Nature 440, 1064–1068 (2006); doi:10.1038/nature04656 Owing to an error in the production process, some details were omitted from the advance online publication version of this Corrigendum: this is the complete version. When our Letter was under consideration at Nature, we originally showed co-immunoprecipitation between caspase-1 and wild-type caspase-12 or catalytically inactive caspase-12 (C299A) as part of Fig.

The structure-activity relationship of a novel series of 8-biarylnaphthyridinones acting as type 4 phosphodiesterase (PDE4) inhibitors for the treatment of long-term memory loss and mild cognitive impairment is described herein. The manuscript describes a new paradigm for the development of PDE4 inhibitor targeting CNS indications. This effort led to the discovery of the clinical candidate MK-0952, an intrinsically potent inhibitor (IC(50)=0.6 nM) displaying limited whole blood activity (IC(50)=555 nM). Supporting in vivo results in two preclinical efficacy tests and one test assessing adverse effects are also reported. The comparative profiles of MK-0952 and two other Merck compounds are described to validate the proposed hypothesis.

The SAR study of a series of 6-aryloxymethyl-8-aryl substituted quinolines is described. Optimization of the series led to the discovery of compound 26b, a highly potent (IC50=0.6nM) and selective PDE4D inhibitor with a 75-fold selectivity over the A, B, and C subtypes and over 18,000-fold selectivity against other PDE family members. Rat pharmacokinetics and tissue distribution are also summarized.

Abstract Caspases ( Cysteinyl ASPartate‐specific proteASE ) are cysteine proteases involved in cell death. The caspase family is comprised of 12 proteins in humans, seven in Drosophila melanogaster and a single protein in Caenorhabditis elegans . All caspases consist of three structural domains: a prodomain, a large subunit and a small subunit. The catalytically active enzymes are formed either by proteolytic cleavage of the subunits, or through a proximity‐induced activation process involving the prodomain. The cleavage site of caspase substrates is indicated by the positions P4, P3, P2 and P1. P1 is always aspartate, while amino acids in the other positions are highly variable. By cleaving over 1000 substrates in the cell, caspase activation results in the complete dismantling and ultimate death of the cell. Caspase activity can be regulated at a pharmacological level and has become a therapeutic target for the treatment of many diseases. Key Concepts Caspases (Cysteinyl ASPartate‐specific proteASE) are the key enzymes regulating and executing apoptosis. They include several proteases acting either at the upstream regulatory events, or in the effector phase of cell death. Three groups of caspases have been classified and are involved either in inflammation or cell death. Caspases cleave over 1000 proteins within the cell causing the morphological events of cell death. Both natural (cellular, bacterial and viral) and pharmacological inhibitors of caspases have been identified offering novel therapeutic approaches to inflammatory and apoptotic pathways.

Substituted 8-arylquinoline analogs bearing alkyl-linked side chain were identified as potent inhibitors of type 4 phophodiesterase. These compounds address the potential liabilities of the clinical candidate L-454560. The pharmacokinetic profile of the best analogs and the in vivo efficacy in an ovalbumin-induced bronchoconstriction assay in conscious guinea pigs are reported.

Cell death is essential for a plethora of physiological processes, and its deregulation characterizes numerous human diseases. Thus, the in-depth investigation of cell death and its mechanisms constitutes a formidable challenge for fundamental and applied biomedical research, and has tremendous implications for the development of novel therapeutic strategies. It is, therefore, of utmost importance to standardize the experimental procedures that identify dying and dead cells in cell cultures and/or in tissues, from model organisms and/or humans, in healthy and/or pathological scenarios. Thus far, dozens of methods have been proposed to quantify cell death-related parameters. However, no guidelines exist regarding their use and interpretation, and nobody has thoroughly annotated the experimental settings for which each of these techniques is most appropriate. Here, we provide a nonexhaustive comparison of methods to detect cell death with apoptotic or nonapoptotic morphologies, their advantages and pitfalls. These guidelines are intended for investigators who study cell death, as well as for reviewers who need to constructively critique scientific reports that deal with cellular demise. Given the difficulties in determining the exact number of cells that have passed the point-of-no-return of the signaling cascades leading to cell death, we emphasize the importance of performing multiple, methodologically unrelated assays to quantify dying and dead cells.

Neurodegeneration often has disease connotations. However, it is also a developmental process for fine sculpting of the nervous system. One signalling cascade might mediate the process in both circumstances. During embryonic development, nerve cells are made in excess numbers, and many of these neurons and their long extensions (axons) are then culled in a phase of natural degeneration. Nikolaev et al. now identify the apoptosis-inducing protein DR6 (death receptor 6) as a regulator of this process, acting by binding to APP (β-amyloid precursor protein), a transmembrane protein of unknown function that has been implicated in Alzheimer's disease through human genetics. Unlike classical neuronal apoptosis, which requires caspase 3, DR6/APP-induced degeneration requires caspase 6 activation. This work suggests that an extracellular fragment of APP, acting via DR6 and caspase 6, may contribute to neural degeneration in Alzheimer's disease.

Inflammatory caspases are important effectors of innate immunity. Caspase-12 , of the inflammatory caspase subfamily, is expressed in all mammals tested to date, but has acquired deleterious mutation in humans. A single-nucleotide polymorphism introduces a premature stop codon in caspase-12 in the majority of the population. However, in 20% of African descendants, caspase-12 is expressed and sensitizes to infections and sepsis. Here, we examined the modalities by which human caspase-12 confers susceptibility to infection. We have generated a fully humanized mouse that expresses the human caspase-12 rare variant (Csp-12L) in a mouse casp-12 −/− background. Characterization of the humanized mouse uncovered sex differences in Csp-12L expression and gender disparity in innate immunity to Listeria monocytogenes infection. The Csp-12L transgene completely reversed the knockout resistance-to-infection phenotype in casp-12 −/− males. In contrast, it had a marginal effect on the response of female mice. We found that estrogen levels modulated the expression of caspase-12. Csp-12L was expressed in male mice but its expression was repressed in female mice. Administration of 17-β-estradiol (E2) to humanized male mice had a direct suppressive effect on Csp-12L expression and conferred relative resistance to infection. Chromatin immunoprecipitation experiments revealed that caspase-12 is a direct transcriptional target of the estrogen receptor alpha (ERα) and mapped the estrogen response element (ERE) to intron 7 of the gene. We propose that estrogen-mediated inhibition of Csp-12L expression is a built-in mechanism that has evolved to protect females from infection.

Caspase-12 is a dominant-negative regulator of caspase-1 (IL-1beta-converting enzyme) and an attenuator of cytokine responsiveness to septic infections. This molecular role for caspase-12 appears to be akin to the role of cFLIP in regulating caspase-8 in the extrinsic cell death pathway; however, unlike cFLIP/Usurpin, we demonstrate here that caspase-12 is catalytically competent. To examine these catalytic properties, rat caspase-12 was cloned, and the recombinant enzyme was used to examine the cleavage of macromolecular and synthetic fluorogenic substrates. Although caspase-12 could mediate autoproteolytic maturation of its own proenzyme, in both cis and trans, it was not able to cleave any other polypeptide substrate, including other caspase proenzymes, apoptotic substrates, cytokine precursors, or proteins in the endoplasmic reticulum that normally undergo caspase-mediated proteolysis. The dearth of potential substrates for caspase-12 also was confirmed by whole-cell diagonal-gel analysis. Autolytic cleavage within the caspase-12 proenzyme was mapped to a single site at the large-small subunit junction, ATAD(319), and this motif was recognized by caspase-12 when incorporated into synthetic fluorogenic substrates. The specific activity of caspase-12 with these substates was several orders of magnitude lower than caspases-1 and -3, highlighting its relative catalytic paucity. In intact cells, caspase-12 autoproteolysis occurred in the inhibitory complex containing caspase-1. We propose that the proteolytic activity of caspase-12 is confined to its own proenzyme and that autocleavage within the caspase-1 complex may be a means for temporal limitation of the inhibitory effects of caspase-12 on proinflammatory cytokine maturation.

The structure-activity relationship of a novel series of 8-biarylquinolines acting as type 4 phosphodiesterase (PDE4) inhibitors is described herein. Prototypical compounds from this series are potent and non-selective inhibitors of the four distinct PDE4 (IC50 < 10 nM) isozymes (A-D). In a human whole blood in vitro assay, they inhibit (IC50 < 0.5 mu M) the LPS-induced release of the cytokine TNF-alpha. Optimized inhibitors were evaluated in vivo for efficacy in an ovalbumin-induced bronchoconstriction model in conscious guinea pigs. Their propensity to produce an emetic response was evaluated by performing pharmacokinetic studies in squirrel monkeys. This work has led to the identification of several compounds with excellent in vitro and in vivo profiles, including a good therapeutic window of efficacy over emesis. (c) 2008 Elsevier Ltd. All rights reserved.

Huntington's disease (HD) is associated with a range of cellular consequences including selective neuronal death and decreased levels of neurogenesis. Ultimately, these altered processes are dependent upon proteins that interact with Huntingtin (Htt) such as the Huntingtin-interacting protein 1 (Hip1) which has a reduced binding preference to expanded Htt. These effects are similar to those observed with modified Notch signal transduction. As Hip1 plays a key role in endocytosis and intracellular transport, and activation of the Notch signal requires both, we investigated putative links between Hip1 and Notch signaling in flies. We have identified two forms of Hip1 that may be produced through the use of alternative first exons: a version of Hip1 with a lipid-binding ANTH domain and Hip1DeltaANTH lacking this domain. The directed expression of Hip1 decreases, while expression of Hip1DeltaANTH increases, the density of sensory microchaetae on the dorsal notum, a classical model of neurogenesis. A reduction in microchaetae density associated with Notch(Microchaetae Deficient (MCD)) (N(MCD) ) alleles is sensitive to both Hip1 and Hip1DeltaANTH levels, as are the bristle phenotypes generated by misexpression of deltex, a key mediator of Notch signaling. Genetic studies further demonstrate that the observed effects of Hip1 and of Hip1DeltaANTH are sensitive to achaete gene dosage while insensitive to the levels of E(Spl), suggesting a non-canonical Notch neurogenic signal through a deltex-dependent pathway. The novel role we describe for Hip1 in Notch-mediated neurogenesis provides a functional link between Notch signaling and proteins related to HD.

Type 4 phosphodiesterases (PDE4) inhibitors are emerging therapeutics in the treatment of a number of chronic disorders including asthma, chronic obstructive pulmonary disease (COPD) and cognitive disorders. This study delineates the preclinical profile of L-454,560, which is a potent, competitive and preferential inhibitor of PDE4A, 4B, and 4D with IC50 values of 1.6, 0.5 and 1.2nM, respectively. In contrast to the exclusive binding of cilomilast and the preferential binding of roflumilast to the PDE4 holoenzyme state (Mg2+-bound form), L-454,560 binds to both the apo-(Mg2+-free) and holoenzyme states of PDE4. The intrinsic enzyme potency for PDE4 inhibition by L-454,560 also results in an effective blockade of LPS-induced TNFα formation in whole blood (IC50=161nM) and is comparable to the human whole blood potency of roflumilast. The cytokine profile of inhibition of L-454,560 is mainly a Th1 profile with significant inhibition of IFNγ and no detectable inhibition of IL-13 formation up to 1μM. L-454,560 was also found to be efficacious in two models of airway hyper-reactivity, the ovalbumin (OVA) sensitized and challenged guinea pig and the ascaris sensitized sheep model. Furthermore, L-454560 was also effective in improving performance in the delayed matching to position (DMTP) version of the Morris watermaze, at a dose removed from that associated with potential emesis. Therefore, L-454,560 is a novel PDE4 inhibitor with an overall in vivo efficacy profile at least comparable to roflumilast and clearly superior to cilomilast.

Caspase-3 is a cysteinyl protease that mediates apoptotic cell death. Its inhibition may have an important impact on the treatment of several degenerative diseases. Here we report the synthesis of reversible inhibitors via a solid-support palladium-catalyzed amination of 3-bromopyrazinones and the discovery of a pan-caspase reversible inhibitor.

Sepsis target Proteases of the caspase family not only help cells to die (by apoptosis) but also process inflammatory cytokines. Caspase-12 has now been found to inhibit caspase-1, diminishing the production of pro-inflammatory cytokines. Mice lacking caspase-12 are therefore better able to combat bacteria than normal mice and become resistant to peritonitis and septic shock. This makes caspase-12 a potential drug target in the treatment of sepsis.

The formin homology (FH) proteins play a crucial role in cytoskeleton remodelling during many essential processes. In this study, we demonstrate for the first time that the formin-homology-domain-containing protein FHOD1 is cleaved by caspase-3 at the SVPD616 site during apoptosis. Using confocal microscopy, we further demonstrate that while full length FHOD1 is mostly cytoplasmic, the FHOD1 N-terminal cleavage product is diffusely localized throughout the cytoplasm and the nucleoplasm, whereas the C-terminal cleavage product is almost exclusively nuclear with some nucleolar localization. Finally, using a run-on transcription assay we show that the C-terminal FHOD1 cleavage product has the ability to inhibit RNA polymerase I transcription when overexpressed in HeLa cells as shown by blockage of BrUTP incorporation.

Hippi functions as an adapter protein that mediates pro-apoptotic signaling from poly-glutamine-expanded huntingtin, an established cause of Huntington disease, to the extrinsic cell death pathway. To explore other functions of Hippi we generated Hippi knock-out mice. This deletion causes randomization of the embryo turning process and heart looping, which are hallmarks of defective left–right (LR) axis patterning. We report that motile monocilia normally present at the surface of the embryonic node, and proposed to initiate the break in LR symmetry, are absent on Hippi−/− embryos. Furthermore, defects in central nervous system development are observed. The Sonic hedgehog (Shh) pathway is downregulated in the neural tube in the absence of Hippi, which results in failure to establish ventral neural cell fate. Together, these findings demonstrate a dual role for Hippi in cilia assembly and Shh signaling during development, in addition to its proposed role in apoptosis signal transduction in the adult brain under pathogenically stressful conditions.