Effect-based monitoring of two rivers under urban and agricultural influence reveals a range of biological activities in sediment and water extracts.

Chemical contaminants, such as pesticides, pharmaceuticals and industrial compounds are ubiquitous in surface water and sediment in areas subject to human activity. While targeted chemical analysis is typically used for water and sediment quality monitoring, there is growing interest in applying effect-based methods with in vitro bioassays to capture the effects of all active contaminants in a sample. The current study evaluated the biological effects in surface water and sediment from two contrasting catchments in Aotearoa New Zealand, the highly urbanised Whau River catchment in Tamaki Makaurau (Auckland) and the urban and mixed agricultural Koreti (New River) Estuary catchment. Two complementary passive sampling devices, Chemcatcher for polar chemicals and polyethylene (PED) for non-polar chemicals, were applied to capture a wide range of contaminants in water, while composite sediment samples were collected at each sampling site. Bioassays indicative of induction of xenobiotic metabolism, receptor-mediated effects, genotoxicity, cytotoxicity and apical effects were applied to the water and sediment extracts. Most sediment extracts induced moderate to strong estrogenic and aryl hydrocarbon (AhR) activity, along with moderate toxicity to bacteria. The water extracts showed similar patterns to the sediment extracts, but with lower activity. Generally, the polar Chemcatcher extracts showed greater estrogenic activity, photosynthesis inhibition and algal growth inhibition than the non-polar PED extracts, though the PED extracts showed greater AhR activity. The observed effects in the water extracts were compared to available ecological effect-based trigger values (EBT) to evaluate the potential risk. For the polar extracts, most sites in both catchments exceeded the EBT for estrogenicity, with many sites exceeding the EBTs for AhR activity and photosynthesis inhibition. Of the wide range of endpoints considered, estrogenic activity, AhR activity and herbicidal activity appear to be the primary risk drivers in both the Whau and Koreti Estuary catchments.

Inhibition of interleukin-1beta converting enzyme (ICE or caspase 1) by aspartyl acyloxyalkyl ketones and aspartyl amidooxyalkyl ketones.

A series of acyloxyalkyl and amidooxyalkyl ketones appended to a carbobenzyloxy aspartic acid core have been prepared. The most potent of these new inhibitors was 4i with a K(i) of 0.5 microM. These two series provide an improved understanding of the binding requirements for the hydrophobic prime side of ICE.

Succinic acid amides as P2-P3 replacements for inhibitors of interleukin-1beta converting enzyme (ICE or caspase 1).

Succinic acid amides have been found to be effective P2-P3 scaffold replacements for peptidic ICE inhibitors. Heteroarylalkyl fragments occupying the P4 position provided access to compounds with nM affinities. Utilization of an acylal prodrug moiety was required to overcome biopharmaceutical issues which led to the identification of 17f, a potential clinical candidate.

Identification of a selective thieno[2,3-c]pyridine inhibitor of COT kinase and TNF-alpha production.

COT (Tpl2 in mice) is a serine/threonine MAP3 kinase that regulates production of TNF-alpha and other pro-inflammatory cytokines such as IL-1beta via the ERK/MAP kinase pathway. As TNF-alpha and IL-1beta are clinically validated targets for therapeutic intervention in rheumatoid arthritis (RA), blocking COT provides a potential avenue for amelioration of disease. Herein we describe identification of a cellular active selective small molecule inhibitor of COT kinase.

Discovery of thieno[2,3-c]pyridines as potent COT inhibitors.

Evaluation of hit chemotypes from high throughput screening identified a novel series of 2,4-disubstituted thieno[2,3-c]pyridines as COT kinase inhibitors. Structural modifications exploring SAR at the 2- and 4-positions resulting in inhibitors with improved enzyme potency and cellular activity are disclosed.

Comparative analysis of various in vitro COT kinase assay formats and their applications in inhibitor identification and characterization.

Cancer osaka thyroid (COT) is a member of the mitogen-activated protein kinase kinase kinase family of enzymes and plays a pivotal role in tumor necrosis factor-alpha production in macrophages. Consequently, COT is considered to be a promising target for antiinflammatory drug discovery. We describe here the development of in vitro COT assays in several formats and the advantages and disadvantages of each. A cascade assay requires very small amounts of enzyme and can provide a useful tool for high-throughput screening, but it is not desirable for compound mechanistic studies due to complicated kinetics. Direct assays are superior to cascade assays and are suitable for both compound screening and mechanistic studies. Among the direct assays, the homogeneous time-resolved fluorescence (HTRF) format is preferred over the radiometric format due to the robustness, throughput, and ease of use of the HTRF format. When the physiological protein substrate MEK1 (MAP/Erk kinase 1) was used to determine inhibitor potencies, false positives were observed due to compound interference by binding to MEK1. Using a MEK1 peptide substrate, these false positives were eliminated. In addition, we describe a simple method to study the ATP competitiveness of compounds. The knowledge gained through our studies with COT, and the methods described for our assays and compound mechanistic studies, can be readily applied to other kinase targets.

Purification and kinetic characterization of recombinant human mitogen-activated protein kinase kinase kinase COT and the complexes with its cellular partner NF-kappa B1 p105.

Cancer osaka thyroid (COT), a human MAP 3 K, is essential for lipopolysaccharide activation of the Erk MAPK cascade in macrophages. COT 30--467 is insoluble, whereas low levels of COT 30--397 can be expressed, but this protein is unstable. However, both COT 30--467 and COT 30--397 are expressed in a soluble and stable form when produced in complex with the C-terminal half of p105. The k(cat) of COT 30--397 is reduced approximately 47--fold in the COT 30--467/p105 Delta N complex. COT prefers Mn(2+) to Mg(2+) as the ATP metal cofactor, exhibiting an unusually high ATP K(m) in the presence of Mg(2+). When using Mn(2+) as the cofactor, the ATP K(m) is reduced to a level typical of most kinases. In contrast, the binding affinity of COT for its other substrate MEK is cofactor independent. Our results using purified proteins indicate that p105 binding improves COT solubility and stability while down-regulating kinase activity, consistent with cellular data showing that p105 functions as an inhibitor of COT.

Targeted deletion of caspase-1 reduces early mortality and left ventricular dilatation following myocardial infarction.

Objective. - Mice with targeted deletion of caspase-1 (interleukin-1beta (IL-1beta)-converting enzyme) lack the active forms of IL-1beta and IL-18, two cytokines implicated in maladaptive ventricular remodeling following cardiac injury. We, therefore, investigated the extent of ventricular dilation in caspase-1-knockout (KO) mice. Methods and results. - Transthoracic echocardiography was performed at days 1, 4, and 9 following left anterior descending artery ligation in caspase-1-KO and wild-type (WT) control animals, including M-mode and short-axis imaging at both mid-papillary and apical levels. Although initial post-operative mortality was lower in KO than in WT animals (21.4% WT, 12.0% KO, P < 0.001), there was no difference in mortality between 24 h and 9 d (P = n.s.). Caspase-1 KOs exhibited significantly less mid-papillary ventricular dilatation at days 4 and 9 compared to day 1 post-myocardial infarction (MI) (P < 0.05). Caspase-1 KOs also had a marked (50%) reduction in the level of matrix metalloproteinase 3 (MMP-3), although no significant changes occurred in other MMPs or in tissue inhibitors of metalloproteinase 1 levels by immunoblot analysis. Although IL-beta plasma levels were not detectable, both IL-18 levels and the rate of apoptosis in remodeling, non-infarcted muscle were significantly higher in WT compared to caspase-1-KO animals.Conclusion. - Mice lacking caspase-1 exhibited both improved peri-infarct survival and a decreased rate of ventricular dilatation, possibly due in part to a decrease in MMP-3 activity, IL-18 production, and a reduction in the rate of apoptosis after experimental MI.

betaTrCP-mediated proteolysis of NF-kappaB1 p105 requires phosphorylation of p105 serines 927 and 932.

NF-kappaB1 p105 functions both as a precursor of NF-kappaB1 p50 and as a cytoplasmic inhibitor of NF-kappaB. Following the stimulation of cells with tumor necrosis factor alpha (TNF-alpha), the IkappaB kinase (IKK) complex rapidly phosphorylates NF-kappaB1 p105 on serine 927 in the PEST region. This phosphorylation is essential for TNF-alpha to trigger p105 degradation, which releases the associated Rel/NF-kappaB subunits to translocate into the nucleus and regulate target gene transcription. Serine 927 resides in a conserved motif (Asp-Ser(927)-Gly-Val-Glu-Thr-Ser(932)) homologous to the IKK target sequence in IkappaBalpha. In this study, TNF-alpha-induced p105 proteolysis was revealed to additionally require the phosphorylation of serine 932. Experiments with IKK1(-/-) and IKK2(-/-) double knockout embryonic fibroblasts demonstrate that the IKK complex is essential for TNF-alpha to stimulate phosphorylation on p105 serines 927 and 932. Furthermore, purified IKK1 and IKK2 can each phosphorylate a glutathione S-transferase-p105(758-967) fusion protein on both regulatory serines in vitro. IKK-mediated p105 phosphorylation generates a binding site for betaTrCP, the receptor subunit of an SCF-type ubiquitin E3 ligase, and depletion of betaTrCP by RNA interference blocks TNF-alpha-induced p105 ubiquitination and proteolysis. Phosphopeptide competition experiments indicate that betaTrCP binds p105 more effectively when both serines 927 and 932 are phosphorylated. Interestingly, however, betaTrCP affinity for the IKK-phosphorylated sequence on p105 is substantially lower than that on IkappaBalpha. Thus, it appears that reduced p105 recruitment of betaTrCP and subsequent ubiquitination may contribute to delayed p105 proteolysis after TNF-alpha stimulation relative to that for IkappaBalpha.

Structure-based design of nonpeptide inhibitors of interleukin-1beta converting enzyme (ICE, caspase-1).

A novel class of reversible inhibitors of Interleukin-1beta-converting enzyme (ICE, caspase-1) were discovered by iterative structure-based design. Guided by the X-ray crystal structure of analogues 1, 7 and 10 bound to ICE, we have designed a nonpeptide series of small molecule inhibitors. These compounds incorporate an arylsulfonamide moiety which replaces Val-His unit (P3-P2 residues) amino acids of the native substrate. The synthesis of the core structure, structure-activity relationships (SARs), and proposed binding orientation based on molecular modeling studies for this series of ICE inhibitors are described.