Multiplexed assays by high-content imaging for assessment of GPCR activity.

G-protein-coupled receptors (GPCR) participate in many disease pathways and represent the largest family of therapeutic targets. Thus, great investments are made to discover drugs modulating GPCR-mediated events. Among functional assays for screening GPCRs, the Transfluor imaging assay is based on redistribution of cytosolic beta-arrestin to an activated GPCR and has become widely used in high-content screening.

The target of ezetimibe is Niemann-Pick C1-Like 1 (NPC1L1).

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.

TLR2 recognizes a bacterial lipopeptide through direct binding.

The TLRs play an important role in the initiation of cellular innate immune responses to a wide range of bacterial products, including LPS and lipoproteins. Although rapid progress has been made on signaling functions of activated TLRs, the molecular mechanisms that lead to TLR activation are still poorly understood. We report in this study that the extracellular domain of TLR2 interacts directly with synthetic bacterial lipopeptide (sBLP), a potent analog of bacterial lipoproteins.

Niemann-Pick C1 Like 1 (NPC1L1) is the intestinal phytosterol and cholesterol transporter and a key modulator of whole-body cholesterol homeostasis.

Niemann-Pick C1 Like 1 (NPC1L1) is a protein localized in jejunal enterocytes that is critical for intestinal cholesterol absorption. The uptake of intestinal phytosterols and cholesterol into absorptive enterocytes in the intestine is not fully defined on a molecular level, and the role of NPC1L1 in maintaining whole body cholesterol homeostasis is not known. NPC1L1 null mice had substantially reduced intestinal uptake of cholesterol and sitosterol, with dramatically reduced plasma phytosterol levels.

A small molecule alpha4beta1/alpha4beta7 antagonist differentiates between the low-affinity states of alpha4beta1 and alpha4beta7: characterization of divalent cation dependence.

An alpha4beta1/alpha4beta7 dual antagonist, 35S-compound 1, was used as a model ligand to study the effect of divalent cations on the activation state and ligand binding properties of alpha4 integrins. In the presence of 1 mM each Ca2+/Mg2+, 35S-compound 1 bound to several cell lines expressing both alpha4beta1 and alpha4beta7, but 2S-[(1-benzenesulfonyl-pyrrolidine-2S-carbonyl)-amino]-4-[4-methyl-2S-(methyl-[2-[4-(3-o-tolyl-ureido)-phenyl]-acetyl]-amino) pentanoylamino]-butyric acid (BIO7662), a specific alpha4beta1 antagonist, completely inhibited 35S-compound 1 binding, suggesting that alpha4beta1 was responsible for the observed binding. 35S-Compound 1 bound RPMI-8866 cells expressing predominantly alpha4beta7 with a KD of 1.

Small molecule antagonists of complement receptor type 3 block adhesion and adhesion-dependent oxidative burst in human polymorphonuclear leukocytes.

The leukocyte integrin complement receptor type 3 (CR3, Mac-1, CD11b/CD18) is the predominant beta(2) integrin receptor of polymorphonuclear leukocytes (PMNs). This cell surface receptor plays a central role in innate immunity against pathogens as well as being a major cellular effector of inflammation and tissue injury. Two small molecules, compounds 1 and 2, have been identified, that interact with CR3 and prevent CR3 from binding to its natural ligand, C3bi.

Simvastatin reduces neointimal thickening in low-density lipoprotein receptor-deficient mice after experimental angioplasty without changing plasma lipids.

Background: Statins exert antiinflammatory and antiproliferative actions independent of cholesterol lowering. To determine whether these actions might affect neointimal formation, we investigated the effect of simvastatin on the response to experimental angioplasty in LDL receptor-deficient (LDLR-/-) mice, a model of hypercholesterolemia in which changes in plasma lipids are not observed in response to simvastatin.

Methods And Results: Carotid artery dilation (2.

Alpha(4)beta(7)/alpha(4)beta(1) dual integrin antagonists block alpha(4)beta(7)-dependent adhesion under shear flow.

The alpha(4) integrin, alpha(4)beta(7), plays an important role in recruiting circulating lymphocytes to the gastrointestinal tract, where its ligand mucosal addressin cell adhesion molecule-1 (MAdCAM-1) is preferentially expressed on high endothelial venules (HEVs). Dual antagonists of alpha(4)beta(1) and alpha(4)beta(7), N-(2,6-dichlorobenzoyl)-(L)-4-(2',6'-bis-methoxyphenyl)phenylalanine (TR14035) and N-(N-[(3,5-dichlorobenzene)sulfonyl]-2-(R)-methylpropyl)-(D)-phenylalanine (compound 1), were tested for their ability to block the binding of alpha(4)beta(7)-expressing cells to soluble ligand in suspension and under in vitro and in vivo shear flow. Compound 1 and TR14035 blocked the binding of human alpha(4)beta(7) to an (125)I-MAdCAM-Ig fusion protein with IC(50) values of 2.

Deficiency in sPLA(2) does not affect HDL levels or atherosclerosis in mice.

Secretory non-pancreatic phospholipase A(2) (sPLA(2)) has been implicated in inflammation and has been found in human atherosclerotic lesions. To test the effect of sPLA(2) deficiency on atherosclerosis, C57BL/Ks mice (apoE(+/+) and PLA(2)(++) were bred with C57BL/6 apoE knockout mice which are sPLA(2)(--) due to a spontaneous mutation. Sibling pairs of mice (apoE(--)/sPLA(2)(++) and apoE(--)/sPLA(2)(--)) on high fat Western diets were dissected at 22 weeks.

Toll-like receptor 2 (TLR2) mediates activation of stress-activated MAP kinase p38.

Early events in the response of cells to lipopolysaccharide (LPS) include activation of NF-kappaB and stress-activated MAP kinase p38. Recent studies have shown that the human Toll-like receptor 2 (TLR2) mediates activation of NF-kappaB in response to commercial preparations of LPS (comLPS), membrane lipoproteins, and Gram-positive bacterial products. Here, we show that expression of TLR2 in human embryonic kidney 293 cells enabled p38 phosphorylation in response to comLPS, a synthetic bacterial lipoprotein, and B.

ApoE(-/-) mice develop atherosclerosis in the absence of complement component C5.

Previous studies have suggested that the terminal complex of complement may contribute to the pathogenesis of atherosclerosis. C5b-9 complexes colocalize with the extracellular lipid in the aortic intima of hypercholesterolemic rabbits, and C6-deficient rabbits develop less atherosclerosis than controls. To test the role of complement in atherosclerosis in a different animal model, C5 deficient (C5def) mice were cross-bred with atherosclerosis susceptible apoE(-/-) mice, generating mice deficient in both apoE and C5 and control apoE(-/-) mice.

Simvastatin has anti-inflammatory and antiatherosclerotic activities independent of plasma cholesterol lowering.

Inhibitors of 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase, such as simvastatin, lower circulating cholesterol levels and prevent myocardial infarction. Several studies have shown an unexpected effect of HMG-CoA reductase inhibitors on inflammation. Here, we confirm that simvastatin is anti-inflammatory by using a classic model of inflammation: carrageenan-induced foot pad edema.

Deficiency in inducible nitric oxide synthase results in reduced atherosclerosis in apolipoprotein E-deficient mice.

Inducible NO synthase (iNOS) present in human atherosclerotic plaques could contribute to the inflammatory process of plaque development. The role of iNOS in atherosclerosis was tested directly by evaluating the development of lesions in atherosclerosis-susceptible apolipoprotein E (apoE)-/- mice that were also deficient in iNOS. ApoE-/- and iNOS-/- mice were cross-bred to produce apoE-/-/iNOS-/- mice and apoE-/-/iNOS+/+ controls.

A target for cholesterol absorption inhibitors in the enterocyte brush border membrane.

Uptake of cholesterol by the intestinal absorptive epithelium can be selectively blocked by specific small molecules, like the sterol glycoside, L-166,143. Furthermore, (3)H-labeled L-166,143 administered orally to hamsters binds specifically to the intestinal mucosa, suggesting the existence of a cholesterol transporter. Using autoradiography, the binding site of (3)H-L-166,143 in the hamster small intestine was localized to the very apical aspect of the absorptive epithelial cells.

Intestinal absorption of cholesterol is mediated by a saturable, inhibitable transporter.

Although the mechanism by which dietary cholesterol is absorbed from the intestine is poorly understood, it is generally accepted that cholesterol is absorbed from bile acid micelles in the jejunum. Once inside the enterocytes, cholesterol is esterified by the action of acyl-coenzyme A:cholesterol acyltransferase (ACAT), assembled into chylomicrons, and secreted into the lymph. In this work, mechanistic aspects of cholesterol absorption were probed using compounds that block cholesterol absorption in hamsters.

Infectious agents are not necessary for murine atherogenesis.

Recent work has revealed correlations between bacterial or viral infections and atherosclerotic disease. One particular bacterium, Chlamydia pneumoniae, has been observed at high frequency in human atherosclerotic lesions, prompting the hypothesis that infectious agents may be necessary for the initiation or progression of atherosclerosis. To determine if responses to gram-negative bacteria are necessary for atherogenesis, we first bred atherosclerosis-prone apolipoprotein (apo) E(-/)- (deficient) mice with animals incapable of responding to bacterial lipopolysaccharide.

Activation of peroxisome proliferator-activated receptor gamma does not inhibit IL-6 or TNF-alpha responses of macrophages to lipopolysaccharide in vitro or in vivo.

We have investigated the potential use of peroxisome proliferator-activated receptor gamma (PPARgamma) agonists as anti-inflammatory agents in cell-based assays and in a mouse model of endotoxemia. Human peripheral blood monocytes were treated with LPS or PMA and a variety of PPARgamma agonists. Although 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2) at micromolar concentrations significantly inhibited the production of TNF-alpha and IL-6, four other high affinity PPARgamma ligands failed to affect cytokine production.

15-Deoxy-Delta12,1412,14-prostaglandin J2 inhibits the beta2 integrin-dependent oxidative burst: involvement of a mechanism distinct from peroxisome proliferator-activated receptor gamma ligation.

15-Deoxy-Delta12,14-PGJ2 (dPGJ2) is a bioactive metabolite of the J2 series that has been identified as a ligand for peroxisome proliferator-activated receptor gamma (PPARgamma) and has received attention for its potential antiinflammatory effects. Because neutrophils express cell-surface receptors for PGs, the effect of dPGJ2 was tested on an inflammatory response that should not require PPARgamma, the oxidative burst made by adherent human neutrophils. dPGJ2 inhibited adhesion-dependent H2O2 production with an IC50 of 1.

A fluorescent cholesterol analog traces cholesterol absorption in hamsters and is esterified in vivo and in vitro.

The fluorescent cholesterol analog 22-(N-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3beta-ol (fluoresterol) was characterized as a tool for exploring the biochemistry and cell biology of intestinal cholesterol absorption. Hamsters absorbed fluoresterol in a concentration- and time-dependent manner, with an efficiency of about 15-30% that of cholesterol. Fluoresterol absorption was blocked by compounds known to inhibit cholesterol absorption, implying that fluoresterol interacts with those elements of the normal pathway for cholesterol absorption on which the inhibitors act.

Internalization of monomeric lipopolysaccharide occurs after transfer out of cell surface CD14.

Lipopolysaccharide (LPS) fluorescently labeled with boron dipyrromethane (BODIPY) first binds to the plasma membrane of CD14-expressing cells and is subsequently internalized. Intracellular LPS appears in small vesicles near the cell surface and later in larger, punctate structures identified as the Golgi apparatus. To determine if membrane (m)CD14 directs the movement of LPS to the Golgi apparatus, an mCD14 chimera containing enhanced green fluorescent protein (mCD14-EGFP) was used to follow trafficking of mCD14 and BODIPY-LPS in stable transfectants.

Role of stress-activated mitogen-activated protein kinase (p38) in beta 2-integrin-dependent neutrophil adhesion and the adhesion-dependent oxidative burst.

Bacterial LPS elicits both rapid activation of the stress-activated MAP kinase p38 in polymorphonuclear leukocytes (PMN) and rapid adhesion of the PMN to ligands for the leukocyte integrin CD11b/CD18. The functional correlation between these two events was examined. The time course for tyrosine phosphorylation of p38 in PMN in response to 10 ng/ml LPS in 1% normal human serum was consistent with participation in signaling for leukocyte integrin-dependent adhesion, with transient phosphorylation peaking at 10 to 20 min.

Sensitive responses of leukocytes to lipopolysaccharide require a protein distinct from CD14 at the cell surface.

Responses of leukocytes to low concentrations of LPS require the expression of membrane-associated CD14 (mCD14) on the cell surface; mCD14 is, however, a glycosyl-phosphatidylinositol-anchored protein and, therefore, a poor candidate for transducing signals across the plasma membrane. The role of other cell surface proteins in responses of leukocytes to LPS was tested by measuring IL-6 secretion of cultured human monocytes and adhesion of PMN in response to LPS after treatment of the cells with trypsin. Trypsin abolished leukocyte integrin-mediated adhesion of PMN in response to LPS, but the trypsinized cells remained responsive to the alternate agonists TNF, formyl peptide, and PMA.

Potential role of membrane internalization and vesicle fusion in adhesion of neutrophils in response to lipopolysaccharide and TNF.

Human polymorphonuclear leukocytes (PMN) respond to LPS with strongly increased integrin-mediated adhesion. While the first step of this process has been identified as the interaction of LPS with CD14 on the cell surface, subsequent steps remain to be elucidated. The experiments presented here suggest that monomeric LPS is internalized in vesicles, and uptake may be required for signaling.