Human lipoxygenase exists in two forms named
Human 15-lipoxygenase exists in two forms, named 15-LOX-1 (also named 12/15-LOX, 15-LO-1) and 15-LOX-2. Several reports indicate that 15-LOX-1 has a pathophysiological role in respiratory inflammatory diseases, in particular asthma. Increased activity of 15-LOX-1 is displayed in the respiratory tract in patients with asthma. 15-LOX-1 is expressed in distinct sets of human cells, for example, airway epithelial cells, eosinophils, mast cells, dendritic cells and macrophages, and an increased expression of the enzyme is often observed in inflammatory diseases, especially those driven by IL-4/IL-13. Studies of both human airway epithelial cells and in rodents indicate that 15-LOX-1 might be involved in mucus production in the respiratory tract., Increased expression and/or activity of 15-LOX-1 have also been demonstrated in Alzheimer’s disease, indicating a possible pathophysiological function in this condition., There are also reports indicating a role of 15-LOX-1 in cell differentiation and maturation of erythrocytes. Some studies also suggest a role of 15-LOX-1 in neoplasia such as prostate cancer and colon carcinoma cells. Finally, inhibition of 15-LOX-1 has been investigated for the treatment of stroke. The predominant arachidonic ionomycin metabolite formed via the 15-LOX-1 pathway is 15()-hydroperoxyeicosatetraenoic acid (15()-HPETE) which in turn can be reduced to 15()-hydroxyeicosatetraenoic acid (15()-HETE) (). Increased expression of 15-LOX-1 in the respiratory tract and elevated levels of 15-HETE in broncoalveolar lavage fluid have been found in asthmatic patients compared to control subjects., In addition, 15()-HPETE can also be converted to the epoxide eoxin A (EXA). This metabolite can be conjugated with glutathione yielding EXC, which in turn can be converted to EXD and EXE (). The biosynthesis of eoxins has been demonstrated in eosinophils, mast cells, nasal polyps, and in the Hodgkin lymphoma cell line L1236. Eoxins have also been found in exhaled breath condensates from children with asthma. Although our knowledge of the biological role of the eoxins is still limited, these mediators have been shown to increase the permeability of an endothelial cell monolayer in vitro, indicating a pro-inflammatory role. Overexpression of 15-LOX-1 in cultured human lung epithelial cells has been shown to stimulate the expression and release of chemokines. Recently, it was reported that 15-LOX-1 activity was greater in eosinophils derived from patients with severe asthma or aspirin-intolerant asthma (AIA) compared to eosinophils isolated from patients with mild asthma or healthy controls. There are numerous publications regarding 15-LOX-1 inhibitors, but many studies are conducted using 15-LOX-1 from non-human species, most notably soybean (see, e.g., Refs. , , ). Such studies are of limited relevance for developing compounds for treatment of human disorders. The groups at Parke–Davis/Warner–Lambert (now Pfizer), , , , , , and Bristol-Myers Squibb, , , used the rabbit reticulocyte 15-LOX-1 for their primary screen, followed by a cell-based assay with recombinant 15-LOX-1 or a rabbit in vivo model to design compounds for the treatment of inflammation and/or atherosclerosis. More recently, Maloney and Holman used 15-LOX-1 in their primary screen to develop molecular tools and compounds aimed for anti-stroke therapy. A selection of 15-LOX-1 inhibitors reported by the groups mentioned above are depicted in . Herein we describe some of our efforts to develop 15-LOX-1 inhibitors for the potential use in the treatment of asthma and chronic obstructive pulmonary disease (COPD). Parts of this work, including experimental procedures, have been disclosed in patent applications., To find a suitable starting point we attempted several approaches: HTS, virtual screening and synthesis of analogues of known lipoxygenase inhibitors. The HTS approach was found to be the most fruitful so the others were soon abandoned. A chemical library comprising a diverse set of 50000 compounds were assayed as inhibitors of 15-LOX-1 at 10μM using linoleic acid (LA) as the substrate. Although there is a difference in the absolute value when AA or LA is used as a substrate, the cost and chemical stability favoured LA in the hit finding phase. However, we would like to emphasize the importance of using 15-LOX-1 in the primary screen, either in an enzyme- or cell-based assay, and then (or concomitantly) ascertain if the compounds also inhibit the proper lipoxygenase from the species that will be used in the in vivo model. For example, compounds reported to be highly active as inhibitors of the rabbit enzyme in our hands did not always show activity when tested in our human enzyme- and cell-based assays. Thus, using, for example, rabbit reticulocyte 15-LO in the primary screen may lead to erroneous conclusions and to the rejection of good inhibitors of human 15-LOX-1.