FLAP/5-lipoxygenase-activating protein (IC50 = 1.6 nM)

SB203580 and Quiflaponodium (MK591) prevent human PBMC cells from proliferating when exposed to SEB. Three genes that are increased by SEB—cathepsin L, IL-17, and guanylate binding protein (GBP)-2—are downregulated by quilaponodium (MK591) [1]. Treatment with quiflapon sodium (MK591) causes apoptosis within hours. Additionally, quilaponodium causes the stress kinase c-Jun N-terminal kinase (JNK) to activate quickly. JNK is crucial to the process of apoptosis. Prostate cancer cells undergo apoptosis when exposed to quilapon sodium, however PI3K-Akt or ERK are not inhibited. Furthermore, Quifflapon sodium and LY294002 work in concert to cause prostate cancer cells to undergo apoptosis [2]. Sp1 is unaffected by quilapon sodium (MK591), but the cAMP response element binding protein is[4].

In contrast to the untreated hyperoxia group, the mice in the hyperoxia group given Quiflapon sodium (MK591) (20, 40 mg/kg) demonstrated aberrant alveolarization, while the alveolarization in the treated group resembled that of room air controls. However, no inflammation is present [3]. When Aβ immunopositive areas from placebo and Quiflapon sodium (MK591) (320 mg/kg) treatment groups were compared, the treated mice's amyloid load was statistically significantly lower. Moreover, quilapon sodium dramatically lowers brain levels of IL-1β. The steady-state levels of overall CREB and its Ser133 phosphorylated version were statistically significantly lower in mice given Quiflapon sodium [4].

MK591 is a synthetic compound which specifically inhibits the activity of 5-Lox and is currently under development for the treatment of asthma. We observed that human prostate cancer cells treated with MK591 undergo apoptosis within hours of treatment. Apoptosis involves severe morphological alteration, externalization of phosphatidyl-serine, cleavage of PARP, and degradation of chromatin-DNA. MK591 also induced rapid activation of the stress kinase, c-Jun N-terminal kinase (JNK), which plays an important role in the apoptosis process. The phosphatidylinositol 3'-kinase-Akt/protein kinase B (PI3K/Akt) axis is a well-known pro-survival pathway which prevents apoptosis through defined anti-apoptotic mechanisms in a variety of cancer cells. Interestingly, we observed that MK591 triggers apoptosis in prostate cancer cells without inhibition of PI3K-Akt, or ERK. Moreover, it was observed that MK591 and LY294002 (an inhibitor of PI3K) exert synergistic effect in inducing apoptosis in prostate cancer cells. Altogether, these findings indicate that 5-Lox inhibition-induced apoptosis in prostate cancer cells occurs without inhibition of PI3K-Akt, or ERK, and suggest for the existence of an Akt- and ERK-independent survival mechanism(s) in these cancer cells maintained via signals generated by metabolites of 5-Lox.[2]

---

A series of performed inhibitor studies identified a specific inhibitor of 5-LO (MK591), which has the ability to block JNK, MAPK, p38kinase and 5-LO signaling-cascades and drastically reducing the activity of pro-inflammatory cytokine TNF-alpha. Further evaluation of MK591 utilizing cell proliferation assays in PBMCs, human proximal tubule cells and in vivo studies (monkey) showed a decrease in cell proliferation. The inhibitory effect of MK591 was reconfirmed at a genetic level through the utilization of a set of SEB specific genes. Signaling activities, inhibitor studies, cellular analysis and gene expression analysis in unison illustrated the significance of pathway interconnectors such as 5-LO as well as inhibiting such inter-connectors (using MK591) in SEB induced human PBMCs.[1]

For each experiment, equal numbers of cells were plated in six-well plates; 24 h later media were removed and fresh media containing either MK-591 (1 μM, 10 μM or 25 μM) or vehicle were added. After incubation for 24 h, supernatants were collected for Aβ and LDH measurement, and cell pellets harvested in lytic buffer for immunoblot analyses as described in the previous paragraphs. For transfection studies, N2A-APPswe cells were transfected with 1 μg Myc-tagged mΔE-Notch-1 complementary DNA overnight by using Lipofectamine 2000 (Invitrogen). The media were removed and fresh media containing MK-591, L685,458 or vehicle were added. After incubation for 24 h, cells lysates were collected NICD expression levels assayed by western blot analysis.[4]

Starting at 7 months of age, mice were randomized to receive MK-591 (40 mg/kg weight) (n = 11) or vehicle (n = 9) in their chow diet for 8 months until they were 15 months old. Considering that each mouse eats on average 5 g/day of chow diet and the diet is formulated for 320 mg MK-591 per kg diet (Harlan Teklad, WI, USA), the final dose of the active drug was approximately 40 mg/kg weight/day. During the study, mice in both groups gained weight regularly, and no significant difference in weight was detected between the two groups. No macroscopic effect on the overall general health was observed in the animals receiving the active treatment. Post-mortem examination showed no sign of macroscopic pathology in any of the organs considered (spleen, liver, thymus, ileum).[4]

[1]. Effect of 5-lipoxygenase inhibitor MK591 on early molecular and signaling events induced by staphylococcal enterotoxin B in human peripheral blood mononuclear cells. FEBS J. 2008 Jun;275(12):3088-98.

[2]. MK591, a leukotriene biosynthesis inhibitor, induces apoptosis in prostate cancer cells: synergistic action with LY294002, an inhibitor of phosphatidylinositol 3'-kinase. Cancer Lett. 2010 May 28;291(2):167-76.

[3]. 5-Lipoxygenase-activating protein (FLAP) inhibitor MK-0591 prevents aberrant alveolarization in newborn mice exposed to 85% oxygen in a dose- and time-dependent manner. Lung. 2011 Feb;189(1):43-50.

[4]. Involvement of 5-lipoxygenase activating protein in the amyloidotic phenotype of an Alzheimer's disease mouse model. J Neuroinflammation. 2012 Jun 14;9:127.

MK-0591 (3-[1-(4-chlorobenzyl)-3-(t-butylthio)-5-(quinolin-2-yl-methoxy)- indol-2-yl]-2,2-dimethyl propanoic acid, previously L-686,708) is a potent inhibitor of leukotriene (LT) biosynthesis in intact human and elicited rat polymorphonuclear leukocytes (PMNLs) (IC50 values 3.1 and 6.1 nM, respectively) and in human, squirrel monkey, and rat whole blood (IC50 values 510, 69, and 9 nM, respectively). MK-0591 had no effect on rat 5-lipoxygenase. MK-0591 has a high affinity for 5-lipoxygenase activating protein (FLAP) as evidenced by an IC50 value of 1.6 nM in a FLAP binding assay and inhibition of the photoaffinity labelling of FLAP by two different photoaffinity ligands. Inhibition of activation of 5-lipoxygenase was shown through inhibition of the translocation of the enzyme from the cytosol to the membrane in human PMNLs. MK-0591 was a potent inhibitor of LT biosynthesis in vivo, first, following ex vivo challenge of blood obtained from treated rats and squirrel monkeys, second, in a rat pleurisy model, and, third, as monitored by inhibition of the urinary excretion of LTE4 in antigen-challenged allergic sheep. Inhibition of antigen-induced bronchoconstriction by MK-0591 was observed in inbred rats pretreated with methysergide, Ascaris-challenged squirrel monkeys, and Ascaris-challenged sheep (early and late phase response). These results indicate that MK-0591 is a potent inhibitor of LT biosynthesis both in vitro and in vivo indicating that the compound will be suitable for assessing the role of leukotrienes in pathological situations.Can J Physiol Pharmacol. 1992 Jun;70(6):799-807.

---

Bronchopulmonary dysplasia is characterized by prolonged oxygen dependency due to compromised gas-exchange capability. This is attributable mainly to inadequate and aberrant alveolarization resulting from insults like hyperoxia. Leukotrienes are associated with hyperoxia-induced inhibition of alveolarization. We hypothesized that a 5-lipoxygenase-activating protein (FLAP) inhibitor given while newborn mice were exposed to 85% oxygen would prevent aberrant alveolarization in a dose- and time-dependent manner. Newborn mice were exposed to either room air or hyperoxia for 14 days. Pups were treated with either vehicle or MK-0591 10, 20, or 40 mg/kg subcutaneously daily for days 1-4, 5-9, or 10-14. On day 14, the lungs were inflated, fixed, and stained for histopathological and morphometric analyses. Hyperoxia groups treated with MK-0591 20 or 40 mg/kg during days P1-P4 or P10-P14 showed alveolarization that resembled that of room air controls while untreated hyperoxia groups showed definite evidence of aberrant alveolarization but no inflammation. In a hyperoxia-exposed newborn mice model, a FLAP inhibitor given during critical window periods may prevent aberration of alveolarization in a dose- and time-dependent manner.Lung. 2011 Feb;189(1):43-50.

C34H34N2O3SCL-.NA+

609.15316

608.188

C, 67.04; H, 5.63; Cl, 5.82; N, 4.60; Na, 3.77; O, 7.88; S, 5.26

147030-01-1

Quiflapon;136668-42-3

23672584

Typically exists as White to off-white solids at room temperature

751.3ºC at 760mmHg

408.2ºC

1.03E-23mmHg at 25°C

7.679

0

5

10

42

879

0

[O-]C(C(C)(C)CC(N1CC2=CC=C(Cl)C=C2)=C(SC(C)(C)C)C3=C1C=CC(OCC4=NC5=CC=CC=C5C=C4)=C3)=O.[Na+]

YPURUCMVRRNPHJ-UHFFFAOYSA-M

InChI=1S/C34H35ClN2O3S.Na/c1-33(2,3)41-31-27-18-26(40-21-25-15-12-23-8-6-7-9-28(23)36-25)16-17-29(27)37(20-22-10-13-24(35)14-11-22)30(31)19-34(4,5)32(38)39;/h6-18H,19-21H2,1-5H3,(H,38,39);/q;+1/p-1

sodium 3-(3-(tert-butylthio)-1-(4-chlorobenzyl)-5-(quinolin-2-ylmethoxy)-1H-indol-2-yl)-2,2-dimethylpropanoate

MK 0591 sodium; MK-0591; MK0591; L 686708; L-686,708; MK 591; MK591; QUIFLAPON SODIUM; 147030-01-1; MK591; Quiflapon (sodium); MK-591; Quiflapon Sodium [USAN]; MK-591 (sodium); MK 591; MK-591

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~50 mg/mL (~82.08 mM)
H2O : < 0.1 mg/mL

Solubility in Formulation 1: ≥ 2.75 mg/mL (4.51 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 27.5 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

Solubility in Formulation 2: ≥ 2.75 mg/mL (4.51 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 27.5 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

---

 (Please use freshly prepared in vivo formulations for optimal results.)