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Abstract-The recent identification of liver X receptors (LXR) as regulators of the cholesterol and phospholipid export pump ABCA1 has raised the possibility that LXR agonists could be developed as HDL-raising agents, possibly also acting on the artery wall to stimulate cholesterol efflux from lipid-laden macrophages. Presently several pharmaceutical companies are working to develop such compounds, which will require finding a path for separating these beneficial effects from the detrimental stimulation of triglyceride synthesis also inherent to LXR agonists. Other challenges to the drug development process include species differences, which makes prediction of in vivo effects of LXR agonists in humans difficult. This review summarizes the present state of knowledge on LXR as a drug target and discusses possible solutions for dissociating the favorable effects of LXR agonists from their unwanted effects.

Organic impurities in compound libraries are known to often cause false-positive signals in screening campaigns for new leads, but organic impurities do not fully account for all false-positive results. We discovered inorganic impurities in our screening library that can also cause positive signals for a variety of targets and/or readout systems, including biochemical and biosensor assays. We investigated in depth the example of zinc for a specific project and in retrospect in various HTS screens at Roche and propose a straightforward counter screen using the chelator TPEN to rule out inhibition caused by zinc.

The LXR nuclear receptors are intracellular sensors of cholesterol excess and are activated by various oxysterols. LXRs have been shown to regulate multiple genes of lipid metabolism, including ABCA1 (formerly known as ABC1). ABCA1 is a lipid pump that effluxes cholesterol and phospholipid out of cells. ABCA1 deficiency causes extremely low high density lipoprotein (HDL) levels, demonstrating the importance of ABCA1 in the formation of HDL. The present work shows that the acetyl-podocarpic dimer (APD) is a potent, selective agonist for both LXR␣ (NR1H3) and LXR␤ (NR1H2). In transient transactivation assays, APD was ϳ1000-fold more potent, and yielded ϳ6-fold greater maximal stimulation, than the widely used LXR agonist 22-(R)-hydroxycholesterol. APD induced ABCA1 mRNA levels, and increased efflux of both cholesterol and phospholipid, from multiple cell types. Gas chromatographymass spectrometry measurements demonstrated that APD stimulated efflux of endogenous cholesterol, eliminating any possible artifacts of cholesterol labeling. For both mRNA induction and stimulation of cholesterol efflux, APD was found to be more effective than was cholesterol loading. Taken together, these data show that APD is a more effective LXR agonist than endogenous oxysterols. LXR agonists may therefore be useful for the prevention and treatment of atherosclerosis, especially in the context of low HDL levels.

Leukotrienes (LTs) are signaling molecules derived from arachidonic acid that initiate and amplify innate and adaptive immunity. In turn, how their synthesis is organized on the nuclear envelope of myeloid cells in response to extracellular signals is not understood. We define the supramolecular architecture of LT synthesis by identifying the activation-dependent assembly of novel multiprotein complexes on the outer and inner nuclear membranes of mast cells. These complexes are centered on the integral membrane protein 5-Lipoxygenase-Activating Protein, which we identify as a scaffold protein for 5-Lipoxygenase, the initial enzyme of LT synthesis. We also identify these complexes in mouse neutrophils isolated from inflamed joints. Our studies reveal the macromolecular organization of LT synthesis.inflammation ͉ multiprotein complex ͉ 5-lipoxygenase ͉ 5-lipoxygenase-activating protein L eukotrienes (LTs) are lipid signaling molecules derived from arachidonic acid (AA) that initiate and amplify innate and adaptive immune responses by regulating the recruitment and activation of leukocytes in inflamed tissues (1-3). Cells employ multiple mechanisms to prevent the inappropriate onset of pro-inflammatory signaling while requiring tightly coupled processes to trigger the generation of pro-inflammatory signaling molecules. The interplay of these processes is epitomized by the synthesis of LTB 4 and LTC 4 . In unstimulated myeloid cells, including mast cells, LT formation is held in abeyance by the cytosolic compartmentalization of cytosolic phospholipase A 2 (cPLA 2 ) (4, 5) and the cytosolic/nucleoplasmic localization of 5-lipoxygenase (5-LO) (6). LT formation is initiated by translocation of cPLA 2 to the Golgi and ER/nuclear envelope to release AA (4, 5). In parallel, 5-LO targets to the inner and outer nuclear membranes to initiate LT synthesis by converting AA to 5-HPETE and LTA 4 , a process that requires the integral nuclear envelope protein 5-lipoxygenase-activating protein (FLAP) (7-9). Although we have shown that LTC 4 synthase and FLAP constitutively interact on the nuclear envelope (10), how or whether 5-LO interacts with these proteins on the nuclear envelope in response to cell signaling is unknown.Understanding how cells couple the reorganization of the LT biosynthetic enzymes to efficient AA utilization is central to understanding the signal transduction pathways that control the synthesis of bioactive lipids derived from AA. Scaffold/docking proteins can localize components of biochemical reactions at membrane interfaces; examples include protein kinase A anchoring proteins (11) and the integral membrane proteins caveolins 1-3 (12). The dependence of cellular LT synthesis on FLAP (7, 8) and its membrane localization (9) make it a conceptually appealing candidate for a 5-LO scaffold. However, no interaction of 5-LO with any membrane protein has been detected.A closely related question is: how do different combinations of extracellular signals lead to LT generation? For example, in mast cells, the engage...

ATP-binding cassette transporter A1 (ABCA1) mediates an active efflux of cholesterol and phospholipids and is mutated in patients with Tangier disease. Expression of ABCA1 may be increased by certain oxysterols such as 22(R)-hydroxycholesterol via activation of the nuclear hormone receptor liver X receptor (LXR). In searching for potential modulators of ABCA1 expression, we have studied the effects of various mevalonate metabolites on the expression of ABCA1 in two human cell lines, THP-1 and Caco-2 cells. Most of the tested metabolites, including mevalonate, geranyl pyrophosphate, farnesyl pyrophosphate, and ubiquinone, failed to significantly change the expression levels of ABCA1. However, treatment with geranylgeranyl pyrophosphate resulted in a dose-and time-dependent reduction of ABCA1 expression. Geranylgeranyl pyrophosphate appears to reduce ABCA1 expression via two different mechanisms. One of these mechanisms is by acting directly as an antagonist of LXR since it reduces the interaction between LXR␣ or -␤ with nuclear coactivator SRC-1. Another mechanism appears to involve activation of the Rho GTP-binding proteins since treatment of Caco-2 cells with inhibitors of geranylgeranyl transferase or the Rho proteins significantly increased the expression and promoter activity of ABCA1. Further studies showed that mutations in the DR4 element of the ABCA1 promoter completely eliminate the inducible activities of these inhibitors. These data indicate that activation of the Rho proteins may change the activation status of LXR.Plasma concentration of high density lipoprotein cholesterol is inversely related to the incidence of coronary heart disease (1, 2). Our understanding of the mechanisms that regulate HDL 1 cholesterol has received a major advance with the elucidation of the cause of Tangier disease (TD). Patients with TD are characterized by near or complete absence of circulating HDL and by the accumulation of cholesteryl esters in many peripheral tissues (3, 4). Recently three groups independently reported identification of the ATP-binding cassette transporter A1 (ABCA1) as the defective gene responsible for TD (5-7). ABCA1 is a member of the ATP-binding cassette superfamily. These proteins couple the energy provided by ATP hydrolysis to the transport of a wide variety of molecules across membranes (8 -11). ABCA1 is thought to mediate the active efflux of cholesterol and phospholipids to apolipoprotein (apo) acceptors, most importantly apoA-I, the major apo of HDL (12, 13). Due to mutations, however, the function of ABCA1 in patients with TD is impaired. Therefore, cellular cholesterol efflux in TD patients is defective, which leads to accumulation of excess cellular cholesterol and defective formation of HDL (14 -16). ABCA1 is widely expressed and is particularly abundant in monocytes and macrophages (17). Studies in macrophages haveshown that the expression of ABCA1 is sterol-dependent (17). Expression of ABCA1 is up-regulated by modified low density lipoprotein and down-regulated by HDL (17, 18). The ...