Substituted 1,2,4-Triazoles as Novel and Selective Inhibitors of Leukotriene Biosynthesis Targeting 5-Lipoxygenase-Activating Protein

5-Lipoxygenase-activating protein (FLAP) is a regulator of cellular leukotriene biosynthesis, which governs the transfer of arachidonic acid (AA) to 5-lipoxygenase for efficient metabolism. Here, the synthesis and FLAP-antagonistic potential of fast synthetically accessible 1,2,4-triazole derivatives based on a previously discovered virtual screening hit compound is described. Our findings reveal that simple structural variations on 4,5-diaryl moieties and the 3-thioether side chain of the 1,2,4-triazole scaffold markedly influence the inhibitory potential, highlighting the significant chemical features necessary for FLAP antagonism. Comprehensive metabololipidomics analysis in activated FLAP-expressing human innate immune cells and human whole blood showed that the most potent analogue 6x selectively suppressed leukotriene B4 formation evoked by bacterial exotoxins without affecting other branches of the AA pathway. Taken together, the 1,2,4-triazole scaffold is a novel chemical platform for the development of more potent FLAP antagonists, which warrants further exploration for their potential as a new class of anti-inflammatory agents.


INTRODUCTION
Leukotrienes (LTs) are a family of bioactive lipid mediators produced from arachidonic acid (AA) through the 5lipoxygenase (5-LO) pathway, which have various inflammatory and vasoactive effects. 1,2The 5-LO branch of the AA cascade produces LTB 4 and cysteinyl (Cys) LTs from an unstable LTA 4 intermediate via the action of LTA 4 hydrolase and LTC 4 synthase, respectively.These bioactive LTs exert their actions via distinct receptors such as BLT 1/2 for LTB 4 and CysLTRs for CysLTs (Figure 1). 3 LTB 4 is pro-inflammatory and acts as a chemoattractant for leukocytes and neutrophils, while cysLTs cause bronchoconstriction, airway edema, and vascular leakage. 1 To date, the most advanced drug class targeting this branch are cysLTR 1 antagonists as antiasthmatic drugs, i.e., montelukast, 4 but this drug class has limited clinical indication as it essentially blocks the action of LTD 4 in the lungs, resulting in decreased inflammation and relaxation of smooth muscle. 5Meanwhile, the development of broad-spectrum inhibitors of LT biosynthesis (LTB 4 , C 4 , D 4 , and E 4 ) is less progressed.Zileuton is the only clinical example of a direct 5-LO inhibitor for asthma treatment, but its use is often limited due to idiosyncratic hepatotoxicity and poor pharmacokinetics. 6In this regard, there remains an ever-increasing demand to search for novel anti-inflammatory drugs that target the LT pathway.
Over the past three decades, there has been great interest in 5-lipoxygenase activating protein (FLAP) as an essential partner of 5-LO, which transfers AA to 5-LO for efficient metabolism. 7FLAP is an indispensable regulatory protein for 5-LO without any enzymatic activity at the nuclear membrane to assemble the LT synthetic complex. 8Hence, it is envisaged that suppression of the FLAP function would selectively intervene with massive, pro-inflammatory, and vasoactive LT production to provide a therapeutic benefit for chronic inflammatory diseases besides asthma. 9,10Indeed, prevailing clinical data imply that FLAP antagonists exert potent and safer anti-LT efficacy in asthma and chronic artery disease, as exemplified by quiflapon, 11,12 veliflapon, 13 fiboflapon, 14,15 and very recently atuliflapon 16,17 (Figure 2).However, despite great interest in this biological target over the past few decades, no FLAP antagonist has so far reached clinical practice. 7,18AP is a homotrimer located in the nuclear membrane comprising four transmembrane helices (α1−α4) in each monomer.Based on the reported FLAP crystal structures, ligands that bind FLAP are largely embedded in the nonpolar center of the trimer, which is located between the α2 and α4 of one FLAP monomer and the α1′ and α2′ of the other FLAP monomer (Figure 3). 19,20Moreover, polar parts of the ligands stick out to the phosphate-exposed solvent accessible protein surface, where the ligands can form salt-bridges/H-bonds with basic side chains such as Lys116 and His28; these, in turn, engage with the anionic phosphate groups of the plasma membrane on the side adjacent to the aqueous cytosol.These ligands have little exposure to the lipid bilayer when bound to FLAP.
−26 Thus, as a result of a recent virtual screening approach for novel chemotypes that interfere with FLAP function, a 1,2,4-triazole derivative (VS-1, Figure 2) was identified as a novel FLAP antagonist that has a distinct scaffold compared to reported FLAP antagonists. 27To further elucidate the structural determinants of this class of compounds for FLAP antagonism, the current work reports the synthesis, biological evaluation, and analysis of structure− activity relationships (SAR) of novel 1,2,4-triazole analogs that could potentially provide further insight into drug development in the AA pathway targeting FLAP.

Chemistry.
To determine the effects of the substitution pattern on 4,5-diaryl subunits and the influence of the thioether side chain of the 1,2,4-triazole core on FLAP antagonism, compounds 6a and its analogs (Table 1, 6b−z) were synthesized by following the synthetic procedure demonstrated in Scheme 1.The general method is straightforward and utilizes the cyclodehydration of thiosemicarbazide intermediates (3), which were conveniently produced from corresponding acyl hydrazides (2), under basic conditions to form corresponding 3-mercapto-4,5disubstituted-1,2,4-triazoles (4). 36,37For the synthesis of compounds with modified spacers by a formal exchange of the sulfur by oxygen, the 3-hydroxy-1,2,4-triazole intermediate 5 was directly prepared by heating 4 in a 50% hydrogen peroxide solution under basic conditions.The target compounds 6a−z were finally generated by simple alkylation of intermediates 4 or 5 with the corresponding alkyl halides in acetonitrile in the presence of triethylamine.The final purity of the target compounds was corroborated by UPLC-MS prior to biological evaluation (purity was >97%).Compound structure elucidation was done through high-resolution mass spectrometry (HRMS) and 1 H-and 13 C NMR spectral data as given in the Supporting Information.

Biological Evaluation and SAR.
Although FLAP is dispensable for 5-LO activity under cell-free conditions, it is essential for cellular LT biosynthesis, and FLAP antagonists do not have inhibitory activity toward 5-LO in cell homogenates. 39,40Hence, for determination of FLAP antagonism, we applied a well-documented "FLAP-dependent" cell-based assay for suppression of 5-LO product (5-H(p)ETE, LTB 4 and its all-trans isomers) formation using human neutrophils stimulated by Ca 2+ -ionophore A23817. 25In addition, a "FLAP- Figure 3. Structural overview of FLAP modeled in the nuclear membrane.−33 Membrane structure and water molecules were added using the predicted membrane orientations from OPM server 34 with the System Builder tool in Maestro. 35ndependent" cell-free 5-LO activity assay using isolated recombinant 5-LO was employed to rule out direct inhibitory effects against 5-LO.
We previously identified the VS-1 (6a in Table 1, IC 50 = 2.18 μM) having a 1,2,4-triazole skeleton as a new chemotype for blocking FLAP function, 27 which lacks typical scaffolds of reported FLAP antagonists. 7,18To deduce SARs around the 1,2,4-triazole skeleton, we randomly prepared analogs of VS-1 (6a), which incorporate changes that would help in elucidating the structural features required to inhibit FLAP function (Table 1).First, we investigated the influence of the 4-methoxyphenyl moiety on the 4-position of the triazole ring (Table 1).Removal of the 4-methoxyphenyl group of 6a resulted in a complete loss of FLAP function (6b, IC 50 = > 10 μM).Therefore, we subsequently examined the effect of the substituent on the phenyl group attached to the 4-position of the triazole ring by casual introduction of different substituents.Moving the methoxy group to the meta position (6c, IC 50 = 1.58 μM) or replacing it with a chloro group (6d, IC 50 = 1.36 μM) was beneficial to enhance the inhibitory potency against 5-LO product formation.Next, SAR was further explored by replacing the methoxy group with polar carboxyl or nitro groups (6e−i), which all abolished the inhibitory activity.This indicated that a phenyl group with rather hydrophobic substituents at the 4-position of the triazole ring is important for FLAP antagonistic activity.It is known that the quinoline ring occurs as a frequently recurring chemical fragment in the architecture of FLAP antagonists (see Figure 2), and simple heteroaryl modifications may also modulate the inhibitory activity of FLAP function. 7,38Thus, we briefly explored the quinoline (6j−k, IC 50 = 3.69−4.17μM), 5-CF 3 -furan-2-yl (6l, IC 50 = 1.42 μM), and 5-Me-pyridin-2-yl (6m, IC 50 ∼ 10 μM) rings as replacements of the benzothiazole ring, which clearly indicated that the antago- b The IC 50 values are given as mean ± SEM of n = 3 determinations.

Scheme 1. Reagents and Conditions
nistic activity of FLAP is sensitive to the nature of the heteroaryl fragment connected to the triazole via a thio-methyl linker at the 3-position.In addition, we briefly examined the thioether spacer by replacing it with an ether, which also caused a decrease in inhibitory activity (6n−o, IC 50 = 3.0−6.3μM).
Having elucidated the necessity of the 4-methoxyphenyl ring and the thio-methyl-benzothiazole side arm, we then investigated the effect of the hydrophobic thiophene ring on the 5-position of the 1,2,4-triazole core.While complete removal of the thiophene ring impairs the inhibitory potency (6p, IC 50 = > 10 μM), the activity was restored by reinstallation of an aromatic phenyl ring (6q, IC 50 = 1.28 μM).These results directed us further to conduct SAR studies around the 5-phenyl of the triazole, while keeping the other parts of the molecule intact.While the 4-chlorophenyl group at this position failed to substantially inhibit 5-LO product formation (6r, ∼ 20% inhibition at 10 μM), the activity was refurbished by replacing the 4-chloro with the more polar 4nitrile substituent (6s, IC 50 = 1.92 μM) that is an H-bond acceptor group with a rodlike geometry and a minuscule steric demand. 41Homologation of the nitrile in 6s to acetonitrile gave 6t, although with a decreased potency (IC 50 = 3.62 μM).In addition, replacing the nitrile group with an H-bond acceptor carboxyl group in 6u diminished the inhibitory potency (40% inhibition at 10 μM).Moreover, while keeping the 4-acetonitrile group at 5-phenyl of the triazole intact, we introduced the 4-chlorophenyl or phenyl groups to the 4position of the triazole core, which resulted in more active compounds (6v−w, IC 50 = 1.35−1.51μM).Lastly, moving the acetonitrile substituent of 6w to the 3-position at 6x, both with a phenyl group at the 4-position of the triazole core, further enhanced the inhibitory activity (IC 50 = 1.15 μM), leading to the most potent derivative in the series.Therefore, we selected 6x for comprehensive analysis of its potency toward 5-LO product formation and its selectivity among various enzymes in the AA cascade, as well as its effects on de novo-biosynthesized lipid mediators that critically regulate both inflammation and resolution, by employing targeted liquid chromatography− tandem mass spectrometry-based metabololipidomics. 42

6x Modulates Lipid Mediator Signature Profiles in Activated 5-LO-Rich Immune Cells and in Human
Blood.Previous results showed that FLAP antagonists, such as MK886, can effectively inhibit LT formation but also affect other branches in the lipid mediator (LM) network, namely, 12/15-LO or COX-1/2 pathways. 42To investigate the potential redirection of LM formation due to FLAP antagonism, we studied the impact of 6x on modulation of LM profiles of relevant cells by performing comprehensive LM metabololipidomics using UPLC-MS-MS (Figure 4).
5-LO-expressing innate immune cells, namely, neutrophils, M1-and M2-monocyte-derived macrophages (MDMs), which also possess various other LOs and COX enzymes, 43 were pretreated with 6x (3 μM) for 30 min before exposing to Staphylococcus aureus-conditioned medium (SACM, 1%) for 90 min, which contains bacterial exotoxins that are suitable stimuli to activate all relevant LM pathways in these cells. 44Formed LM derived from AA in 6x-treated M2-MDM are shown by quantitative LM pathway analysis, revealing potent suppression of all 5-LO/FLAP-mediated LMs, while others such as COX and 12/15-LO products remained unaffected (Figure 4A).Notably, we observed cell type-specific inhibition of 5-LO/ FLAP-mediated products such as LTB 4 , t-LTB 4, and 5-HETE.Thus, in neutrophils and M1-MDM cells expressing abundant FLAP, 6x potently inhibited 5-LO/FLAP product formation (Figure 4B,C and Table 2), while in M2-MDMs, the efficiency of 6x was reduced in accordance with minor FLAP expression in this MDM phenotype 42 (Figure 4D and Table 2).Interestingly, the potency to inhibit proinflammatory LTB 4 was higher than the suppression of 5-HETE formation in both M1-and M2-MDMs (Table 2).Moreover, 6x suppressed EPAderived 5-LO/FLAP products such as 5-HEPE effectively, whereas DHA-derived 7-HDHA was less reduced (Table 2).In all investigated cell types, we found that 6x did not affect other prominent LM such as 12-HETE, 15-HETE, or PGE 2 , representing 12-LO, 15-LO, and COX pathways, respectively (Figure 4B−D and Table 2).Besides inhibition in isolated cells, we also investigated the ability of 6x to suppress 5-LO/ FLAP-mediated LT formation in human whole blood.Therefore, we pretreated freshly withdrawn human blood with 6x (30 μM) for 30 min prior to stimulation with SACM (3%) for 90 min.6x decreased LTB 4 levels by around 50%, while other 5-LO/FLAP-, 12/15-LO-, or COX-mediated products are essentially unchanged, suggesting a lower potency of 6x as a free drug in human whole blood (Figure 4E and Table 2).Concerning the selectivity of 6x toward FLAP over 5-LO, 6x did not inhibit 5-LO product formation in cell-free assays using isolated human recombinant 5-LO, whereas the direct 5-LO inhibitor zileuton used as a reference showed potency in this respect (Figure S1).
Considering the potential cytotoxicity, 6x did not affect the membrane integrity after treatment of M1-MDMs for 90 min (Figure S2).These results indicate that 6x indeed exerts its inhibitory effect on 5-LO product formation by selectively targeting FLAP.

Molecular Docking and Dynamics Simulations of 6x with FLAP.
To provide further information on the interaction of 6x with FLAP, we conducted docking studies in combination with molecular dynamic (MD) simulations using the recently reported FLAP crystal structure (2.35 Å, PDB code: 6VGC). 20The ligand binding site of FLAP is situated in the phosphate-exposed region of the protein, and ligands that bind FLAP largely embed themselves in the nonpolar center of the trimer between the α helices of the two adjacent monomers. 20In this respect, the docking-derived FLAP-6x complex (Figure S3) was submitted to 200 ns long MD simulations (four replicates, Figure S4A) after embedding the starting complex within the membrane (taken from OPM Database 34 ).Considering the docking/MD calculations previously reported for FLAP-ligand complexes 21,27,45 and shown in this study, the binding specificity of 6x to FLAP is characterized by several interactions (Figure 5).Simulation analysis revealed that the existing interactions of 6x from docking modes were generally conserved during MD runs (Figure S3B).Accordingly, the nitrile group of 6x makes direct (10%) as well as water-mediated hydrogen bond interactions with the side chain of Lys116 (11%) and His28 (12%) at the phosphate-exposed binding region.In addition, the neighboring phenyl groups of 6x engage in π−π stacking interactions with the imidazole ring of His28 (18%) and Phe123 (68%), which further stabilizes 6x at the binding site.Moreover, the side chain benzothiazole group of 6x tucks into a deep hydrophobic pocket with surrounding residues such as Ala27, Ala63, Tyr112, Ile113, Phe114, and Lue120 and makes π−π interactions with the aromatic residues of Tyr112 and Phe114 (32 and 27%, respectively).It seems that aromatic and hydrophobic interactions between 6x and the nonpolar binding area of the FLAP binding site appear to be the main driving force for ligand binding, while the polar nitrile pendant presumably contributes little toward potency.Since potent FLAP inhibitors such as MK-591 are known to form strong polar interactions with Lys116 and His28 residues at the highly charged phosphate-exposed region of the binding site, the moderate activity of 6x is likely due to the inability to bind this area effectively.Consequently, more favorable substitutions of the phenyl ring at 6x could be a valuable tool for developing new analogs with greater potency, and further simulation analysis revealed that the existing interactions of 6x from docking modes were generally conserved during MD runs (Figure S3 and S4).

CONCLUSIONS
Through a methodical process of synthesis and biological assessment of VS-1 analogs, we successfully established that the rapidly accessible 3,4,5-trisubstituted-1,2,4-triazole core is a new and essential framework for this novel class of FLAP antagonists, without any direct inhibitory effect on 5-LO activity.Our SAR results showed that the substitution pattern on either of the vicinal diaryl groups as well as the nature of the heterocyclic ring at the thioether side chain significantly influence the potency of compounds (Figure 6), and future explorations are needed to determine a more appropriate substitution pattern around these features to improve the potency of the compounds by increasing favorable binding interactions at the FLAP binding site.In addition, using human neutrophils and macrophages as experimental models, we established that 6x displayed selective suppression of 5-LO product formation due to the antagonism of FLAP, without interfering with other branches in the AA cascade such as COX, 12/15-LO, and CYP450.
Taken together, 6x represents a novel lead structure of LT biosynthesis inhibitors with unprecedented selectivity in human neutrophils and macrophages activated under pathophysiologically relevant conditions.Thus, based on its unique pharmacologic profile, the most potent analog 6x was identified as a viable tool compound for future studies.
In conclusion, our results prove the ability of the 1,2,4triazole core as an attractive new platform in the rational design of potential anti-LT drugs and provide valuable insight into the chemical features functional for the design of future members of this new class of FLAP antagonists.

EXPERIMENTAL SECTION
4.1.Chemistry.The starting materials, reagents, and solvents were obtained from BLD Pharm (BLD Pharmatech Ltd., Shanghai, China), ABCR (abcr GmbH, Karlsruhe, Germany), and Merck Chemicals (Merck KGaA, Darmstadt, Germany).The reaction progress was monitored by TLC using Merck silica gel Aluminum TLC plates, silica gel coated with fluorescent indicator F 254 , and visualized under UV.The melting points were determined by an SMP50 model automatic melting point apparatus (Stuart, Staffordshire, ST15 OSA, UK).The purification of the compounds was carried out on RediSep silica gel columns (12 and 24 g) using the Combiflash Rf Automatic Flash Chromatography System (Teledyne-Isco, Lincoln, NE, USA) or Buchi Pure C-815 Automatic Flash Chromatography System with UV and ELSD detectors using prepacked Buchi EcoFlex and FlashPure silica gel columns (12, 24, 40 g).The purity of the compounds was confirmed by thin-layer chromatography and UPLC/MS-TOF analyses.The 1 H and 13 C APT NMR spectra of the synthesized compounds were taken in DMSO-d 6 on Bruker Avance Neo 500 MHz and Bruker DPX-400 MHz High-Performance Digital FT-NMR Spectrometers.All chemical shift values (δ) were recorded as ppm, and coupling constants were reported in Hertz (Hz).HRMS spectra of the compounds were obtained on a Waters LCT Premier XE UPLC/MS-TOF system (Waters Corporation) using an Aquity BEH C18 column (2.1 × 100 mm 1.7 μM, flow rate: 0.3 mL/min) as the stationary phase, and CH 3 CN:H 2 O (1% → 90%) containing 0.1% formic acid as the mobile phase.Synthetic methods and experimental data for all intermediate compounds can be found in the Supporting Information.dine) (1 equiv), respectively.The mixture was stirred at room temperature for 24 h.The solvent was removed, and the crude product was dissolved with 5 mL of methanol and poured into 100 mL of water.The precipitate was filtered off to give a crude solid, which was purified by automated-flash chromatography on silica gel eluting with a gradient of 0% → 60% EtOAc in n-hexane.

Determination of FLAP-Dependent 5-LO Product
Formation in Intact Neutrophils for SAR.Human neutrophils (5 × 10 6 /mL in PBS containing 1 mM CaCl 2 and 0.1% glucose) were preincubated with 0.1% DMSO (vehicle) or with the compounds at 37 °C for 15 min.After addition of 2.5 μM A23187 (Cayman Chemical/Biomol GmbH, Hamburg, Germany), the reaction was incubated for 10 min at 37 °C and then stopped by addition of 1 mL of methanol, and 30 μL of 1 N HCl plus 200 ng of PGB 1 and 500 μL of PBS were added.Samples were then prepared by solid phase extraction on C18columns (100 mg, UCT, Bristol, PA), and 5-LO products (LTB 4 and its trans-isomers, and 5-H(p)ETE) were analyzed in the presence of internal standard PGB 1 by RP-HPLC and UV detection as reported elsewhere. 48ell and whole blood incubations were for LM metabololipidomics analysis.

LM Metabololipidomics by UPLC-MS-MS.
Samples obtained from MDM, neutrophils, and whole blood containing deuterated LM standards were kept at −20 °C for at least 60 min to allow protein precipitation.The extraction of LM was performed as recently published. 42In brief, after centrifugation (1200 × g; 4 °C; 10 min), acidified H 2 O (9 mL; final pH = 3.5) was added, and samples were extracted on solid phase cartridges (Sep-Pak Vac 6 cm 3 500 mg/6 mL C18; Waters, Milford, MA, USA).Samples were loaded on the cartridges after equilibration with methanol followed by H 2 O.After being washed with H 2 O and n-hexane, samples were eluted with methyl formate (6 mL).The solvent was fully evaporated using an evaporation system (TurboVap LV, Biotage, Uppsala, Sweden) and the residue was resuspended in 150 μL methanol/water (1:1, v/v) for UPLC-MS-MS analysis.LM were analyzed with an Acquity UPLC system (Waters, Milford, MA, USA) and a QTRAP 5500 Mass Spectrometer (ABSciex, Darmstadt, Germany) equipped with a Turbo V Source and electrospray ionization.LM were eluted using an ACQUITY UPLC BEH C18 column (1.7 μm, 2.1 mm × 100 mm; Waters, Eschborn, Germany) heated at 50 °C with a flow rate of 0.3 mL/min and a mobile phase consisting of methanol−water− acetic acid at a ratio of 42:58:0.01 (v/v/v) that was ramped to 86:14:0.01 (v/v/v) over 12.5 min and then to 98:2:0.01 (v/v/ v) for 3 min. 42The QTRAP 5500 was run in negative ionization mode using scheduled multiple reaction monitoring (MRM) coupled with information-dependent acquisition.The scheduled MRM window was 60 s, optimized LM parameters were adopted, 42 with a curtain gas pressure of 35 psi.The retention time and at least six diagnostic ions for each LM were confirmed by means of an external standard for each and every LM (Cayman Chemical/Biomol GmbH).Quantification was achieved by calibration curves for each LM; linear calibration curves were obtained and gave r 2 values of 0.998 or higher.The limit of detection for each targeted LM was determined as described. 42For UPLC-MS-MS analysis, the quantification limit was 3 pg/sample, and this value was taken to express the fold increase for samples where the LM was not detectable (n.d.).
4.3.Computational Studies.4.3.1.Molecular Docking.All publicly released FLAP structures (PDB codes: 2Q7M, 2Q7R, 19 6VGC, 6VGI 20 ) were considered for selection of the most appropriate crystal to be used in the modeling studies.6VGC was selected based on the crystal resolution, generating the lowest RMSD variance in the binding poses of each cocrystallized ligand.The designed compounds were drawn by using Maestro interface. 35Chemical states and atom types at pH 7.0 ± 2.0 were assigned with OPLS4 force field by utilizing LigPrep for the ligand structures, 51 and Protein Preparation Wizard for the protein structures. 52van der Waals radius scaling factor and partial charge cutoffs were left with the default values, 1.0 and 0.25.Docking simulations were issued with Glide in standard precision (SP) mode. 53,54The highestranking pose was selected for the generation of MD simulations.
4.3.2.Molecular Dynamics.Four copies (200 ns) were conducted after generating the system with System Builder utility.The SPC model was used for water molecules, and POPC atoms were used for generating the lipid bilayer.The membrane was positioned by obtaining the coordinates from the OPM database. 34Coulombic interaction cut off was applied as 9.0 Å.The simulation system was neutralized with Na + ions.The atom types were issued with the OPLS4 force field.Later, the simulations were run with Desmond. 55The simulations started with a multistep relaxation protocol; (i) for the first step of the minimization, Brownian Dynamics was applied with NVT ensemble by the heating system to 10 K with small timesteps and applying restraints on solute heavy atoms for 100 ps.(ii) Subsequently, relaxation continued at 100 K with the H 2 O Barrier and Brownian NPT ensemble, membrane restrained in the z axis and also protein restrained.(iii) Next, the same approach was applied, but this time with the NPgT ensemble.(iv) Later, the whole restraints were removed, and the simulation was run with NPT ensemble at 300 K for 200 ns.Simulations were analyzed by utilizing Simulation Interaction Diagram interface of Maestro 35 and Gromacs 56 scripts.

■ ASSOCIATED CONTENT
The authors declare that they have no competing interests.Abdurrahman Olgȃeçis founder and CEO of Evias Pharmaceutical R&D Ltd.

Figure 2 .
Figure 2. Chemical structures of FLAP antagonists that entered clinical trials and the virtual screening hit compound VS-1.

Figure 4 .
Figure 4. Modulation of lipid mediator profiles in activated 5-LO-rich immune cells and whole blood by 6x.(A) Quantitative LM pathway analysis and effects of 6x in exotoxin-stimulated M2-MDM.Node size represents the mean values in pg/2 × 10 6 cells, and intensity of color denotes the fold change of 6x-versus vehicle-treated cells for each LM; n = 3.Effects of 6x on the bioactive LTs and PGE 2 produced in human neutrophils (B), M1-MDM (C), M2-MDM (D), and freshly drawn human whole blood (E).Statistical analysis was done by matched one-way ANOVA with Tukey's multiple comparison test, *p < 0.05, **p < 0.01, ***p < 0.001, 6x vs control.

Table 2 .
Modulation of Lipid Mediator Profiles in Activated 5-LO-Rich Immune Cells and Whole Blood by 6x a a Human neutrophils (PMNL), M1-MDM, and M2-MDM were preincubated with 3 μM 6x or vehicle (0.1% DMSO) for 30 min before stimulation with 1% SACM for 90 min at 37 °C.Freshly drawn human whole blood was preincubated with 30 μM 6x or vehicle (0.1% DMSO) for 30 min before stimulation with 1% SACM for 90 min at 37 °C.Formed LM were isolated from the supernatants by SPE and analyzed by UPLC-MS-MS.Data are given as mean ± SEM and as fold-change ('f') versus vehicle.

Figure 5 .
Figure 5. Protein−ligand interactions and their occupancy values obtained by 200 ns of molecular dynamic simulations conducted with FLAP-6x complex.Residues in green sticks represent the A chain, and cyan sticks represent the B chain.Compound 6x is shown in orange sticks.POPC unit of the membrane is shown as white sticks.Interaction types and their representations are shown in the legend.