Temporal Regulation of the Bacterial Metabolite Deoxycholate during Colonic Repair Is Critical for Crypt Regeneration.

Colonic wound repair is an orchestrated process, beginning with barrier re-establishment and followed by wound channel formation and crypt regeneration. Elevated levels of prostaglandin E2 (PGE2) promote barrier re-establishment; however, we found that persistently elevated PGE2 hinders subsequent repair phases. The bacterial metabolite deoxycholate (DCA) promotes transition through repair phases via PGE2 regulation. During barrier re-establishment, DCA levels are locally diminished in the wound, allowing enhanced PGE2 production and barrier re-establishment. However, during transition to the wound channel formation phase, DCA levels increase to inhibit PGE2 production and promote crypt regeneration. Altering DCA levels via antibiotic treatment enhances PGE2 levels but impairs wound repair, which is rescued with DCA treatment. DCA acts via its receptor, farnesoid X receptor, to inhibit the enzyme cPLA2 required for PGE2 synthesis. Thus, colonic wound repair requires temporally regulated signals from microbial metabolites to coordinate host-associated signaling cascades. VIDEO ABSTRACT.

Exploration of cathepsin S inhibitors characterized by a triazole P1-P2 amide replacement.

This paper details exploration of a class of triazole-based cathepsin S inhibitors originally reported by Ellman and co-workers. SAR studies involving modifications across the whole inhibitor provide a perspective on the strengths and weaknesses of this class of inhibitors. In addition, we put the unique characteristics of this class of compounds into perspective with other classes of cathepsin S inhibitors.

Essential role for IKKß in production of type 1 interferons by plasmacytoid dendritic cells.

Plasmacytoid dendritic cells (pDCs) are characterized by their ability to produce high levels of type 1 interferons in response to ligands that activate TLR7 and TLR9, but the signaling pathways required for IFN production are incompletely understood. Here we exploit the human pDC cell line Gen2.2 and improved pharmacological inhibitors of protein kinases to address this issue. We demonstrate that ligands that activate TLR7 and TLR9 require the TAK1-IKKß signaling pathway to induce the production of IFNß via a pathway that is independent of the degradation of IκBα. We also show that IKKß activity, as well as the subsequent IFNß-stimulated activation of the JAK-STAT1/2 signaling pathway, are essential for the production of IFNα by TLR9 ligands. We further show that TLR7 ligands CL097 and R848 fail to produce significant amounts of IFNα because the activation of IKKß is not sustained for a sufficient length of time. The TLR7/9-stimulated production of type 1 IFNs is inhibited by much lower concentrations of IKKß inhibitors than those needed to suppress the production of NFκB-dependent proinflammatory cytokines, such as IL-6, suggesting that drugs that inhibit IKKß may have a potential for the treatment of forms of lupus that are driven by self-RNA and self-DNA-induced activation of TLR7 and TLR9, respectively.

Novel cross-talk within the IKK family controls innate immunity.

Members of the IKK {IκB [inhibitor of NF-κB (nuclear factor κB)] kinase} family play a central role in innate immunity by inducing NF-κB- and IRF [IFN (interferon) regulatory factor]-dependent gene transcription programmes required for the production of pro-inflammatory cytokines and IFNs. However, the molecular mechanisms that activate these protein kinases and their complement of physiological substrates remain poorly defined. Using MRT67307, a novel inhibitor of IKKϵ/TBK1 (TANK {TRAF [TNF (tumour-necrosis-factor)-receptor-associated factor]-associated NF-κB activator}-binding kinase 1) and BI605906, a novel inhibitor of IKKß, we demonstrate that two different signalling pathways participate in the activation of the IKK-related protein kinases by ligands that activate the IL-1 (interleukin-1), TLR (Toll-like receptor) 3 and TLR4 receptors. One signalling pathway is mediated by the canonical IKKs, which directly phosphorylate and activate IKKϵ and TBK1, whereas the second pathway appears to culminate in the autocatalytic activation of the IKK-related kinases. In contrast, the TNFα-induced activation of the IKK-related kinases is mediated solely by the canonical IKKs. In turn, the IKK-related kinases phosphorylate the catalytic subunits of the canonical IKKs and their regulatory subunit NEMO (NF-κB essential modulator), which is associated with reduced IKKα/ß activity and NF-κB-dependent gene transcription. We also show that the canonical IKKs and the IKK-related kinases not only have unique physiological substrates, such as IκBα, p105, RelA (IKKα and IKKß) and IRF3 (IKKϵ and TBK1), but also have several substrates in common, including the catalytic and regulatory (NEMO and TANK) subunits of the IKKs themselves. Taken together, our studies reveal that the canonical IKKs and the IKK-related kinases regulate each other by an intricate network involving phosphorylation of their catalytic and regulatory (NEMO and TANK) subunits to balance their activities during innate immunity.

Substituted 2H-isoquinolin-1-ones as potent Rho-kinase inhibitors: part 2, optimization for blood pressure reduction in spontaneously hypertensive rats.

Phenylglycine substituted isoquinolones 1 and 2 have previously been described as potent dual ROCK1/ROCK2 inhibitors. Here we describe the further SAR of this series to improve metabolic stability and rat oral exposure. Piperidine analog 20 which demonstrates sustained blood pressure normalization in an SHR blood pressure reduction model was identified through this effort.

Substituted 2H-isoquinolin-1-ones as potent Rho-kinase inhibitors: part 3, aryl substituted pyrrolidines.

The discovery and SAR of a series of beta-aryl substituted pyrrolidine 2H-isoquinolin-1-one inhibitors of Rho-kinase (ROCK) derived from 2 is herein described. SAR studies have shown that aryl groups in the beta-position are optimal for potency. Our efforts focused on improving the ROCK potency of this isoquinolone class of inhibitors which led to the identification of pyrrolidine 32 which demonstrated a 10-fold improvement in aortic ring (AR) potency over 2.

Substituted 2H-isoquinolin-1-one as potent Rho-Kinase inhibitors. Part 1: Hit-to-lead account.

Two closely related scaffolds were identified through an uHTS campaign as desirable starting points for the development of Rho-Kinase (ROCK) inhibitors. Here, we describe our hit-to-lead evaluation process which culminated in the rapid discovery of potent leads such as 22 which successfully demonstrated an early in vivo proof of concept for anti-hypertensive activity.

BI 5700, a Selective Chemical Inhibitor of IκB Kinase 2, Specifically Suppresses Epithelial-Mesenchymal Transition and Metastasis in Mouse Models of Tumor Progression.

Increasing evidence suggests that processes termed epithelial-mesenchymal transitions (EMTs) play a key role in therapeutic resistance, tumor recurrence, and metastatic progression. NF-κB signaling has been previously identified as an important pathway in the regulation of EMT in a mouse model of tumor progression. However, it remains unclear whether there is a broad requirement for this pathway to govern EMT and what the relative contribution of IKK family members acting as upstream NF-κB activators is toward promoting EMT and metastasis. To address this question, we have used a novel, small-molecule inhibitor of IκB kinase 2 (IKK2/IKKß), termed BI 5700. We investigated the role of IKK2 in a number of mouse models of EMT, including TGFß-induced EMT in the mammary epithelial cell line EpRas, CT26 colon carcinoma cells, and 4T1 mammary carcinoma cells. The latter model was also used to evaluate in vivo activities of BI 5700.We found that BI 5700 inhibits IKK2 with an IC(50) of 9 nM and was highly selective as compared to other IKK family members (IKK1, IKKε, and TBK1) and other kinases. BI 5700 effectively blocks NF-κB activity in EpRas cells and prevents TGFß-induced EMT. In addition, BI 5700 reverts EMT in mesenchymal CT26 cells and prevents EMT in the 4T1 model. Oral application of BI 5700 significantly interferes with metastasis after mammary fat-pad injection of 4T1 cells, yielding fewer, smaller, and more differentiated metastases as compared to vehicle-treated control animals. We conclude that IKK2 is a key regulator of both the induction and maintenance of EMT in a panel of mouse tumor progression models and that the IKK2 inhibitor BI 5700 constitutes a promising candidate for the treatment of metastatic cancers.

Hit to lead account of the discovery of bisbenzamide and related ureidobenzamide inhibitors of Rho kinase.

A highly selective series of bisbenzamide inhibitors of Rho-associated coiled-coil forming protein kinase (ROCK) and a related ureidobenzamide series, both identified by high throughput screening (HTS), are described. Details of the hit validation and lead generation process, including structure-activity relationship (SAR) studies, a selectivity assessment, target-independent profiling (TIP) results, and an analysis of functional activity using a rat aortic ring assay are discussed.

Identification of a novel class of succinyl-nitrile-based Cathepsin S inhibitors.

The synthesis and in vitro activities of a series of succinyl-nitrile-based inhibitors of Cathepsin S are described. Several members of this class show nanomolar inhibition of the target enzyme as well as cellular potency. The inhibitors displaying the greatest potency contain N-alkyl substituted piperidine and pyrrolidine rings spiro-fused to the alpha-carbon of the P1 residue.

Discovery of potent and selective PKC-theta inhibitors.

An uHTS campaign was performed to identify selective inhibitors of PKC-theta. Initial triaging of the hit set based on selectivity and historical analysis led to the identification of 2,4-diamino-5-nitropyrimidines as potent and selective PKC-theta inhibitors. A homology model and initial SAR is presented demonstrating that a 2-arylalkylamino substituent in conjunction with suitable 4-diamino substituent are essential for achieving selectivity over many kinases. Additional hit to lead profiling is presented on selected compounds.

Infrared echoes near the supernova remnant Cassiopeia A.

Two images of Cassiopeia A obtained at 24 micrometers with the Spitzer Space Telescope over a 1-year time interval show moving structures outside the shell of the supernova remnant to a distance of more than 20 arc minutes. Individual features exhibit apparent motions of 10 to 20 arc seconds per year, independently confirmed by near-infrared observations. The observed tangential velocities are at roughly the speed of light. It is likely that the moving structures are infrared echoes, in which interstellar dust is heated by the explosion and by flares from the compact object near the center of the remnant.

Simultaneous multichannel mass-specific detection for high-performance liquid chromatography using an array detector sector-field mass spectrometer.

The use of a separation step, such as liquid chromatography, prior to inductively coupled plasma mass spectrometry (ICP-MS) has become a common tool for highly selective and sensitive analyses. This type of coupling has several benefits including the ability to perform speciation analysis or to remove isobaric interferences. Several limitations of conventional instruments result from the necessity to scan or pulse the mass spectrometer to obtain a complete mass spectrum. When the instrument is operated in such a non-continuous manner, duty cycle is reduced, resulting in poorer absolute limits of detection. Additionally, with scanning instruments, spectral skew can be introduced into the measurement, limiting quantitation accuracy. To address these shortcomings, a high-performance liquid chromatograph has been coupled to an ICP-MS capable of continuous sample introduction and simultaneous multimass detection. These features have been realized with a novel detector array, the focal plane camera. Instrument performance has been tested for both speciation analysis and for the elimination of isobaric interferences. Absolute limits of detection in the sub picogram to tens of picograms regime are obtainable, while the added mass dimension introduced by simultaneous detection dramatically increases chromatographic peak capacity.

Characterization of a focal plane camera fitted to a Mattauch-Herzog geometry mass spectrograph. 2. Use with an inductively coupled plasma.

A novel charge-sensitive detector array, termed the focal plane camera (FPC), has been coupled to a Mattauch-Herzog mass spectrograph (MHMS) with an inductively coupled plasma ionization source. The FPC employs an array of gold Faraday cups, each with its own charge-integrating circuit that allows the simultaneous detection of several m/z ratios. The ion-sampling interface of the MHMS has been redesigned to provide better heat transfer away from the sampler and skimmer cones and to reduce the negative effects of turbulent gas flows around the plasma. The instrument has produced limits of detection in the tens to hundreds of parts per quadrillion regime and isotope ratio accuracy and precision of 5% error and 0.007% RSD, respectively. Limits of detection with the FPC are comparable to those obtained with a single-channel secondary electron multiplier (SEM). However, the isotope ratio accuracy and precision are better with the FPC than when the SEM is employed. The dynamic range has been shown to be linear over 7 orders of magnitude.

Characterization of a focal plane camera fitted to a Mattauch-Herzog geometry mass spectrograph. 1. Use with a glow-discharge source.

A Mattauch-Herzog geometry mass spectrograph (MHMS) has been equipped with a novel array detector, the focal plane camera (FPC). The FPC consists of an array of gold Faraday cups, each coupled to its own integrator, with interrogation of the integrators performed by a multiplexer. The initial coupling of this instrument with a pin-type glow discharge source has provided limits of detection in the single to hundreds of nanograms per gram regime; isotope ratio accuracy and precision better than 5% error and 0.2% RSD, respectively; and a linear dynamic range of at least 6 orders of magnitude. A current weakness of the FPC is its pixel size, which limits both sensitivity and baseline resolution (to R = 130). The minimum data acquisition time for multiple images at present is 1 ms/image, with a dead time of 3.2 ms between images, which will limit the ability of the FPC to monitor extremely short transient signals.