Pontus Forsell

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.

On the expression of cytosolic calcium-independent phospholipase A2 (88 kDa) in immature and mature myeloid cells and its role in leukotriene synthesis in human granulocytes

The human calcium‐independent phospholipase A2 (iPLA2; 88 kDa) has recently been cloned (Larsson, P.K.A., Claesson, H.‐E. and Kennedy, B.P. (1998) J. Biol. Chem. 272, 207–214). Here we demonstrate the expression of the human iPLA2 mRNA and its splice variants in blood progenitor cells, immature leukemic cells and mature granulocytes. Chromatographical resolvable iPLA2 activity was found in the cytosolic fraction of granulocytes and the activity was inhibited by the iPLA2 inhibitor bromoenol lactone. This drug also inhibited leukotriene synthesis in human granulocytes, induced by low concentration of calcium ionophore A23187 (0.10–0.15 μM) or opsonized zymosan. These results suggest that iPLA2 is involved in the regulation of the pool of arachidonic acid destined for leukotriene synthesis in human granulocytes.

Cloning, purification and characterization of non-human primate 12/15-lipoxygenases

The enzyme 15-lipoxygenase-1 (15-LO-1) possesses mainly 15-LO activity and has so far only been described in human cells and rabbit reticulocytes. The animal ortholog, except rabbit reticulocytes, is an enzyme with predominantly a 12-lipoxygenase activity, commonly referred to as 12/15-LO. We describe herein the characterization of the 12/15-LOs in Macaca mulatta (rhesus monkey) and in Pongo pygmaeus (orang-utan). The rhesus and the orang-utan enzymes have mainly 12-lipoxygenase and 15-lipoxygenase activity, respectively, and they display 94% and 98% identity to the human 15-LO-1 protein. The rhesus enzyme was functionally different from the human enzyme with respect to substrate utilization in that anandamide was used differently and that the rhesus enzymes positional specificity could be affected by the substrate concentration. Furthermore, genomic data indicate that chimpanzees express an enzyme with mainly 15-lipoxygenase activity whereas marmosets express an enzyme with mainly 12-LO activity. Taken together, the switch during evolution from a 12-lipoxygenating enzyme in lower primates to a 15-lipoxygenating enzyme in higher primates and man might be of importance for the biological function of this enzyme.

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.

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.

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 IC50 determinations.

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.

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 − 14C] 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.

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.

Chapter Five - Stimulating Neurotrophin Receptors in the Treatment of Neurodegenerative Disorders

Abstract Neurodegenerative disorders, such as Alzheimers disease, Parkinsons disease, and Huntingtons disease are currently lacking disease-modifying treatments. A substantial body of evidence links increases in neurotrophin (NT) signaling in neurodegenerative disorders with biological mechanisms that could have a positive effect on disease outcome. NTs like NGF and BDNF signal through Trk receptors. This signaling is of high importance to support neuronal survival, differentiation, neurogenesis, and synaptic plasticity. The poor pharmacokinetics of NTs makes them unsuitable as drugs. In addition, the NTs have pleiotropic effects that may give side effects. Therefore, the identification of small molecules that promote NT signaling is of increasing interest. Such small-molecule activators of NT signaling will support neuronal survival in the CNS, and thus they could possibly function as disease modifiers. Herein, we review the current status of small-molecule compounds that are able to activate NT receptors.