Neuroprotective and Disease-Modifying Effects of the Triazinetrione ACD856, a Positive Allosteric Modulator of Trk-Receptors for the Treatment of Cognitive Dysfunction in Alzheimer's Disease.

The introduction of anti-amyloid monoclonal antibodies against Alzheimer's disease (AD) is of high importance. However, even though treated patients show very little amyloid pathology, there is only a modest effect on the rate of cognitive decline. Although this effect can possibly increase over time, there is still a need for alternative treatments that will improve cognitive function in patients with AD. Therefore, the purpose of this study was to characterize the triazinetrione ACD856, a novel pan-Trk positive allosteric modulator, in multiple models to address its neuroprotective and potential disease-modifying effects. The pharmacological effect of ACD856 was tested in recombinant cell lines, primary cortical neurons, or animals. We demonstrate that ACD856 enhanced NGF-induced neurite outgrowth, increased the levels of the pre-synaptic protein SNAP25 in PC12 cells, and increased the degree of phosphorylated TrkB in SH-SY5Y cells. In primary cortical neurons, ACD856 led to increased levels of phospho-ERK1/2, showed a neuroprotective effect against amyloid-beta or energy-deprivation-induced neurotoxicity, and increased the levels of brain-derived neurotrophic factor (BDNF). Consequently, administration of ACD856 resulted in a significant increase in BDNF in the brains of 21 months old mice. Furthermore, repeated administration of ACD856 resulted in a sustained anti-depressant effect, which lasted up to seven days, suggesting effects that go beyond merely symptomatic effects. In conclusion, the results confirm ACD856 as a cognitive enhancer, but more importantly, they provide substantial in vitro and in vivo evidence of neuroprotective and long-term effects that contribute to neurotrophic support and increased neuroplasticity. Presumably, the described effects of ACD856 may improve cognition, increase resilience, and promote neurorestorative processes, thereby leading to a healthier brain in patients with AD.

Neuronal cell-based high-throughput screen for enhancers of mitochondrial function reveals luteolin as a modulator of mitochondria-endoplasmic reticulum coupling.

BACKGROUND: Mitochondrial dysfunction is a common feature of aging, neurodegeneration, and metabolic diseases. Hence, mitotherapeutics may be valuable disease modifiers for a large number of conditions. In this study, we have set up a large-scale screening platform for mitochondrial-based modulators with promising therapeutic potential. RESULTS: Using differentiated human neuroblastoma cells, we screened 1200 FDA-approved compounds and identified 61 molecules that significantly increased cellular ATP without any cytotoxic effect. Following dose response curve-dependent selection, we identified the flavonoid luteolin as a primary hit. Further validation in neuronal models indicated that luteolin increased mitochondrial respiration in primary neurons, despite not affecting mitochondrial mass, structure, or mitochondria-derived reactive oxygen species. However, we found that luteolin increased contacts between mitochondria and endoplasmic reticulum (ER), contributing to increased mitochondrial calcium (Ca2+) and Ca2+-dependent pyruvate dehydrogenase activity. This signaling pathway likely contributed to the observed effect of luteolin on enhanced mitochondrial complexes I and II activities. Importantly, we observed that increased mitochondrial functions were dependent on the activity of ER Ca2+-releasing channels inositol 1,4,5-trisphosphate receptors (IP3Rs) both in neurons and in isolated synaptosomes. Additionally, luteolin treatment improved mitochondrial and locomotory activities in primary neurons and Caenorhabditis elegans expressing an expanded polyglutamine tract of the huntingtin protein. CONCLUSION: We provide a new screening platform for drug discovery validated in vitro and ex vivo. In addition, we describe a novel mechanism through which luteolin modulates mitochondrial activity in neuronal models with potential therapeutic validity for treatment of a variety of human diseases.

Identification of amino acid residues of nerve growth factor important for neurite outgrowth in human dorsal root ganglion neurons.

Nerve growth factor (NGF) is an essential neurotrophic factor for the development and maintenance of the central and the peripheral nervous system. NGF deficiency in the basal forebrain precedes degeneration of basal forebrain cholinergic neurons in Alzheimer's disease, contributing to memory decline. NGF mediates neurotrophic support via its high-affinity receptor, the tropomyosin-related kinase A (TrkA) receptor, and mediates mitogenic and differentiation signals via the extracellular signal-regulated protein kinases 1 and 2 (ERK1/2). However, the molecular mechanisms underlying the different NGF/TrkA/ERK signalling pathways are far from clear. In this study, we have investigated the role of human NGF and three NGF mutants, R100E, W99A and K95A/Q96A, their ability to activate TrkA or ERK1/2, and their ability to induce proliferation or differentiation in human foetal dorsal root ganglion (DRG) neurons or in PC12 cells. We show that the R100E mutant was significantly more potent than NGF itself to induce proliferation and differentiation, and significantly more potent in activation of ERK1/2 in DRG neurons. The W99A and K95A/Q96A mutants, on the other hand, were less effective than the wild-type protein. An unexpected finding was the high efficacy of the K95A/Q96A mutant to activate TrkA and to induce differentiation of DRG neurons at elevated concentrations. These data demonstrate an NGF mutant with improved neurotrophic properties in primary human neuronal cells. The R100E mutant represents an interesting candidate for further drug development in Alzheimer's disease and other neurodegenerative disorders.

N-Substituted pyrazole-3-carboxamides as inhibitors of human 15-lipoxygenase.

High-throughput screening was used to find selective inhibitors of human 15-lipoxygenase-1 (15-LOX-1). One hit, a 1-benzoyl substituted pyrazole-3-carboxanilide (1a), was used as a starting point in a program to develop potent and selective 15-LOX-1 inhibitors.

3-Substituted pyrazoles and 4-substituted triazoles as inhibitors of human 15-lipoxygenase-1.

Investigation of 1N-substituted pyrazole-3-carboxanilides as 15-lipoxygenase-1 (15-LOX-1) inhibitors demonstrated that the 1N-substituent was not essential for activity or selectivity. Additional halogen substituents on the pyrazole ring, however, increased activity. Further development led to triazole-4-carboxanilides and 2-(3-pyrazolyl) benzoxazoles, which are potent and selective 15-LOX-1 inhibitors.

Epigenetic and transcriptional control of the 15-lipoxygenase-1 gene in a Hodgkin lymphoma cell line.

Lipoxygenases oxidatively metabolize polyunsaturated fatty acids to a rich spectrum of biologically active metabolites. The present study aimed at delineating the transcriptional and epigenetic mechanisms leading to 15-lipoxygenase-1 (15-LOX-1) expression in the Hodgkin lymphoma (HL) cell line L1236. Examination of the 15-LOX-1 5' promoter region demonstrated three putative binding sites for signal transducer and activator of transcription (STAT6) within the proximal 1200 base pairs relative to the start codon. Analysis by serial promoter deletions and STAT6 binding site mutations indicated that all three STAT6 binding sites are required for full activation of the 15-LOX-1 promoter. Chromatin immunoprecipitation assay demonstrated that these regions were occupied by STAT6 in L1236 (15-LOX-1 positive) but not in L428 (15-LOX-1 negative) cultured HL cells. Furthermore, DNA hypomethylation and histone hyperacetylation were detectable within the core promoter region of 15-LOX-1 only in L1236 cells but not L428 cells. Taken together, our data indicate that STAT6 activation and chromatin remodeling by DNA demethylation and histone acetylation are crucial for transcriptional activation of 15-LOX-1 in cultured HL cells. These prerequisites are fulfilled in the L1236 cell line, but not in the L428 cell line.

Antidepressant effects of novel positive allosteric modulators of Trk-receptor mediated signaling - a potential therapeutic concept?

BACKGROUND: Major depressive disorder (MDD) is defined as a complex mental disorder which is characterized by a pervasive low mood and aversion to activity. Several types of neurotransmitter systems e.g. serotonergic, glutamatergic and noradrenergic systems have been suggested to play an important role in the origination of depression, but neurotrophins such as brain derived neurotrophic factor (BDNF) have also been implicated in the disease process. OBJECTIVES: The purpose of this study was to examine the effects of a newly developed class of molecules, characterized as positive allosteric modulators of neurotrophin/Trk receptor mediated signaling (Trk-PAM), on neurotransmitter release and depression-like behavior in vivo. METHODS: The effect of and possible interaction of neurotrophin/Trk signaling pathways with serotonergic and glutamatergic systems in the modulation of depression-related responses was studied using newly developed Trk-PAM compounds (ACD855, ACD856 and AC26845), as well as ketamine and fluoxetine in the forced swim test (FST) in rodents. Moreover, in vivo microdialysis in freely moving rats was used to assess changes in neurotransmitter levels in the rat. RESULTS: The results from the study show that several different compounds, which all potentiate Trk-receptor mediated signaling, display antidepressant-like activity in the FST. Moreover, the data also indicate that the effects of both fluoxetine and ketamine in the FST, both used in clinical practice, are mediated via BDNF/TrkB signaling, which could have implications for novel therapies in MDD. CONCLUSIONS: Trk-PAMs could provide an interesting avenue for the development of novel therapeutics in this area.

Neurotrophin-targeted therapeutics: A gateway to cognition and more?

Neurotrophins, such as brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF), are small proteins expressed in the brain and peripheral tissues, which regulate several key aspects of neuronal function, including neurogenesis, synaptic plasticity and neuroprotection, but also programmed cell death. This broad range of effects is a result of a complex downstream signaling pathway, with differential spatial and temporal activation patterns further diversifying their physiological effects. Alterations in neurotrophin levels, or known polymorphisms in neurotrophin genes, have been linked to a variety of disorders, including depression and Alzheimer's disease (AD). Historically, their therapeutic potential in these disorders has been hampered by the lack of suitable tool molecules for clinical studies. However, recent advancements have led to the development of new therapeutic candidates, which are now in clinical testing.

Applicability of the triad concept for the positional specificity of mammalian lipoxygenases.

The nomenclature of lipoxygenases (LOXs) is partly based on the positional specificity of arachidonic acid oxygenation, but there is no unifying concept explaining the mechanistic basis of this enzyme property. According to the triad model, Phe-353, Ile-418, and Ile-593 of the rabbit 12/15-LOX form the bottom of the substrate-binding pocket, and introduction of less space-filling residues at either of these positions favors arachidonic acid 12-lipoxygenation. The present study was aimed at exploring the validity of the triad concept for two novel primate 12/15-LOX (Macaca mulatta and Pongo pygmaeus) and for five known members of the mammalian LOX family (human 12/15-LOX, mouse 12/15-LOX, human 15-LOX2, human platelet type 12-LOX, and mouse (12R)-LOX). The enzymes were expressed as N-terminal His tag fusion proteins in E. coli, the potential sequence determinants were mutated, and the specificity of arachidonic acid oxygenation was quantified. Taken together, our data indicate that the triad concept explains the positional specificity of all 12/15-LOXs tested (rabbit, human, M. mulatta, P. pygmaeus, and mouse). For the new enzymes of M. mulatta and P. pygmaeus, the concept had predictive value because the positional specificity predicted on the basis of the amino acid sequence was confirmed experimentally. The specificity of the platelet 12-LOX was partly explained by the triad hypothesis, but the concept was not applicable for 15-LOX2 and (12R)-LOX.

Identification of Novel Positive Allosteric Modulators of Neurotrophin Receptors for the Treatment of Cognitive Dysfunction.

Alzheimer's disease (AD) is the most common neurodegenerative disorder and results in severe neurodegeneration and progressive cognitive decline. Neurotrophins are growth factors involved in the development and survival of neurons, but also in underlying mechanisms for memory formation such as hippocampal long-term potentiation. Our aim was to identify small molecules with stimulatory effects on the signaling of two neurotrophins, the nerve growth factor (NGF) and the brain derived neurotrophic factor (BDNF). To identify molecules that could potentiate neurotrophin signaling, 25,000 molecules were screened, which led to the identification of the triazinetrione derivatives ACD855 (Ponazuril) and later on ACD856, as positive allosteric modulators of tropomyosin related kinase (Trk) receptors. ACD855 or ACD856 potentiated the cellular signaling of the neurotrophin receptors with EC50 values of 1.9 and 3.2 or 0.38 and 0.30 µM, respectively, for TrkA or TrkB. ACD855 increased acetylcholine levels in the hippocampus by 40% and facilitated long term potentiation in rat brain slices. The compounds acted as cognitive enhancers in a TrkB-dependent manner in several different behavioral models. Finally, the age-induced cognitive dysfunction in 18-month-old mice could be restored to the same level as found in 2-month-old mice after a single treatment of ACD856. We have identified a novel mechanism to modulate the activity of the Trk-receptors. The identification of the positive allosteric modulators of the Trk-receptors might have implications for the treatment of Alzheimer's diseases and other diseases characterized by cognitive impairment.

Interaction of human 15-lipoxygenase-1 with phosphatidylinositol bisphosphates results in increased enzyme activity.

15-lipoxygenase-1 (15-LO-1) can oxygenate both free fatty acids and fatty acids bound to membrane phospholipids. The regulation of the activity of membrane associated 15-LO-1 is poorly understood. Here we demonstrate that calcium ionophore stimulates the translocation of 15-LO-1 to the plasma membrane in human dendritic cells. In a protein-lipid overlay assay, 15-LO-1 was capable of interacting with several phosphoinositides. In the presence of calcium, addition of phosphatidylinositol-4.5-bisphosphate (PI(4.5)P(2)) or PI(3.4)P(2) to the vesicles containing arachidonic acid, led to the formation of approximately three times more 15-HETE than vesicles without phosphoinositides and up to seven times more 15-HETE than vesicles without both calcium and phosphoinositides. The Vmax was unchanged but the apparent Km of 15-LO-1 towards arachidonic acid was significantly lower in the presence of PI(4.5)P(2) or PI(3.4)P(2) in the vesicles in comparison to vesicles with PC only. Taken together, this report demonstrates that human 15-LO-1 binds to PI(4.5)P(2) and PI(3.4)P(2) and that these phospholipids stimulate enzyme activity in the presence of calcium in a vesicle based assay.

The expression of cytosolic phospholipase A2 and biosynthesis of leukotriene B4 in acute myeloid leukemia cells.

Leukotrienes (LT) exert stimulatory effects on myelopoiesis, beside their inflammatory and immunomodulating effects. Here, we have studied the expression and activity of the enzymes involved in the synthesis of leukotriene B4 (LTB4) in acute myeloid leukemia (AML) cells (16 clones) and G-CSF mobilized peripheral blood CD34+ cells. CD34+ cells from patients with non-myeloid malignancies expressed cytosolic phospholipase A2 (cPLA2), 5-lipoxygenase activating protein (FLAP), and leukotriene A4 (LTA4) hydrolase but not 5-lipoxygenase (5-LO). The enzyme cPLA2 was abundantly expressed in AML cells and the activity of the enzyme was high in certain AML clones. The expression of 5-LO, FLAP, and LTA4 hydrolase in AML clones was in general lower than in healthy donor polymorphonuclear leukocytes (PMNL). The calcium ionophore A23187-induced release of [14C] arachidonic acid (AA) in AML cells was low, compared with PMNL, and did not correlate with the expression of cPLA2 protein. Biosynthesis of LTB4, upon calcium ionophore A23187 activation, was only observed in five of the investigated AML clones and only three of the most differentiated clones produced similar amounts of LTB4 as PMNL. The capacity of various cell clones to produce LTs could neither be explained by the difference in [1-(14)C] AA release nor 5-LO expression. Taken together, these results indicate that LT synthesis is under development during early myelopoiesis and the capacity to produce LTs is gained upon maturation. High expression of cPLA2 in AML suggests a putative role of this enzyme in the pathophysiology of this disease.

Polychlorinated biphenyls induce arachidonic acid release in human platelets in a tamoxifen sensitive manner via activation of group IVA cytosolic phospholipase A2-alpha.

Polychlorinated biphenyls (PCBs) are stable compounds commonly found in nature as environmental pollutants. PCBs can affect the endocrine function of hormones such as steroid-hormones. Also, PCBs are known to be inducers of arachidonic acid release in various cells. We report, here, the effects of PCBs on eicosanoid formation, arachidonic acid release and cytosolic phospholipase A2-alpha (cPLA2-alpha) activation in human platelets. Ortho-substituted PCBs induced a time and dose-dependent release of arachidonic acid and the concomitant formation of 12(S)-hydroxy-5,8-cis-10-trans-14-cis-eicosatetraenoic acid (12-HETE) and 12(S)-hydroxy-5-cis-8,10-trans-heptadecatrienoic acid (12-HHT) in human platelets. The release of arachidonic acid and the formation of 12-HETE was completely blocked by the cPLA2-alpha inhibitors AACOCF3 or pyrrolidine-1. PCB-treatment of platelets demonstrated that the cPLA2-alpha protein as well as PLA2 activity translocated to the membrane fraction, independent of a rise in intracellular Ca2+. Furthermore, electrophoretic gel mobility shift analysis of cPLA2-alpha on SDS-PAGE demonstrated a PCB-dependent phosphorylation of cPLA2-alpha. The effects of 17beta-estradiol and two structurally unrelated anti-estrogens, nafoxidin and tamoxifen on PCB-induced arachidonic acid release in platelets were also investigated. Both nafoxidin and tamoxifen inhibited PCB-induced arachidonic acid release as well as 12-HETE and 12-HHT formation. Interestingly, platelets incubated with PCBs did not aggregate despite the fact that robust release of arachidonic acid was observed. In summary, these results demonstrate that certain PCBs induce activation of cPLA2-alpha independent of a rise in intracellular calcium and a robust release of arachidonic acid release with resulting eicosanoid formation in human platelets.

The battle of Alzheimer's Disease - the beginning of the future Unleashing the potential of academic discoveries.

Alzheimer's Disease (AD) is the most common form of dementia, affecting approximately 36 million people worldwide. To date there is no preventive or curative treatment available for AD, and in absence of major progress in therapeutic development, AD manifests a concrete socioeconomic threat. The awareness of the growing problem of AD is increasing, exemplified by the recent G8 Dementia Summit, a meeting held in order to set the stage and steer the compass for the future. Simultaneously, and paradoxically, we have seen key players in the pharmaceutical industry that have recently closed or significantly decreased their R&D spending on AD and other CNS disorders. Given the pressing need for new treatments in this area, other actors need to step-in and enter this drug discovery arena complementing the industrial efforts, in order to turn biological and technological progress into novel therapeutics. In this article, we present an example of a novel drug discovery initiative that in a non-profit setting, aims to integrate with both preclinical and clinical academic groups and pharmaceutical industry to explore the therapeutic potential of new concepts in patients, using novel biology, state of the art technologies and rapid concept testing.

The use of TrkA-PathHunter assay in high-throughput screening to identify compounds that affect nerve growth factor signaling.

The TrkA-PathHunter cell-based assay was used in high-throughput screening (HTS) to identify compounds that inhibit nerve growth factor (NGF)/TrkA signaling. The assay was conducted in a 384-well format, and typical Z' values during HTS ranged from 0.3 to 0.8. The reproducibility of IC50 values was good, and the use of cryopreserved cells was well tolerated, as judged by assay parameters such as Z' and S/B and by comparison of IC50 values obtained with cells in culture. During hit deconvolution, TrkA-kinase inhibitors were identified with ATP-competitive as well as non-ATP-competitive mechanisms of action. Furthermore, other mechanisms of action such as NGF and TrkA antagonists were also identified. Because of the different molecular mechanisms identified, it is possible that subsequent optimization work to increase affinity and selectivity might lead to compounds that could have a better chance to evoke clinical efficacy without the adverse effects observed for nonselective TrkA inhibitors.

Metabolism of anandamide into eoxamides by 15-lipoxygenase-1 and glutathione transferases.

Human 15-lipoxygenase-1 (15-LO-1) can metabolize arachidonic acid (ARA) into pro-inflammatory mediators such as the eoxins, 15-hydroperoxyeicosatetraenoic acid (HPETE), and 15-hydroxyeicosatetraenoyl-phosphatidylethanolamine. We have in this study investigated the formation of various lipid hydroperoxide by either purified 15-LO-1 or in the Hodgkin lymphoma cell line L1236, which contain abundant amount of 15-LO-1. Both purified 15-LO-1 and L1236 cells produced lipid hydroperoxides more efficiently when anandamide (AEA) or 2-arachidonoyl-glycerol ester was used as substrate than with ARA. Furthermore, L1236 cells converted AEA to a novel class of cysteinyl-containing metabolites. Based on RP-HPLC, mass spectrometry and comparison to synthetic products, these metabolites were identified as the ethanolamide of the eoxin (EX) C(4) and EXD(4). By using the epoxide EXA(4)-ethanol amide, it was also found that platelets have the capacity to produce the ethanolamide of EXC(4) and EXD(4). We suggest that the ethanolamides of the eoxins should be referred to as eoxamides, in analogy to the ethanolamides of prostaglandins which are named prostamides. The metabolism of AEA into eoxamides might engender molecules with novel biological effects. Alternatively, it might represent a new mechanism for the termination of AEA signalling.

Development of a fluorescent intensity assay amenable for high-throughput screening for determining 15-lipoxygenase activity.

15-Lipoxygenase-1 catalyzes the introduction of molecular oxygen into polyunsaturated fatty acids to form a lipid hydroperoxide. The authors have developed an assay for the detection of lipid hydroperoxides formed by human 15-lipoxygenase (15-LO) in enzyme or cellular assays using either a 96-well or a 384-well format. The assays described take advantage of the ability of lipid hydroperoxides to oxidize nonfluorescent diphenyl-1-pyrenylphosphine (DPPP) to a fluorescent phosphine oxide. Oxidation of DPPP yields a fluorescent compound, which is not sensitive to temperature and is stable for more than 2 h. The assay is sensitive toward inhibition and robust with a Z' value of 0.79 and 0.4 in a 96- and 384-well format, respectively, and thus amenable for high-throughput screening. The utility of DPPP as a marker for 15-lipoxygenase activity was demonstrated with both enzyme- and cell-based assays for the identification of hits and to determine potency by IC(50) determinations.

Basal activation of p70S6K results in adipose-specific insulin resistance in protein-tyrosine phosphatase 1B -/- mice.

Although protein-tyrosine phosphatase 1B (PTP-1B) is a negative regulator of insulin action, adipose tissue from PTP-1B-/- mice does not show enhanced insulin-stimulated insulin receptor phosphorylation. Investigation of glucose uptake in isolated adipocytes revealed that the adipocytes from PTP-1B-/- mice have a significantly attenuated insulin response as compared with PTP-1B+/+ adipocytes. This insulin resistance manifests in PTP-1B-/- animals older than 16 weeks of age and could be partially rescued by adenoviral expression of PTP-1B in null adipocytes. Examination of adipose signaling pathways found that the basal p70S6K activity was at least 50% higher in adipose from PTP-1B-/- mice compared with wild type animals. The increased basal activity of p70S6K in PTP-1B-/- adipose correlated with decreases in IR substrate-1 protein levels and insulin-stimulated Akt/protein kinase B activity, explaining the decrease in insulin sensitivity even as insulin receptor phosphorylation was unaffected. The insulin resistance of the of the PTP-1B-/- adipocytes could also be rescued by treatment with rapamycin, suggesting that in adipose the loss of PTP-1B results in basal activation of mTOR (mammalian target of rapamycin) complex 1 leading to a tissue-specific insulin resistance.

Transcriptional regulation of 15-lipoxygenase expression by promoter methylation.

15-Lipoxygenase type 1 (15-LO), a lipid-peroxidating enzyme implicated in physiological membrane remodeling and the pathogenesis of atherosclerosis, inflammation, and carcinogenesis, is highly regulated and expressed in a tissue- and cell-type-specific fashion. It is known that interleukins (IL) 4 and 13 play important roles in transactivating the 15-LO gene. However, the fact that they only exert such effects on a few types of cells suggests additional mechanism(s) for the profile control of 15-LO expression. In the present study, we demonstrate that hyper- and hypomethylation of CpG islands in the 15-LO promoter region is intimately associated with the transcriptional repression and activation of the 15-LO gene, respectively. The 15-LO promoter was exclusively methylated in all examined cells incapable of expressing 15-LO (certain solid tumor and human lymphoma cell lines and human T lymphocytes) while unmethylated in 15-LO-competent cells (the human airway epithelial cell line A549 and human monocytes) where 15-LO expression is IL4-inducible. Inhibition of DNA methylation in L428 lymphoma cells restores IL4 inducibility to 15-LO expression. Consistent with this, the unmethylated 15-LO promoter reporter construct exhibited threefold higher activity in A549 cells compared to its methylated counterpart. Taken together, demethylation of the 15-LO promoter is a prerequisite for the gene transactivation, which contributes to tissue- and cell-type-specific regulation of 15-LO expression.