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

Jonathan M Blevitt,Michael D Hack,Krystal Herman,Leon Chang,John M Keith,Tara Mirzadegan,Navin L Rao,Alec D Lebsack,Marcos E Milla

doi:10.1074/jbc.m116.725325

Jonathan M Blevitt, Michael D Hack + Show 7 more

Open Access

https://doi.org/10.1074/jbc.m116.725325

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Abstract

5-Lipoxygenase activating protein (FLAP) plays a critical role in the metabolism of arachidonic acid to leukotriene A4, the precursor to the potent pro-inflammatory mediators leukotriene B4 and leukotriene C4 Studies with small molecule inhibitors of FLAP have led to the discovery of a drug binding pocket on the protein surface, and several pharmaceutical companies have developed compounds and performed clinical trials. Crystallographic studies and mutational analyses have contributed to a general understanding of compound binding modes. During our own efforts, we identified two unique chemical series. One series demonstrated strong inhibition of human FLAP but differential pharmacology across species and was completely inactive in assays with mouse or rat FLAP. The other series was active across rodent FLAP, as well as human and dog FLAP. Comparison of rodent and human FLAP amino acid sequences together with an analysis of a published crystal structure led to the identification of amino acid residue 24 in the floor of the putative binding pocket as a likely candidate for the observed speciation. 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.

Highlights

Been the subject of multiple drug discovery efforts [5, 6], with several inhibitors reaching proof of concept in small clinical trials [7,8,9]
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 MK-886, MK-591, and AM-803 [15, 21, 22]
Comparative Modeling of the MK-591 Binding Pocket in Human and Mouse FLAP Orthologs and Functional Assessment of Binding Mode—The SAR shown in Fig. 2 yielded a simple pharmacophore needed for activity of the biaryl amino-heteroarenes

Summary

Experimental Procedures

Preparation of FITC-labeled MK-591—MK-591 (30 mg) 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-dimethylaminopropyl)carbodiimide, 5 ␮l (1 equivalent) of N-methylmorpholine, and 25 mg (1 equivalent) of 5-(((2-(carbohydrazino) methyl)thio)acetyl)aminofluores-

Differential Pharmacology of Novel FLAP Inhibitors

Results

Discussion