A promising target that is involved in the

A promising target that is involved in the biosynthesis of the precursor LTA4 is the 5-LO-activating protein (FLAP), which in vivo is seemingly indispensable for LT formation [8], [9]. FLAP, a member of the MAPEG family (membrane-associated proteins in eicosanoid and glutathione metabolism), is an integral nuclear membrane protein lacking enzymatic activity but it is playing a crucial role in LT formation in various intact leukocytes [8], [10]. Upon activation, 5-LO redistributes from soluble cellular pools to the perinuclear region and interacts with FLAP at the nuclear membrane [11], [12]. FLAP facilitates AA substrate transfer to 5-LO [13], [14] and AA is required for assembling the 5-LO/FLAP complex. In fact, FLAP inhibitors efficiently inhibit 5-LO product formation in intact TCS JNK 5a which can be overcome, at least in part, by supplementation of high concentrations of exogenous AA [9], [11], [14], [15], [16]. Notably, FLAP inhibitors fail to inhibit 5-LO activity in cell-free assays [17], [18], implying that FLAP is operative in 5-LO product biosynthesis only in the cellular context. The crystal structure of FLAP provided substantial insights into the binding site of AA and also clarified how FLAP inhibitors (i.e., MK591) might compete with AA from binding to FLAP [19]. Moreover, we recently showed that FLAP may help to coordinate 5-LO membrane binding with unblocking active AA-binding site access that is corked by Y181 and F177 [14]. Together, a dynamic 5-LO/FLAP complex is crucial for LTA4 biosynthesis and represents, besides direct targeting of 5-LO enzymatic activity, a valuable target for pharmacological inhibitors in order to effectively and selectively intervene with LT-related disorders. Within the frame of a recent screening approach for novel chemotypes that antagonize FLAP using a combined ligand- and structure based pharmacophore model [20], compound 1 (2-[4-(4-chlorophenyl)-3-methyl-1,2-oxazol-5-yl)-5-[(2-methylphenyl)methoxy)phenol, A was identified as moderate inhibitor of 5-LO product biosynthesis in human neutrophils (IC50=4.4μM), with unclear mode of action. In a follow-up study aiming to improve the LT biosynthesis inhibitory potency of compound 1 through interference with FLAP, the derivative BRP-187 (4-(4-chlorophenyl)-5-[4-(quinolin-2-ylmethoxy)phenyl]isoxazol-3-carboxylic acid, A was obtained that potently inhibited cellular 5-LO product formation with an IC50=0.24μM and >30-fold lower activity against 5-LO directly in cell-free assays [21]. Here we (i) provide a comprehensive analysis of BRP-187 on 5-LO product biosynthesis in vitro and in vivo, (ii) reveal that this compound impedes the 5-LO/FLAP complex assembly, and (iii) demonstrate potent anti-inflammatory effectiveness of BRP-187 in a LT-related animal model of inflammation.
Materials and methods
Results
Discussion We here present the 4,5-diarylisoxazol-3-carboxylic acid derivative BRP-187 as highly potent inhibitor of cellular 5-LO product biosynthesis in human PMNL and monocytes with IC50 of 7–10nM upon cell activation by LPS and fMLP that are pathophysiological relevant stimuli [34]. In mouse zymosan-induced peritonitis, BRP-187 exhibits marked effectiveness as LT biosynthesis inhibitor in vivo, and effectively suppressed vascular permeability and infiltration of leukocytes which are the major biological functions of cysLTs and LTB4, respectively [2]. Our mechanistic studies using PLA suggest that BRP-187 acts primarily by preventing the 5-LO/FLAP complex assembly, a determinant for cellular LT biosynthesis [2], [8], but affects 5-LO enzymatic activity only at micromolar concentrations, and fails to interfere with cellular AA release or cellular viability. Our results suggest that BRP-187 apparently interacts with both, 5-LO and FLAP. Assignment of a given compound as FLAP inhibitor is hampered by the circumstance that (i) FLAP is operative only in intact cells and (ii) FLAP possesses no enzymatic activity or any function that can be easily monitored as read-out reflecting functional FLAP interference. Nevertheless, we provide several lines of evidence that BRP-187 interferes with the function of FLAP. First, our previous docking studies and MD simulations using the 3D structure of FLAP (PDB code 2Q7M) reveal concrete molecular interactions of BRP-187 with amino acids in the AA-binding pocket and thus, strongly support FLAP binding [21]. Second, the potent suppression of cellular 5-LO product biosynthesis in PMNL and monocytes was clearly impaired by provision of excess of exogenous AA, which is a typical feature for FLAP inhibitors [15], [29], [42]. Thus, FLAP facilitates access of endogenous AA to 5-LO [13], [16], [19] and supplementation of exogenous AA can circumvent the requirement for FLAP. Third, and strikingly, BRP-187 prevented the agonist-induced 5-LO/FLAP complex assembly in monocytes and PMNL analyzed by a previously established PLA [11], a method that allows studying in-situ analysis of intracellular protein-protein interactions [27]. This effect of BRP-187 was not due to general blockade of 5-LO translocation to the perinuclear region, even when higher concentrations of BRP187 (or MK886 as control) were used as compared to the PLA experiments. This is in contrast, to hyperforin that prevents membrane-binding of 5-LO via interaction with the C2-like domain [43]. Of note, MK886, a drug that was originally used as probe to identify FLAP [36], revealed the same pattern: preventing 5-LO/FLAP complex formation without blocking 5-LO translocation. In this respect, the direct 5-LO inhibitor zileuton (used as control) was not effective in either experiment, as expected and shown before [11].