Characterization of beta2-glycoprotein I binding to phospholipid membranes.

The plasma protein beta2-glycoprotein I (beta2-GPI) is a major target of autoantibodies in patients with the antiphospholipid syndrome. To understand the physiological function of beta2-GPI and its potential role in the pathophysiology of the antiphospholipid syndrome, the binding of beta2-GPI to phospholipid membranes was characterized. The interaction of beta2-GPI with unilamellar vesicles containing varying amounts of acidic phospholipids with phosphatidylcholine (PC) was measured at equilibrium via relative light scattering. Analysis of binding isotherms gave apparent Kd values ranging from approximately 5.0 to 0.5 microM over a range of 5-20 mol % anionic phospholipid. Inhibition of binding by increasing ionic strength and Ca2+ ions suggests that binding is primarily electrostatic. These data indicate that beta2-GPI binding to membranes with physiological anionic phospholipid content is relatively weak in comparison to plasma coagulation proteins, suggesting that beta2-GPI does not function as a physiological anticoagulant based on its phospholipid-binding properties.

New low-density lipoprotein receptor upregulators acting via a novel mechanism.

The synthesis and biological activity of a new series of benzamides and related compounds that upregulate the expression of the low-density lipoprotein (LDL) receptor in human hepatocytes (HepG2 cells) by a novel mechanism are described. The lead compound, N-[5-[(3-cyclohexylpropionyl)amino]-2-methylphenyl]-4-hydroxybe nzamide (1, RPR102359), increased the expression of the LDL receptors in HepG2 cells by 80% when tested at a concentration of 3 microM. Mevinolin (lovastatin) was found to increase the LDL receptor expression by 70% at the same concentration. In contrast to mevinolin, 1 was found to have no effect on cholesterol biosynthesis in liver homogenates or in HepG2 cells at doses where substantial upregulation of the LDL receptor was observed and thus stimulated LDL receptor expression by a novel mechanism.

"Anticardiolipin" autoantibodies recognize beta 2-glycoprotein I in the absence of phospholipid. Importance of Ag density and bivalent binding.

"Anticardiolipin" autoantibodies (aCL) bind to anionic phospholipids only in the presence of beta 2-glycoprotein I (beta 2GPI), a phospholipid-binding plasma protein. The exact role of beta 2GPI in the antigenic specificity of these autoantibodies is unclear, however. Experiments were performed to determine whether aCL recognize beta 2GPI in the absence of phospholipid or neo-Ags formed as a consequence of the beta 2GPI-phospholipid interaction. Although aCL+ IgG fractions did not bind to beta 2GPI alone in ELISAs that used standard polystyrene immunoassay plates, significant specific binding was detected when beta 2GPI was coated on gamma-irradiated ("high binding") polystyrene plates. This difference was associated with the greater density of beta 2GPI immobilized on the gamma-irradiated plates. Fab' fragments of patient IgG demonstrated little or no binding to immobilized beta 2GPI in ELISA, indicating a critical role for Ab bivalency. Inhibition studies of three aCL+ IgG fractions confirmed their specificity for beta 2GPI and demonstrated low affinity binding to fluid-phase beta 2GPI (Kd values of approximately 10(-5) M). aCL binding to beta 2GPI was not a result of phospholipid contamination of the assays, as determined by microphosphate assay and by lipid extraction of IgG and beta 2GPI preparations. In summary, these experiments indicate that IgG aCL are intrinsically low affinity Abs to beta 2GPI. Ab binding to beta 2GPI on a microtiter plate or anionic phospholipid membrane is dependent upon the marked increase in avidity provided by engagement of both Ag binding sites of a given IgG molecule. The data support the hypothesis that phospholipid-bound beta 2GPI is the physiologic target of aCL.

Acyl-CoA:cholesterol O-acyl transferase (ACAT) inhibitors. 1. 2-(Alkylthio)-4,5-diphenyl-1H-imidazoles as potent inhibitors of ACAT.

A potent, bioavailable ACAT inhibitor may have beneficial effects in the treatment of atherosclerosis by (i) reducing the absorption of dietary cholesterol, (ii) reducing the secretion of very low density lipoproteins into plasma from the liver, and (iii) preventing the transformation of arterial macrophages into foam cells. We have found that a mevalonate derivative 2, which contains a 4,5-diphenyl-1H-imidazol-2-yl moiety, inhibits rat hepatic microsomal ACAT in vitro and produces a significant hypocholesterolemic effect in the cholesterol-fed rat. Structure-activity relationships for analogues of 2 demonstrate that the 4,5-diphenyl-1H-imidazole moiety is a pharmacophore for inhibition of rat microsomal ACAT.