Sphingosine-1-phosphate (S1P), the phosphorylated metabolite of D-sphingosine, binds to five G protein-coupled receptors (S1P1-S1P5) and regulates a plethora of biological actions (GARCIA et al., J. Clin. Invest, 108:689-701 (2001); ISHII et al., Annu. Rev. Biochem., 73:321-354 (2004)). In particular, the prototypical S1P1 receptor is essential for vascular maturation during development and promotes endothelial cell migration, angiogenesis and barrier functions (LIU et al., J. Clin. Invest, 106:951-961 (2000); PAIK et al., J. Biol Chem., 276:11830-11837 (2001); LEE et al., Cell, 99:301-312 (1999)). Thus, S1P is required for maintenance of the barrier property of the lung endothelium (CAMERER et al., J. Clin. Invest, 119:1871-1879 (2009)). Plasma S1P, which is derived from several cellular sources (PAPPU et al., Science, 316:295-298 (2007); VENKATARAMAN et al., Circ. Res., 102:669-676 (2008)), is associated with high density lipoprotein (HDL) (˜65%) and albumin (˜35%) (AOKI et al., J. Biochem., 138:47-55 (2005); ARGRAVES et al., J. Lipid Res., 48:2325-2333 (2007)). HDL-induced vasorelaxation as well as barrier-promoting and pro-survival actions on the endothelium have been attributed to S1P signalling (KIMURA et al., J. Biol Chem., 281:37457-37467 (2006); NOFER et al., J. Clin. Invest, 113:569-581 (2004); ARGRAVES et al., J. Biol Chem., 283:25074-25081 (2008)). Hence, much of the endothelium-protective actions of HDL may be due to the actions of S1P on the endothelial S1P receptors. The molecular nature of the S1P binding to HDL and interaction to S1P receptors, however, has not been characterized. The molecular structure of S1P is displayed in FIG. 6.
Apolipoprotein M (apoM) is a ˜22-kDa HDL-associated apolipoprotein and a member of the lipocalin family of proteins which mainly resides in the plasma HDL fraction (XU et al., J. Biol Chem., 274:31286-31290 (1999)). Mature apoM (human apoM, SEQ ID NO: 1, and murine apoM, SEQ ID NO: 2) retains its signal peptide, which serves as a lipid anchor attaching apoM to the phospholipid layer of the lipoproteins, thereby keeping it in the circulation and preventing filtration of apoM in the kidney (CHRISTOFFERSEN et al., J. Biol Chem., 283:18765-18772 (2008)). The biological functions of apoM are only partly understood and the mechanisms by which it enables these functions are unknown. Studies in apoM gene-modified mice suggest that apoM has antiatherogenic effects, possibly related in part to the ability of apoM to increase cholesterol efflux from macrophage-foam cells, to increased preβ-HDL formation and to anti-oxidative effects (CHRISTOFFERSEN et al., J. Lipid Res., 47:1833-1843 (2006); CHRISTOFFERSEN et al., J. Biol Chem., 283:1839-1847 (2008); WOLFRUM et al., Nat. Med., 11:418-422 (2005)). The recent elucidation of the crystal structure of recombinant human apoM demonstrated a typical lipocalin fold characterized by an eight-stranded antiparallel β barrel that encloses an internal binding pocket, which likely facilitates binding of small lipophilic ligands (SEVVANA et al., J. Mol. Biol, 393:920-936 (2009)). Indeed, the recombinant apoM, which was expressed in E. coli, was found to cocrystallize with myristic acid (SEVVANA et al., J. Mol. Biol, 393:920-936 (2009)). This illustrated that apoM can bind lipid compounds with fatty acid side chains, and in vitro binding experiments demonstrated that S1P displaced the myristic acid with an IC50 of 0.90 μM (SEVVANA et al., J. Mol. Biol, 393:920-936 (2009)).
USPTO Backgrounds
USPTO Backgrounds