PF-5901

The cysteinyl leukotrienes (CysLTs), i.e. LTC₄, LTD₄ and LTE₄, are a family of proinflammatory agents synthesized from the arachidonic acid. In target cells, these lipid mediators bind to the cysteinyl leukotriene receptors (CysLTR), a family of seven transmembrane G-protein coupled receptors. The CysLT₁R is a validated target for treatment of pulmonary diseases and several selective antagonists for this receptor, including montelukast, zafirlukast and pranlukast, have shown effective in the management of asthma. Nevertheless, others CysLT₁R antagonists, such as the alpha-pentyl-3-[2-quinolinylmethoxy] benzyl alcohol (REV5901), have been extensively characterized without reaching sufficient priority for clinical development. Since drug reposition is an efficient approach for maximizing investment in drug discovery, we have investigated whether CysLT₁R antagonists might exert off-target effects. In the report we demonstrate that REV5901 interacts with GPBAR1, a well characterized cell membrane receptor for secondary bile acids. REV5901 transactivates GPBAR1 in GPBAR1-transfected cells with an EC₅₀ of 2.5 µM and accommodates the GPBAR1 binding site as shown by in silico analysis. Exposure of macrophages to REV5901 abrogates the inflammatory response elicited by bacterial endotoxin in a GPBAR1-dependent manner. In vivo, in contrast to montelukast, REV5901 attenuates inflammation and immune dysfunction in rodent models of colitis. The beneficial effects exerted by REV5901 in these models were abrogated by GPBAR1 gene ablation, confirming that REV5901, a shelved CysLT₁R antagonist, is a GPBAR1 ligand. These data ground the basis for the development of novel hybrid ligands designed for simultaneous modulation of CysTL₁R and GPBAR1.

Embryonic stem (ES) cells can differentiate into various kinds of cells, such as endothelial and hematopoietic cells. In addition, some evidence suggests that inflammatory mediators such as leukotrienes (LTs), which include the 5-lipoxygenase (LOX) family, can regulate endothelial cell differentiation. In the present study, the eicosanoid precursor arachidonic acid (AA) stimulated vasculogenesis of ES cells by increasing the number of fetal liver kinase-1+ vascular progenitor cells as well as vascular structures positive for platelet endothelial cell adhesion protein-1 and vascular endothelial cadherin. The stimulation of vasculogenesis and expression of the rate-limiting enzyme in the LT signaling pathway, 5-LOX-activating protein (FLAP), was blunted upon treatment with the FLAP inhibitors AM643 and REV5901. Vasculogenesis was significantly restored upon exogenous addition of LTs. Downstream of FLAP, the LTB4 receptor (BLT1) blocker U75302, the BLT2 receptor blocker LY255283 as well as the cysteinyl LT blocker BAY-u9773 inhibited vasculogenesis of ES cells. AA treatment of differentiating ES cells increased reactive oxygen species (ROS) generation, which was not affected upon either FLAP or cyclooxygenase-2 inhibition. Prevention of ROS generation by either the free radical scavengers vitamin E and N-(2-mercaptopropionyl)glycine or the NADPH oxidase inhibitor VAS2870 downregulated vasculogenesis of ES cells and blunted the provasculogenic effect of AA. In summary, our data demonstrate that proinflammatory AA stimulates vasculogenesis of ES cells via the LT pathway by mechanisms involving ROS generation.