsatile Stimulation Determines Timing and Specificity of NF-κB-Dependent Transcription

The nuclear factor κB (NF-κB) transcription factor regulates cellular stress responses and the immune response to infection. NF-κB activation results in oscillations in nuclear NF-κB abundance. To define the function of these oscillations, we treated cells with repeated short pulses of tumor necrosis factor–α at various intervals to mimic pulsatile inflammatory signals. At all pulse intervals that were analyzed, we observed synchronous cycles of NF-κB nuclear translocation. Lower frequency stimulations gave repeated full-amplitude translocations, whereas higher frequency pulses gave reduced translocation, indicating a failure to reset. Deterministic and stochastic mathematical models predicted how negative feedback loops regulate both the resetting of the system and cellular heterogeneity. Altering the stimulation intervals gave different patterns of NF-κB–dependent gene expression, which supports the idea that oscillation frequency has a functional role.

Potent Reversible Inhibition of Myeloperoxidase by Aromatic Hydroxamates

Background: Myeloperoxidase causes oxidative damage in many inflammatory diseases. Results: New substituted aromatic hydroxamates are identified as potent, selective, and reversible inhibitors of MPO. Conclusion: Binding affinities of hydroxamates to the heme pocket determine the potency of inhibition. Significance: Compounds that bind tightly to the active site of myeloperoxidase have potential as therapeutically useful inhibitors of oxidative stress. The neutrophil enzyme myeloperoxidase (MPO) promotes oxidative stress in numerous inflammatory pathologies by producing hypohalous acids. Its inadvertent activity is a prime target for pharmacological control. Previously, salicylhydroxamic acid was reported to be a weak reversible inhibitor of MPO. We aimed to identify related hydroxamates that are good inhibitors of the enzyme. We report on three hydroxamates as the first potent reversible inhibitors of MPO. The chlorination activity of purified MPO was inhibited by 50% by a 5 nm concentration of a trifluoromethyl-substituted aromatic hydroxamate, HX1. The hydroxamates were specific for MPO in neutrophils and more potent toward MPO compared with a broad range of redox enzymes and alternative targets. Surface plasmon resonance measurements showed that the strength of binding of hydroxamates to MPO correlated with the degree of enzyme inhibition. The crystal structure of MPO-HX1 revealed that the inhibitor was bound within the active site cavity above the heme and blocked the substrate channel. HX1 was a mixed-type inhibitor of the halogenation activity of MPO with respect to both hydrogen peroxide and halide. Spectral analyses demonstrated that hydroxamates can act variably as substrates for MPO and convert the enzyme to a nitrosyl ferrous intermediate. This property was unrelated to their ability to inhibit MPO. We propose that aromatic hydroxamates bind tightly to the active site of MPO and prevent it from producing hypohalous acids. This mode of reversible inhibition has potential for blocking the activity of MPO and limiting oxidative stress during inflammation.

NF-κB signalling is inhibited by glucocorticoid receptor and STAT6 via distinct mechanisms

NF-κB transcription factors are involved in the cellular response to stress, and are regulated by inhibitor (IκB) proteins, which prevent NF-κB-mediated transcription by maintaining NF-κB in the cytoplasm. Proteins from other pathways are also known to regulate NF-κB negatively, notably the glucocorticoid receptor (GR) and IL-4-responsive STAT6. Both pathways were shown to inhibit NF-κB-mediated transcription, by expressing either STAT6 or GR and activating the respective pathways. Using fluorescent fusion proteins, we show that GR alters the timing of activated p65 NF-κB nuclear occupancy by increasing the export rate of p65 and is independent of whether GR is present as a dimer or monomer. Expression of STAT6 was also shown to alter p65 nuclear occupancy but appeared to affect the import rate and hence the overall maximal level of p65 translocation. Activating STAT6 with IL-4 prior to activating NF-κB significantly increased this inhibition. Investigation of IκBa showed that activated STAT6 inhibited TNFα-mediated IκBa phosphorylation and degradation, whereas GR activation did not alter IκBαkinetics. This demonstrates a clear separation of two distinct mechanisms of inhibition by STAT6 and GR upon the NF-κB pathway.

Hit-to-lead studies: the discovery of potent, orally bioavailable triazolethiol CXCR2 receptor antagonists.

A Hit-to-Lead optimisation programme was carried out on the high throughput screening hit, the triazolethiol 1, resulting in the discovery of the potent, orally bioavailable triazolethiol CXCR2 receptor antagonist 45.

Plant lectins are novel Toll-like receptor agonists.

The T cell mitogen and plant glycoprotein, phytohaemagglutinin (PHA), is commonly used to stimulate peripheral blood mononuclear cell (PBMC) preparations to produce IL-2, IL-5, GM-CSF and IFN-γ and so provide an assay to detect immunosuppressants like FK506 and anti-inflammatories such as PDE IV inhibitors. During the early discovery of novel TLR agonists for the treatment of asthma we initially showed that PHA-L is a specific human TLR4 agonist, devoid of effects on equivalent TLR4 null cells. This TLR4 agonism was not due to LPS contamination of PHA-L, as polymyxin B was ineffective and unlike PHA-L, LPS did not stimulate TLR5 or TLR2/6. Also this specific PHA-L agonism of TLR4 was shown for different PHA forms, for example, PHA-P. This TLR lectin pharmacology finding was further explored by testing a broader panel of plant lectin representatives for agonism against a suite of hrTLR cell reporter assays (2/6, 3, 4, 5, 7, 8 and 9). Soybean agglutinin (SBA), concanavalin A (ConA) and PHA lectin family members only stimulated extracellular TLRs (2/6, 4 and 5) probably due to lack of intracellular access, whilst other lectins were either pan-active (WGA) or inactive (AIL). Interestingly SBA only stimulated TLR4, ConA, TLR2/6 and PHA-L, TLR2/6, 4 and 5. As each lectin family exhibits different sugar ligand specificity for interaction, these results suggest that the pharmacology of this TLR agonism is encoded by the lectins carbohydrate recognition motifs and the appropriate surface presentation of these motifs on different TLRs.

Discovery of small molecule human FPR1 receptor antagonists

A novel series of small molecule C5a antagonists is reported. In particular, in vitro metabolic studies and solution based combinatorial synthesis are demonstrated as useful tools for the rapid identification of antagonists with low in vitro clearance. Members of this series specifically inhibited the binding of (125)I-labeled C5a to human recombinant C5a receptor (C5aR). In functional cell assays these compounds displayed surmountable antagonism against C5a and did not demonstrate any detectable agonist activity.

Development and Validation of a Simple Cell-Based Fluorescence Assay for Dipeptidyl Peptidase 1 (DPP1) Activity

Dipeptidyl peptidase 1 (DPP1) (EC 3.4.14.1; also known as cathepsin C, cathepsin J, dipeptidyl aminopeptidase, and dipeptidyl aminotransferase) is a lysosomal cysteinyl protease of the papain family involved in the intracellular degradation of proteins. Isolated enzyme assays for DPP1 activity using a variety of synthetic substrates such as dipeptide or peptide linked to amino-methyl-coumarin (AMC) or other fluorophores are well established. There is, however, no report of a simple whole-cell-based assay for measuring lysosomal DPP1 activity other than the use of flow cytometry (fluorescence-activated cell sorting) or the use of invasive activity-based probes or the production of physiological products such as neutrophil elastase. The authors investigated a number of DPP1 fluorogenic substrates that have the potential to access the lysosome and enable the measurement of DPP1 enzyme activity in situ. They describe the development and evaluation of a simple noninvasive fluorescence assay for measuring DPP1 activity in fresh or cryopreserved human THP-1 cells using the substrate H-Gly-Phe-AFC (amino-fluoro-coumarin). This cell-based fluorescence assay can be performed in a 96-well plate format and is ideally suited for determining the cell potency of potential DPP1 enzyme inhibitors.

Development of a high-throughput human rhinovirus infectivity cell-based assay for identifying antiviral compounds

Asthma and chronic obstructive pulmonary disease exacerbations are associated with human rhinovirus (HRV) lung infections for which there are no current effective antiviral therapies. To date, HRV infectivity of cells in vitro has been measured by a variety of biochemical and immunological methods. This paper describes the development of a high-throughput HRV infectivity assay using HeLa OHIO cells and a chemiluminescent-based ATP cell viability system, CellTiter-Glo from Promega, to measure HRV-induced cytopathic effect (CPE). This CellTiter-Glo assay was validated with standard antiviral agents and employed to screen AstraZeneca compounds for potential antiviral activity. Compound potency values in this assay correlated well with the quantitative RT-PCR assay measuring HRV infectivity and replication in human primary airway epithelial cells. In order to improve pan-HRV screening capability, compound potency was also measured in the CellTiter-Glo assay with a combination of 3 different HRV serotypes. This HRV serotype combination assay could be used to identify quickly compounds with desirable broad spectrum antiviral activity.

Discovery of pyrazoles as novel FPR1 antagonists

A series of pyrazole inhibitors of the human FPR1 receptor have been identified from high throughput screening. The compounds demonstrate potent inhibition in human neutrophils and attractive physicochemical and in vitro DMPK profiles to be of further interest.

Novel cobalt complex inhibitors of mitochondrial calcium uptake.

Reperfusion of the ischaemic myocardium leads to intracellular calcium overload followed by mitochondrial dysfunction, resulting in insufficient energy supply and ultimately myocardial necrosis. Ruthenium red (RR), a potent mitochondrial calcium uptake inhibitor, prevents this disruption to mitochondrial metabolism and improves post reperfusion recovery. This therefore suggested that mitochondrial calcium influx is an attractive target for the treatment of reperfusion injury. However, RR is unsuitable for therapeutic use, so we undertook a search for novel compounds which inhibit mitochondrial calcium uptake. The most potent compounds discovered were simple tris(ethylenediamine) transition metal complexes and dinuclear Co complexes. The structure-activity relationship (SAR) of these small molecules has helped to define the structural requirements for inhibition of calcium transport by outlining the size and charge dependency of the interactive site on the mitochondrial calcium uniporter.