Identification of novel benzimidazole derivatives as inhibitors of leukotriene biosynthesis by virtual screening targeting 5-lipoxygenase-activating protein (FLAP)

Pharmacological suppression of leukotriene biosynthesis by 5-lipoxygenase (5-LO)-activating protein (FLAP) inhibitors is a promising strategy to intervene with inflammatory, allergic and cardiovascular diseases. Virtual screening targeting FLAP based on a combined ligand- and structure-based pharmacophore model led to the identification of 1-(2-chlorobenzyl)-2-(1-(4-isobutylphenyl)ethyl)-1H-benzimidazole (7) as developable candidate. Compound 7 potently suppressed leukotriene formation in intact neutrophils (IC(50)=0.31 μM) but essentially failed to directly inhibit 5-LO suggesting that interaction with FLAP causes inhibition of leukotriene synthesis. For structural optimization, a series of 46 benzimidazole-based derivatives of 7 were synthesized leading to more potent analogues (70-72, 82) with IC(50)=0.12-0.19 μM in intact neutrophils. Together, our results disclose the benzimidazole scaffold bearing an ibuprofen fingerprint as a new chemotype for further development of anti-leukotriene agents.     

IL-5 increases expression of 5-lipoxygenase-activating protein and translocates 5-lipoxygenase to the nucleus in human blood eosinophils.

Cysteinyl-leukotrienes are potent bronchoconstrictor mediators synthesized by the 5-lipoxygenase (5-LO) pathway. Eosinophilopoietic cytokines such as IL-5 enhance cysteinyl-leukotriene synthesis in eosinophils in vitro, mimicking changes in eosinophils from asthmatic patients, but the mechanism is unknown. We hypothesized that IL-5 induces the expression of 5-LO and/or its activating protein FLAP in eosinophils, and that this might be modulated by anti-inflammatory corticosteroids. Compared with control cultures, IL-5 increased the proportion of normal blood eosinophils immunostaining for FLAP (65 +/- 4 vs 34 +/- 4%; p < 0.0001), enhanced immunoblot levels of FLAP by 51 +/- 14% (p = 0.03), and quadrupled ionophore-stimulated leukotriene C4 synthesis from 5.7 to 20.8 ng/106 cells (p < 0.02). IL-5 effects persisted for 24 h and were abolished by cycloheximide and actinomycin D. The proportion of FLAP+ eosinophils was also increased by dexamethasone (p < 0.0001). Neither IL-5 nor dexamethasone altered 5-LO expression, but IL-5 significantly increased 5-LO immunofluorescence localizing to eosinophil nuclei. Compared with normal subjects, allergic asthmatic patients had a greater proportion of circulating FLAP+ eosinophils (46 +/- 6 vs 27 +/- 3%; p < 0.03) and a smaller IL-5-induced increase in FLAP immunoreactivity (p < 0.05). Thus, IL-5 increases FLAP expression and translocates 5-LO to the nucleus in normal blood eosinophils in vitro. This is associated with an enhanced capacity for cysteinyl-leukotriene synthesis and mimics in vivo increases in FLAP expression in eosinophils from allergic asthmatics.     

Cytogenetic and radiation hybrid mapping of human arachidonate 5-lipoxygenase-activating protein (ALOX5AP) to chromosome 13q12

Arachidonate 5-lipoxygenase-activating protein (ALOX5AP) is an arachidonic acid binding protein that has been shown to be critical in the biosynthesis of leukotrienes. We mapped the ALOX5AP gene to the chromosome 13q12 region by cytogenetic mapping, yeast artificial chromosome (YAC) pool screening, and radiation hybrid mapping. It was mapped to YAC contig WC13.2 by YAC pool screening with an unambiguous hit to WI-4874, which is at 78 cR on the radiation hybrid map, 3.36 cR, by radiation hybrid mapping, from WI-4874.     

Isolation and characterization of the human CLC-5 chloride channel gene promoter

The human CLC-5 chloride channel is expressed mainly in the kidney and its mutations cause Dent's disease (a familial renal tubular syndrome with hypercalciuria, tubular proteinuria, rickets, nephrocalcinosis, and eventual renal failure). To gain insight into the regulatory mechanism of CLC-5 expression, a genomic clone that contains the 5'-flanking region of the human CLC-5 gene was isolated and characterized. Two types of 5'-ends of cDNA were isolated by 5'-rapid amplification of cDNA ends, and one of them, approximately 2.1 kbp upstream of ATG-containing exon II, was first identified in human. The major promoter activity was detected in the 5'-flanking region of this newly identified exon Ia. The sequence of the proximal 5'-flanking region contained an activator protein (AP)-1-like site and cAMP-responsive element, but it lacked a TATA box, a GC-rich element, and an SP-1 site. Deletion analysis of the 5'-flanking region showed that the fragments containing the AP-1-like element (TGACTCC) positioned at -38 exhibited high promoter activities in CLC-5 expressing LLC-PK1 cells, but that further deletions not containing this AP-1-like element resulted in a great loss of luciferase activities. Gel-retardation analysis demonstrated the existence of a specific protein binding to this AP-1-like element in LLC-PK1 cells, which seemed to differ from an authentic AP-1. This study clarified the key element of the human CLCN5 promoter, and the mutation in this region could be the cause of Dent's disease.     

Correlation between expression of 5-lipoxygenase-activating protein, 5-lipoxygenase, and cellular leukotriene synthesis

Previous studies involving transfection of cDNAs for 5-lipoxygenase-activating protein (FLAP) and 5-lipoxygenase into osteosarcoma cells have shown that both these proteins are essential for leukotriene synthesis (Dixon, R. A. F., Diehl, R. E., Opas, E., Rands, E., Vickers, P. J., Evans, J. F., Gillard, J. W., and Miller, D. K. (1990) Nature 343, 282-284). In the present study we show that FLAP is present in a variety of cells known to produce leukotrienes, but is absent from a number of cells which do not synthesize leukotrienes. Furthermore, differentiation of the human promyelocytic HL-60 cell line towards granulocytic cells following exposure to dimethylsulfoxide is associated with the concurrent induction of both FLAP and 5-lipoxygenase and an increased capacity to synthesize leukotrienes. Cellular leukotriene synthesis in this system is functionally dependent on FLAP as shown by its inhibition by the leukotriene biosynthesis inhibitor MK-886, a compound which specifically binds to FLAP.     

Parital characterization of the rat anti-encephalitogenic protein (RSCP).

A protein antigenically similar to the anti-encephalitogenic bovine spinal cord protein (BSCP) was detected in saline extracts of rat nervous tissues by immunodiffusion analyses using a rabbit anti-BSCP serum. Rat SCP (RSCP) appears to be evenly distributed throughout all parts of the rat nervous system and occurs also in the thymus, thyroid, and adrenal glands. Although immunodiffusion analyses indicated that RSCP shares some antigenic sites with BSCP, anti-RSCP sera reacted only with RSCP, indicating that the major immunogenic determinants of the RSCP are peculiar to the rat and differ from the immunogenic determinants of human, monkey, rabbit, guinea pig, or bovine SCP. Immunoelectrophoretic analyses of concentrated pastes of rat brain (RB) or rat spinal cord (RSC) in agar at pH 8.6 revealed that RSCP occurs in two molecular forms having the electrophoretic mobilities of a serum beta-globulin and a serum gamma-globulin, respectively. However, gamma-RSCP is the predominant component of extracts of brain or spinal cord. Gamma-RSCP was isolated from RB and RSC by a procedure which involved: a) extraction with 0.05 M ammonium acetate buffer, pH 4.0; b) batch absorption of impurities on CM-52 cellulose; c) batch absorption of RSCP on SP-Sephadex, pH 3.5; d) elution of RSCP from SP-Sephadex, pH 5.5; and finally, e) gel filtration on Sephadex G-50 superfine. Purified gamma-RSCP formed one band when analyzed by polyacrylamide electrophoresis in acid gels containing 8 M urea. In contrast, two bands were always present when gamma-RSCP from brain or spinal cord were subjected to SDS-polyacrylamide electrophoresis in 15% gels. The larger of the two components of brain gamma-RSCP had a m.w. of 12,400 daltons, whereas the two components of spinal cord gamma-RSCP were smaller. The molecular sizes of brain RSCP and spinal cord RSCP was estimated by gel filtration chromatography to be 12,400 daltons. The amino acid compositions of gamma-RSCP prepared from RB or RSC were similar except that gamma-RSCP from RSC contained twice as much half-cystine and a slightly higher proportion of basic amino acid than gamma-RSCP from RB.     

Association between the gene encoding 5-lipoxygenase-activating protein and stroke replicated in a Scottish population

Cardiovascular diseases, including myocardial infarction (MI) and stroke, most often occur on the background of atherosclerosis, a condition attributed to the interactions between multiple genetic and environmental risk factors. We recently reported a linkage and association study of MI and stroke that yielded a genetic variant, HapA, in the gene encoding 5-lipoxygenase-activating protein (ALOX5AP), that associates with both diseases in Iceland. We also described another ALOX5AP variant, HapB, that associates with MI in England. To further assess the contribution of the ALOX5AP variants to cardiovascular diseases in a population outside Iceland, we genotyped seven single-nucleotide polymorphisms that define both HapA and HapB from 450 patients with ischemic stroke and 710 controls from Aberdeenshire, Scotland. The Icelandic at-risk haplotype, HapA, had significantly greater frequency in Scottish patients than in controls. The carrier frequency in patients and controls was 33.4% and 26.4%, respectively, which resulted in a relative risk of 1.36, under the assumption of a multiplicative model (P=.007). We did not detect association between HapB and ischemic stroke in the Scottish cohort. However, we observed that HapB was overrepresented in male patients. This replication of haplotype association with stroke in a population outside Iceland further supports a role for ALOX5AP in cardiovascular diseases.     

Single nucleotide polymorphism analysis and functional characterization of the human Ah receptor (AhR) gene promoter

The aryl hydrocarbon receptor (AhR) mediates biological and toxicological actions of e.g., halogenated aromatic hydrocarbons such as 2,3,7,8-tetrachlorodibenzo-p-dioxin. Although much is known about the biochemical and molecular mechanisms of AhR action, little is known about the control of the expression of the AhR gene itself. Therefore, we aimed at the identification and characterization of regions important for constitutive AhR gene expression. First, we screened 2.6 kb of the 5(')-flanking region of the AhR gene in 91 healthy Caucasian volunteers for naturally occurring genetic variants. Seven variants were detected. However, they do not seem to influence AhR gene expression in lymphocytes. Using a 2.7 kb AhR promoter luciferase reporter gene construct and various deletion constructs, a putative regulatory region was identified and characterized further by electrophoretic mobility shift assays and site-directed mutagenesis. These investigations were confirmed by cotransfection experiments in Drosophila SL2 cells. The obtained results prove an involvement of Sp1 in AhR gene regulation.     

Gene structure and promoter analysis of the human GJB6 gene encoding connexin 30

Connexins (Cx) are the protein subunits of gap junctions, which play an important role in cell-to-cell communication. We characterized the genomic structure of the human GJB6 gene, encoding connexin 30 (C x 30), and showed that it differs from most connexin-encoding genes. GJB6 presents six different exons, some of which can be alternatively spliced. We also mapped a basal promoter sequence active in a human keratinocyte cell line which responds to the activation of the EGF receptor. One of the non-encoding exons of GJB6, which has been described in brain C x 30 cDNA, was not found in cDNA obtained from human keratinocytes, suggesting tissue-specific splicing.