Lung eQTLs to help reveal the molecular underpinnings of asthma.

Genome-wide association studies (GWAS) have identified loci reproducibly associated with pulmonary diseases; however, the molecular mechanism underlying these associations are largely unknown. The objectives of this study were to discover genetic variants affecting gene expression in human lung tissue, to refine susceptibility loci for asthma identified in GWAS studies, and to use the genetics of gene expression and network analyses to find key molecular drivers of asthma. We performed a genome-wide search for expression quantitative trait loci (eQTL) in 1,111 human lung samples. The lung eQTL dataset was then used to inform asthma genetic studies reported in the literature. The top ranked lung eQTLs were integrated with the GWAS on asthma reported by the GABRIEL consortium to generate a Bayesian gene expression network for discovery of novel molecular pathways underpinning asthma. We detected 17,178 cis- and 593 trans- lung eQTLs, which can be used to explore the functional consequences of loci associated with lung diseases and traits. Some strong eQTLs are also asthma susceptibility loci. For example, rs3859192 on chr17q21 is robustly associated with the mRNA levels of GSDMA (P = 3.55 × 10(-151)). The genetic-gene expression network identified the SOCS3 pathway as one of the key drivers of asthma. The eQTLs and gene networks identified in this study are powerful tools for elucidating the causal mechanisms underlying pulmonary disease. This data resource offers much-needed support to pinpoint the causal genes and characterize the molecular function of gene variants associated with lung diseases.

Discovery of MK-0952, a selective PDE4 inhibitor for the treatment of long-term memory loss and mild cognitive impairment.

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 discovery and synthesis of highly potent subtype selective phosphodiesterase 4D inhibitors.

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=0.6 nM) 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.

Alkyl-bridged substituted 8-arylquinolines as highly potent PDE IV inhibitors.

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.

Gender differences in expression of the human caspase-12 long variant determines susceptibility to Listeria monocytogenes infection.

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-beta-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 (ERalpha) 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.

Huntingtin interacting protein 1 can regulate neurogenesis in Drosophila.

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.

Confinement of caspase-12 proteolytic activity to autoprocessing.

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.

Solid-phase analogue synthesis of caspase-3 inhibitors via palladium-catalyzed amination of 3-bromopyrazinones.

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.

Caspase-3 cleaves the formin-homology-domain-containing protein FHOD1 during apoptosis to generate a C-terminal fragment that is targeted to the nucleolus.

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 SVPD(616) 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.

Apoptosis and caspases regulate death and inflammation in sepsis.

Although the prevailing concept has been that mortality in sepsis results from an unbridled hyper-inflammatory cytokine-mediated response, the failure of more than 30 clinical trials to treat sepsis by controlling this cytokine response requires a 'rethink' of the molecular mechanism underpinning the development of sepsis. As we discuss here, remarkable new studies indicate that most deaths from sepsis are actually the result of a substantially impaired immune response that is due to extensive death of immune effector cells. Rectification of this apoptotic-inflammatory imbalance using modulators of caspases and other components of the cell-death pathway have shown striking efficacy in stringent animal models of sepsis, indicating an entirely novel path forward for the clinical treatment of human sepsis.

Hippi is essential for node cilia assembly and Sonic hedgehog signaling.

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.

Cleavage at the caspase-6 site is required for neuronal dysfunction and degeneration due to mutant huntingtin.

Cleavage of huntingtin (htt) has been characterized in vitro, and accumulation of caspase cleavage fragments represents an early pathological change in brains of Huntington's disease (HD) patients. However, the relationship between htt proteolysis and the pathogenesis of HD is unknown. To determine whether caspase cleavage of htt is a key event in the neuronal dysfunction and selective neurodegeneration in HD, we generated YAC mice expressing caspase-3- and caspase-6-resistant mutant htt. Mice expressing mutant htt, resistant to cleavage by caspase-6 but not caspase-3, maintain normal neuronal function and do not develop striatal neurodegeneration. Furthermore, caspase-6-resistant mutant htt mice are protected against neurotoxicity induced by multiple stressors including NMDA, quinolinic acid (QA), and staurosporine. These results are consistent with proteolysis of htt at the caspase-6 cleavage site being an important event in mediating neuronal dysfunction and neurodegeneration and highlight the significant role of htt proteolysis and excitotoxicity in HD.

Enhanced bacterial clearance and sepsis resistance in caspase-12-deficient mice.

Caspases function in both apoptosis and inflammatory cytokine processing and thereby have a role in resistance to sepsis. Here we describe a novel role for a caspase in dampening responses to bacterial infection. We show that in mice, gene-targeted deletion of caspase-12 renders animals resistant to peritonitis and septic shock. The resulting survival advantage was conferred by the ability of the caspase-12-deficient mice to clear bacterial infection more efficiently than wild-type littermates. Caspase-12 dampened the production of the pro-inflammatory cytokines interleukin (IL)-1beta, IL-18 (interferon (IFN)-gamma inducing factor) and IFN-gamma, but not tumour-necrosis factor-alpha and IL-6, in response to various bacterial components that stimulate Toll-like receptor and NOD pathways. The IFN-gamma pathway was crucial in mediating survival of septic caspase-12-deficient mice, because administration of neutralizing antibodies to IFN-gamma receptors ablated the survival advantage that otherwise occurred in these animals. Mechanistically, caspase-12 associated with caspase-1 and inhibited its activity. Notably, the protease function of caspase-12 was not necessary for this effect, as the catalytically inactive caspase-12 mutant Cys299Ala also inhibited caspase-1 and IL-1beta production to the same extent as wild-type caspase-12. In this regard, caspase-12 seems to be the cFLIP counterpart for regulating the inflammatory branch of the caspase cascade. In mice, caspase-12 deficiency confers resistance to sepsis and its presence exerts a dominant-negative suppressive effect on caspase-1, resulting in enhanced vulnerability to bacterial infection and septic mortality.

Correlating the fractional inhibition of caspase-3 in NT2 cells with apoptotic markers using an active-caspase-3 enzyme-linked immunosorbent assay.

A rapid and quantitative method for measuring the activity and fractional inhibition of enzymes within their natural cellular environment remains an unmet need in drug discovery. We describe the use of a nonradioactive quantitative enzyme-linked immunosorbent assay (ELISA) for measuring intracellular caspase activity that is amenable to robotic automation. The ELISA specifically detects active-caspase-3 and was used to correlate the in-cell activity of caspase-3 with the progress of caspase-3-mediated events under varying concentrations of caspase-3 inhibitors in NT2 cells. We examined the cleavage of endogenous substrates (poly(ADP-ribose)polymerase and alphaII-spectrin), the extent of DNA fragmentation, and the autocatalytic removal of the caspase-3 prodomain as markers of caspase-3 activity. To impart inhibition of the downstream markers, a greater level of caspase-3 inhibition was required. Although the functional markers were found not to accurately predict intracellular caspase-3 activity, we found that the inhibition of intracellular caspase-3 was highly correlated (R(2) = 0.96) to the inhibition of DNA fragmentation. Also, by comparing the potency of the different inhibitors against the intracellular enzyme versus the purified enzyme, the effects of inhibitor functional groups on whole-cell activity were addressed.

Axonal dynactin p150Glued transports caspase-8 to drive retrograde olfactory receptor neuron apoptosis.

Olfactory receptor neurons (ORNs) undergo caspase-mediated retrograde apoptosis after target removal (bulbectomy), in which axonal caspase-9 and caspase-3 activation leads to terminal apoptosis in ORN soma of the olfactory epithelium. Here, we show that caspase-8 can act as an initiator of ORN apoptosis after bulbectomy and also after synaptic instability is induced by NMDA-mediated excitotoxic death of ORN target neurons in the olfactory bulb. Caspase-8 and caspase-3 are sequentially activated within ORN presynaptic terminals, and caspase-8 complexes with dynactin p150Glued, (a retrograde motor protein) and is transported retrogradely, preceding axonal caspase-3 activation and apoptosis of ORN cell bodies. Focal in vivo inhibition of initiator caspase activation or microtubule-dependent transport (with Taxol) at the lesioned axon terminus results in a significant reduction in retrograde axonal caspase-8 and caspase-3 activation and inhibition of retrograde ORN death. Caspase-8 activation and retrograde transport after NMDA lesion is similarly reduced in mice null for p75, the low-affinity nerve growth factor receptor. The retrograde apoptosis of ORNs thus involves a novel mechanism that used p75 in the local activation of caspase-8. Once caspase-8 is maximally activated in the presynaptic terminal, it is transported retrogradely by the motor complex dynactin/dynein, a process that can be inhibited focally to inhibit ORN apoptosis after acute axonal lesion. These data have revealed a novel mechanism of retrograde apoptosis, in which caspase-8 complexes directly with axonal dynactin p150Glued to reveal a differential vulnerability of subpopulations of ORNs to undergo apoptosis after axonal damage and the loss of olfactory bulb target neurons.

Lipophilic versus hydrogen-bonding effect in P3 on potency and selectivity of valine aspartyl ketones as caspase 3 inhibitors.

Caspase 3 is a cysteinyl protease that mediates apoptotic cell death. Its inhibition may have an important impact in the treatment of several degenerative diseases. The P1 aspartic acid residue is a required element of recognition for this enzyme that was maintained constant along with the adjacent natural valine as the P2 group. The thiobenzylmethylketone warhead on the aspartate was conveniently handled through solid-phase synthesis allowing modification in the P3 region that eventually led to simpler derivatives with increased potency against caspase 3. The key to such an effect is the introduction of hydroxyl group alpha to the P3 carbonyl.

Novel pyrazinone mono-amides as potent and reversible caspase-3 inhibitors.

The iterative process for the discovery of a series of pyrazinone mono-amides as potent, selective and reversible non-peptide caspase-3 inhibitors (e.g., M826 and M867) is reported. These compounds display potent anti apoptotic activities in a number of cell based systems in vitro as well as in several animal models in vivo.

Caspase-7 expanded function and intrinsic expression level underlies strain-specific brain phenotype of caspase-3-null mice.

Caspase-3-deficient mice of the 129S1/SvImJ (129) strain show severe brain development defects resulting in brain overgrowth and perinatal lethality, whereas on the C57BL/6J (B6) background, these mice develop normally. We therefore sought to identify the strain-dependent ameliorating gene. We biochemically isolated caspase-7 from B6-caspase-3-null (Casp3-/-) tissues as being the enzyme with caspase-3-like properties and capability of performing a caspase-3 surrogate function, apoptotic DNA fragmentation. Moreover, we show that, in contrast to the human enzymes, mouse caspase-7 is as efficient as caspase-3 at cleaving and thus inactivating ICAD (inhibitor of caspase-activated DNase), the inhibitor of apoptotic DNA fragmentation. Low levels of caspase-7 expression and activation correlate with lack of DNA fragmentation in 129-Casp3-/- apoptotic precursor neurons, whereas B6-Casp3-/- cells, which can fragment their DNA, show higher levels of caspase-7 expression and activation. The amount of caspase-7 activation in apoptotic precursor neurons is independent of the presence of caspase-3. Together, our findings demonstrate for the first time a strong correlation between caspase-7 activity, normal brain development, and apoptotic DNA fragmentation in Casp3-/- mice.