A Single Amino Acid Difference between Mouse and Human 5-Lipoxygenase Activating Protein (FLAP) Explains the Speciation and Differential Pharmacology of Novel FLAP Inhibitors

Abstract

On that basis, we tested compounds for binding to human G24A and mouse A24G FLAP mutant variants and compared the data to that generated for wild type human and mouse FLAP. These studies confirmed that a single amino acid mutation was sufficient to reverse the speciation observed in wild type FLAP. In addition, a PK/PD method was established in canines to enable preclinical profiling of mouse-inactive compounds. 5-Lipoxygenase activating protein (FLAP)2 is a key accessory protein in the arachidonic acid metabolism pathway (1). Its function is to present arachidonic acid to 5-lipoxygenase for conversion to leukotriene A 4 and subsequently present leukotriene A 4 to leukotriene C 4 synthase to generate the potent pro-inflammatory mediator leukotriene C 4 (2-4). Because of this critical role in the biosynthesis of leukotrienes, FLAP has been the subject of multiple drug discovery efforts (5, 6), with several inhibitors reaching proof of concept in small clinical trials (7-9).FLAP was originally identified via phenotypic screening for 5-lipoxygenase inhibitors and was described as an 18-kDa membrane protein (10). During the course of that effort, the compound MK-886 was found to bind to the FLAP polypeptide (11). Subsequently, the FLAP cDNA was cloned from multiple species, revealing high sequence homology (12, 13) (Fig. 1). X-ray crystallographic evidence indicated that FLAP exists as a homotrimer, similar to other members of the MAPEG family, with each monomer containing four transmembrane ␣-helices (14). A major compound binding site is embedded within the membrane, formed by the interface of ␣-helices 2 and 4 of one monomer and ␣-helix 1 of the adjacent monomer (14), resulting in three binding sites per trimer. Mutational analysis revealed several key interactions with the FLAP inhibitor MK-591, aiding in the understanding of its binding mode and SAR surrounding this series of indoles (15).During our own high throughput screening efforts, we discovered benzimidazoles and a series of biaryl amino-heteroarenes ( Fig. 2 and Table 2; Refs. 16 -20) with distinct SAR relative to previously reported indole-containing FLAP inhibitors exemplified by 21,22). Unexpectedly, we found that the biaryl amino-heteroarenes lacked activity in rodent whole blood ex vivo and in vivo models. Here we propose that a single amino acid difference in the binding pocket that is conserved in murine, rat, and porcine FLAP is sufficient to render compounds of this series inactive in these species, based on ligand displacement analysis, whole blood activity assays, and computational studies. Because rodents are commonly used for pharmacokinetic and pharmacodynamics studies, we established an alternative path for the preclinical profiling of biaryl amino-heteroarenes and related compounds in canines. Experimental ProceduresPreparation of was suspended in a 2:1 mixture of N,N-dimethylformamide and water to a total volume of 1.5 ml, followed by addition of 34 mg (5 equivalents) of hydroxybenzotriazole, 10 mg (1 equivalent) of 1-ethyl-3-(3-dimeth...

Discovery and Early Clinical Development of an Inhibitor of 5-Lipoxygenase Activating Protein (AZD5718) for Treatment of Coronary Artery Disease

“…With the promising safety profile and no adverse cardiovascular effects observed in dogs for compound 12 , we further investigated its ability to inhibit 5-LO pathway activity when dosed in vivo. The compounds in this series showed no inhibition of 5-LO pathway activity in rodent blood but potently inhibited LTB 4 production in dog blood, and hence dogs were utilized for preclinical PK/PD assessment . Pharmacodynamic actions of 12 were initially assessed by measuring ex vivo LTB 4 production in whole blood.…”

“…The compounds in this series showed no inhibition of 5-LO pathway activity in rodent blood but potently inhibited LTB 4 production in dog blood, and hence dogs were utilized for preclinical PK/ PD assessment. 26 Pharmacodynamic actions of 12 were initially assessed by measuring ex vivo LTB 4 production in whole blood. Full data sets and individual data are provided in the Supporting Information.…”

Thymus algeriensis and Thymus fontanesii: Chemical Composition, In Vivo Antiinflammatory, Pain Killing and Antipyretic Activities: A Comprehensive Comparison

“…Important amino acids forming hydrophobic interactions with the bound ligand include residues Val20, Val21, Gly24, Phe25, Ala27, Tyr112, Ile113, A63, Ile119, Leu120 and Phe123. Few polar interactions are also observed with residues Asn23 from transmembrane helix α1, residues Asp62 and Thr66 from transmembrane helix α2 and residue Lys116 from transmembrane helix α4 of the neighboring FLAP monomer [36].…”

“…The binding site is embedded within the membrane, formed by the interface of α -helices 2 and 4 of one monomer and α -helix 1 of the neighboring monomer. Each ligand molecule is bound to the groove at the interface between two adjacent monomers [36,37].…”

“…The binding site is embedded within the membrane, formed by the interface of α -helices 2 and 4 of one monomer and α -helix 1 of the neighboring monomer. Each ligand molecule is bound to the groove at the interface between two adjacent monomers [36,37]. Important amino acids forming hydrophobic interactions with the bound ligand include residues Val20, Val21, Gly24, Phe25, Ala27, Tyr112, Ile113, A63, Ile119, Leu120 and Phe123.…”