Ezetimibe is a potent inhibitor of cholesterol absorption that has been approved for the treatment of hypercholesterolemia, but its molecular target has been elusive. Using a genetic approach, we recently identified Niemann-Pick C1-Like 1 (NPC1L1) as a critical mediator of cholesterol absorption and an essential component of the ezetimibe-sensitive pathway. To determine whether NPC1L1 is the direct molecular target of ezetimibe, we have developed a binding assay and shown that labeled ezetimibe glucuronide binds specifically to a single site in brush border membranes and to human embryonic kidney 293 cells expressing NPC1L1. Moreover, the binding affinities of ezetimibe and several key analogs to recombinant NPC1L1 are virtually identical to those observed for native enterocyte membranes. KD values of ezetimibe glucuronide for mouse, rat, rhesus monkey, and human NPC1L1 are 12,000, 540, 40, and 220 nM, respectively. Last, ezetimibe no longer binds to membranes from NPC1L1 knockout mice. These results unequivocally establish NPC1L1 as the direct target of ezetimibe and should facilitate efforts to identify the molecular mechanism of cholesterol transport.cholesterol ͉ intestinal brush border membranes

Leukotrienes are proinflammatory products of arachidonic acid oxidation by 5-lipoxygenase that have been shown to be involved in respiratory and cardiovascular diseases. The integral membrane protein FLAP is essential for leukotriene biosynthesis. We describe the x-ray crystal structures of human FLAP in complex with two leukotriene biosynthesis inhibitors at 4.0 and 4.2 angstrom resolution, respectively. The structures show that inhibitors bind in membrane-embedded pockets of FLAP, which suggests how these inhibitors prevent arachidonic acid from binding to FLAP and subsequently being transferred to 5-lipoxygenase, thereby preventing leukotriene biosynthesis. This structural information provides a platform for the development of therapeutics for respiratory and cardiovascular diseases.

Objective-Leukotriene B 4 (LTB 4 ), a product of the 5-lipoxygenase (5-LO) pathway of arachidonic acid metabolism, has been implicated in atherosclerosis. However, the molecular mechanisms for the atherogenic effect of LTB 4 are not well understood. This study is to determine candidate mechanisms. Method and Results-Primary human monocytes were treated with LTB 4 and the supernatant was analyzed for cytokine/chemokine production by an immuno-protein array. This analysis revealed a strong increase of the monocyte chemoattractant protein-1 (MCP-1), a proinflammatory cytokine. See page 1748Recent studies suggest a strong link between LTB 4 pathways with atherosclerosis. For example, human atherogenic plaques produce LTB 4 , 7 and expression of both BLT 1 and BLT 2 increases with the progression of atherosclerotic lesions. 8 Treatment of atherosclerosis-prone mice (apolipoprotein E [ApoE] or low-density lipoprotein receptor [LDLR]-deficient mice) with the BLT 1 -specific antagonist CP-105,696 9,10 markedly decreased lesion size. 11 Interestingly, the anti-atherogenic effects of CP-105,696 were diminished in mice deficient in the chemoattractant monocyte chemotactic protein-1 (MCP-1), 11 indicating a critical role for MCP-1 in mediating the LTB 4 atherogenic signals.MCP-1 is a prototype of the C-C chemokine ␤ subfamily and exhibits the most potent chemotactic activity for monocytes. 12 Overexpression of MCP-1 contributes to the development of atherosclerosis in mouse models. 13 Deficiency of either MCP-1 or its cognate high-affinity receptor C-C chemokine receptor 2 (CCR2) results in a marked decrease in atheromas and fewer monocytes in vascular lesions. 14,15 Additionally, therapeutic gene transfer of a dominantnegative MCP-1 mutant attenuated the development of early atherosclerosis and also limited progression of preexisting atherosclerotic lesions in ApoE-null mice. 16 Despite the critical role played by LTB 4 in atherogenesis, the molecular mechanisms for these activities are poorly understood. In this study, we investigated specifically whether LTB 4 regulates MCP-1 production in primary human monocytes to broaden the mechanistic understanding for LTB 4 -induced atherosclerosis. Our study shows that LTB 4 induced MCP-1 protein by several hundred-fold in primary human monocytes. LTB 4 induced MCP-1 mRNA by 500-fold Original

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