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    • Direct inhibition of LO activity by BRP is

    2023-10-31

    Direct inhibition of 5-LO activity by BRP-187 is clearly evident in cell-free assays using PMNL homogenates and isolated human recombinant 5-LO as enzyme source. In such assays, pure FLAP inhibitors like MK886 are inactive [9], [10], [29], [44]. Wash-out experiments and studies using the nonionic detergent Triton X-100 exclude irreversible 5-LO inhibition and unspecific (lipophilic) aggregate-induced 5-LO interference, respectively. Note that in contrast to direct redox-type and iron-chelating 5-LO inhibitors, the so-called “competitive nonredox-type” 5-LO inhibitors (such as CJ-13,610 [37] or ZM230487 [38]) show a similar pattern of differential efficiency in cell-free and cell-based 5-LO assays, that is, loss of potency in cell-free test systems. Therefore, BRP-187 could act as nonredox-type 5-LO inhibitor with high potency only in intact cells. However, lowering hydroperoxide levels by reconstitution of a reducing milieu (supplementation of GSH or DTT to PMNL homogenates) restores efficient 5-LO inhibition by nonredox-type inhibitors [45], which was not the case for BRP-187. Together, BRP-187 specifically and reversibly interferes with 5-LO, albeit only at 30- to 300-fold higher concentrations as compared to suppression of 5-LO product biosynthesis in intact PMNL or monocytes. Because FLAP is member of the MAPEG family [46], we asked whether BRP-187 may affect also other enzymes out of this class, which in fact is the case for MK886 that blocks LTC4 synthase [40] and mPGES-1 activity [39]. Although BRP-187 failed to inhibit AA release in intact cells (this study) or other AA-converting dioxygenases like COX-1/2 or 12/15-LOs [21], it markedly suppressed mPGES-1 activity and to a lower extent also LTC4 synthase activity. In this respect, the high potency of BRP-187 against human mPGES-1 is remarkable (IC50=0.2μM versus 2.4μM for MK886 [41] in a similar assay) and might be of pharmacological relevance for the treatment of pain and inflammatory conditions. Further cellular and preclinical studies warrant evaluation of (human and rodent) mPGES-1 inhibition in more detail. Despite the obvious value and benefit of anti-LT therapy for many LT-related diseases such as Diclofenac and allergic rhinitis, CVD and cancer, there is still an unmet need for safe and efficient drugs suitable for intervention in pharmacotherapy [47]. In fact, there are currently several clinical trials ongoing with FLAP inhibitors and promising results encourage for preclinical evaluations of novel compounds [10]. Moreover, data from cohort studies suggest a link between CVD and FLAP [48], [49], [50], [51] and increased the interest in developing agents that interfere with FLAP. It also seems that FLAP inhibitors might outperform direct 5-LO inhibitors in in vivo experiments, in preclinical studies, and in clinical trials [10]. In our LT-related model of murine peritonitis induced by zymosan [30], BRP-187 (as well as MK886) was highly efficient in reducing the cysLT levels in vivo after i.p. application and in this respect outperformed the direct 5-LO inhibitor zileuton. Accordingly, one of the major bioactivities of cysLTs, i.e. increase of vascular permeability [52], was significantly blocked under these conditions by BRP-187 but not so by zileuton at the same dose. Along these lines, infiltration of leukocytes into the peritoneal cavity, which is potently elicited by the powerful chemotactic LTB4[53], was inhibited by BRP-187 in the zymosan-induced peritonitis model. Therefore, our data support the concept of FLAP interference as pharmacological therapy of LT-related diseases and highlight BRP-187 as novel type of efficient inhibitor of the FLAP/5-LO complex assembly. MK886, an indole derivate that inhibits LT formation in vitro and in vivo was used as tool/probe to identify FLAP [36], and thus represented the first FLAP inhibitor [18], [54] that was assessed up to phase II clinical studies. Subsequently, the quinoline-class compound BAY X1005 (syn. DG-031) from Bayer [55], and the quinoline-indole hybrid MK591 [56] were presented as FLAP inhibitors that effectively inhibited LT biosynthesis. Although these compounds could enter phase II clinical trials, the clinical evaluations were discontinued for unknown reasons. However, novel derivatives of MK886 such as AM103 and the follow-up compound AM803 (now GSK2190915) were recently developed that potently inhibited LTB4 formation with acceptable pharmacokinetics and preclinical toxicology [44], [57], [58]. GSK2190915 underwent several phase II clinical trials for treatment of asthma with partially promising results.