Activity-based protein profiling reveals off-target proteins of the FAAH inhibitor BIA 10-2474

Abstract

A recent phase 1 trial of the fatty acid amide hydrolase (FAAH) inhibitor BIA 10-2474 led to the death of one volunteer and produced mild-to-severe neurological symptoms in four others. Although the cause of the clinical neurotoxicity is unknown, it has been postulated, given the clinical safety profile of other tested FAAH inhibitors, that off-target activities of BIA 10-2474 may have played a role. Here we use activity-based proteomic methods to determine the protein interaction landscape of BIA 10-2474 in human cells and tissues. This analysis revealed that the drug inhibits several lipases that are not targeted by PF04457845, a highly selective and clinically tested FAAH inhibitor. BIA 10-2474, but not PF04457845, produced substantial alterations in lipid networks in human cortical neurons, suggesting that promiscuous lipase inhibitors have the potential to cause metabolic dysregulation in the nervous system.

Fig. 1. Comparison of human FAAH inhibition by BIA 10-2474, BIA 10-2639, and PF04457845

(A) The structures of BIA 10-2474, the metabolite BIA 10-2639, and PF04457845. (B) Inhibition of human FAAH in HEK293T cell lysates (in vitro) or intact cells (in situ) as measured using an anandamide substrate hydrolysis assay. (C) In vitro and in situ inhibition of human FAAH as measured by competitive gel-based ABPP. Top panels, HEK293T cell lysates (in vitro) or intact cells (in situ) recombinantly expressing human FAAH were pretreated with compound or DMSO (in vitro: 30 min, 37 °C in situ: 4 h, 37 °C). FAAH activity was measured by reactivity with the serine hydrolase-directed probe fluorophosponate-rhodamine and visualization of signals by gel-based ABPP. Bottom panels, Corresponding IC50 curves for gel-based ABPP data shown in Top Panels. N = 3 independent experiments per group.

Fig. 2. Quantitative proteomic analysis of serine hydrolase targets of FAAH inhibitors in human cells

(A, B) MS-based ABPP of serine hydrolase activities in SW620 cells treated with DMSO or FAAH inhibitor. Shown in (A) are BIA 10-2474, BIA 10-2639, and PF04457845 (10 μM, 4 h, 37 °C). Shown in (B) are BIA 10-2474 and PF04457845 (50 μM, 24 h, 37 °C). Data are expressed as median SILAC ratio values for all isotopic peptide pairs quantified per protein from two biological replicates. (C) Confirmation of representative off-targets of BIA 10-2474 by gel-based ABPP of recombinantly expressed enzymes in HEK293T cells.

Fig. 3. BIA 10-2474, but not PF04457845, causes substantial alterations in lipid metabolism in human cortical neurons

(A, B) Cortical neurons were treated with DMSO (A, B), BIA 10-2474 (50 μM) (A) or with PF04457845 (1 μM) (B) and analyzed by MS-based lipidomics after 48 h. X-axis denotes fold change of lipid species in the inhibitor-treated cells vs DMSO-treated cells. Lipidomic data are presented as a volcano plot and lipids with a fold change (FC) threshold of ≥1.20 or ≤0.80 and Benjamini–Hochberg false discovery rate (FDR) ≤ 25% are represented by colored circles distinguished by lipid class. Data represent average values from at least two independent experiments.