Discovery, synthesis and biological evaluation of 2-(4-(N-phenethylsulfamoyl)phenoxy)acetamides (SAPAs) as novel sphingomyelin synthase 1 inhibitors.

Sphingomyelin synthase (SMS) has been proved to be a potential drug target for the treatment of atherosclerosis. However, few SMS inhibitors have been reported. In this paper, structure-based virtual screening was performed on hSMS1. SAPA 1a was discovered as a novel SMS1 inhibitor with an IC50 value of 5.2 µM in enzymatic assay. A series of 2-(4-(N-phenethylsulfamoyl)phenoxy)acetamides (SAPAs) were synthesized and their biological activities toward SMS1 were evaluated. Among them, SAPA 1j was found to be the most potent SMS1 inhibitor with an IC50 value of 2.1 µM in in vitro assay. The molecular docking studies suggested the interaction modes of SMS1 inhibitors and PC with the active site of SMS1. Site-directed mutagenesis validated the involvement of residues Arg342 and Tyr338 in enzymatic sphingomyelin production. The discovery of SAPA derivatives as a novel class of SMS1 inhibitors would advance the development of more effective SMS1 inhibitors.

Generation of general and tissue-specific gene knockout mouse models.

Knockout technology has established the functions of many genes affecting plasma lipid and lipoprotein levels and the development of atherosclerosis. However, many genes remain to be characterized. The ability to produce mice lacking whole-body expression of a given gene is still one of the most powerful techniques available for determining gene function. A complementary approach, underutilized yet vitally important to understanding lipoprotein metabolism, is the ability to create mice with gene deficiency only in a specific tissue. Liver, intestine, and macrophages are the major tissues and cells involved in lipoprotein metabolism and atherosclerosis, and additional tissues such as adipose tissue and brain are also of interest. Thus, feasible approaches to prepare general and tissue-specific gene knockout mouse models are necessary. Here, we describe our general procedure for generating whole-body knockout mice, using as an example the preparation of general (whole-body) phospholipid transfer protein (PLTP) gene knockout mice. We also describe several approaches to generating liver, intestine, and myeloid cell-specific tissue-specific knockout mice, using as an example the preparation of tissue-specific knockout mice for the subunit 2 of serine palmitoyltransferase (SPT), a key enzyme for sphingomyelin de novo synthesis. Bone marrow transplantation is an alternative means of creating myeloid cell-specific knockout mice. The general principles and techniques described here apply to the establishment of other gene knockout mouse models as well. The ability to manipulate gene expression in specific tissues as well as throughout the entire body of the mouse is anticipated to yield novel insights into lipid and lipoprotein metabolism and the development of atherosclerosis.

Sphingomyelin synthase 2 activity and liver steatosis: an effect of ceramide-mediated peroxisome proliferator-activated receptor γ2 suppression.

OBJECTIVE: Sphingolipid de novo biosynthesis is related to nonalcoholic fatty liver disease or hepatic steatosis. However, the mechanism is still unclear. Sphingomyelin synthase (SMS), using ceramide as one of the substrates to produce sphingomyelin, sits at the crossroads of sphingolipid biosynthesis. SMS has 2 isoforms: SMS1 and SMS2. SMS2 is the major isoform in liver. APPROACH AND RESULTS: To investigate the relationship between liver SMS2 activity-mediated sphingolipid changes and hepatic steatosis, we used 2 mouse models: Sms2 liver-specific transgenic and Sms2 knockout mice. We found that Sms2 liver-specific transgenic livers have lower ceramide and higher sphingomyelin, whereas Sms2 knockout livers have higher ceramide and lower sphingomyelin. We also found that liver Sms2 overexpression promoted fatty acid uptake and liver steatosis, whereas Sms2 deficiency had an opposite effect in comparison with their respective controls. Importantly, the exogenous ceramide supplementation to Huh7 cells, a human hepatoma cell line, reduced the expression of peroxisome proliferator-activated receptor γ2 and its target genes, Cd36 and Fsp27. Peroxisome proliferator-activated receptor γ reporter analysis confirmed this phenomenon. Furthermore, peroxisome proliferator-activated receptor γ antagonist treatment significantly decreased triglyceride accumulation in Sms2 liver-specific transgenic liver. CONCLUSIONS: We attributed these effects to ceramide that can suppress peroxisome proliferator-activated receptor γ2, thus reducing the expression of Cd36 and Fsp27 and reducing liver steatosis. After all, SMS2 inhibition in the liver could diminish liver steatosis.

Identification and characterization of dual inhibitors for phospholipid transfer protein and microsomal triglyceride transfer protein.

Phospholipid transfer protein (PLTP) plays an important role in atherogenesis and lipoprotein metabolism. PLTP exerts its functions intracellularly and extracellularly. Both PLTP and microsomal triglyceride transfer protein (MTP) have been shown to regulate the secretion of apolipoprotein B (apoB) in hepatocytes. We have previously reported the characterization of inhibitors that selectively inhibit PLTP activity and reduce apoB secretion in hepatocytes. In the present study, we identified more compounds that inhibit both PLTP and MTP activity to various extents. These dual inhibitors are structurally different from the PLTP-selective inhibitors. In human hepatoma cell lines, dual inhibitors seem to be more effective in reducing apoB secretion than selective PLTP or MTP inhibitors. Furthermore, the dual inhibitors markedly reduced triglyceride secretion from hepatocytes. In the absence of PLTP, the dual inhibitors can further reduce apoB secretion, whereas selective PLTP inhibitors had no effect. We conclude that MTP and PLTP may work coordinately in the process of hepatic apoB assembly and secretion. To avoid liver toxicity mediated by MTP inhibition, selective PLTP inhibitors should be pursued.

Synthesis of a series of novel 2,4,5-trisubstituted selenazole compounds as potential PLTP inhibitors.

Based on a homology-modeled structure of PLTP and characteristic structural features of reported cholesteryl ester transfer protein (CETP) inhibitors, we designed and synthesized a novel series of 2,4,5-trisubstituted selenazole compounds. Biological evaluation reveals that compounds 12 and 17 exhibit favorable PLTP activity, and their IC(50)s are 8 microM and 10 microM, respectively.

Effect of plasma phospholipid transfer protein deficiency on lethal endotoxemia in mice.

Lipopolysaccharides (LPS) are components of Gram-negative bacteria. The cellular response from the host to LPS is mediated through stepwise interactions involving the lipopolysaccharide-binding protein (LBP), CD14, and MD-2, which produces the rearrangement of TLR4. In addition to LBP, the lipid transfer/lipopolysaccharide-binding protein gene family includes the phospholipid transfer protein (PLTP). Here we show that the intravascular redistribution of LPS from the plasma lipoprotein-free fraction toward circulating lipoproteins is delayed in PLTP-deficient mice. In agreement with earlier in vitro studies, which predicted the neutralization of the endotoxic properties of LPS when associated with lipoproteins, significant increases in the plasma concentration of proinflammatory cytokines were found in PLTP-deficient as compared with wild type mice. Similar inflammatory damage occurred in tissues from wild type and PLTP-deficient mice 24 h after one single intraperitoneal injection of LPS but with a more severe accumulation of red blood cells in glomeruli of LPS-injected PLTP-deficient mice. Complementary ex vivo experiments on isolated splenocytes from wild type and PLTP-deficient mice further supported the ability of cell-derived PLTP to prevent LPS-mediated inflammation and cytotoxicity when combined with lipoprotein acceptors. Finally, PLTP deficiency in mice led to a significant increase in LPS-induced mortality. It is concluded that increasing circulating levels of PLTP may constitute a new and promising strategy in preventing endotoxic shock.

Phospholipid transfer protein (PLTP) deficiency reduces brain vitamin E content and increases anxiety in mice.

Vitamin E supplementation constitutes a promising strategy in the prevention of neurodegenerative diseases. Here, we show that a phospholipid transfer protein (PLTP) is widely expressed in the brain where it appears to function as a transfer factor for alpha-tocopherol, the main isomer of vitamin E. PLTP deficiency results in significant depletion of brain alpha-tocopherol in both homozygous (-30.1%, P<0.0002) and heterozygous (-18.0%, P<0.05) PLTP knocked-out mice. Alpha-tocopherol depletion in PLTP-deficient homozygotes is associated with the elevation of lipofuscin (+25% and +450% increases in cortex and substantia nigra, respectively), cholesterol oxides (+54.5%, P<0.05), and cellular peroxides (+32.3%, P<0.01) in the brain. Complete PLTP deficiency in homozygotes is accompanied by increased anxiety as shown by fewer entries (8.3% vs. 44.4% in controls, P<0.01) and less time spent (1.7% vs. 41.3% in controls, P<0.05) in the open arms of an elevated plus-maze, in the absence of locomotor deterioration. Thus, the vitamin E transfer activity of PLTP appears to be a key process in preventing oxidative damage in the brain, and PLTP-deficient mice could be a new model of the contribution of oxidative brain injury in the etiology of neurodegenerative diseases.

Phospholipid transfer protein deficiency protects circulating lipoproteins from oxidation due to the enhanced accumulation of vitamin E.

Vitamin E is a lipophilic anti-oxidant that can prevent the oxidative damage of atherogenic lipoproteins. However, human trials with vitamin E have been disappointing, perhaps related to ineffective levels of vitamin E in atherogenic apoB-containing lipoproteins. Phospholipid transfer protein (PLTP) promotes vitamin E removal from atherogenic lipoproteins in vitro, and PLTP deficiency has recently been recognized as an anti-atherogenic state. To determine whether PLTP regulates lipoprotein vitamin E content in vivo, we measured alpha-tocopherol content and oxidation parameters of lipoproteins from PLTP-deficient mice in wild type, apoE-deficient, low density lipoprotein (LDL) receptor-deficient, or apoB/cholesteryl ester transfer protein transgenic backgrounds. In all four backgrounds, the vitamin E content of very low density lipoprotein (VLDL) and/or LDL was significantly increased in PLTP-deficient mice, compared with controls with normal plasma PLTP activity. Moreover, PLTP deficiency produced a dramatic delay in generation of conjugated dienes in oxidized apoB-containing lipoproteins as well as markedly lower titers of plasma IgG autoantibodies to oxidized LDL. The addition of purified PLTP to deficient plasma lowered the vitamin E content of VLDL plus LDL and normalized the generation of conjugated dienes. The data show that PLTP regulates the bioavailability of vitamin E in atherogenic lipoproteins and suggest a novel strategy for achieving more effective concentrations of anti-oxidants in lipoproteins, independent of dietary supplementation.

Role of hepatic lipase and scavenger receptor BI in clearing phospholipid/free cholesterol-rich lipoproteins in PLTP-deficient mice.

Phospholipid transfer protein knock-out (PLTP0) mice have defective transfer of phospholipids (PL) from triglyceride-rich lipoproteins (TRL) into high-density lipoproteins (HDL). In this study, we examined the role of diet, hepatic lipase (HL) and scavenger receptor BI (SRBI) in determining the accumulation of excess PL and free cholesterol (FC, "surface remnants") in plasma of PLTP0 mice. PL and FC accumulated in the very low-density lipoprotein (VLDL)-LDL region of PLTP0 mice on a highly saturated, coconut oil-based diet, but not on chow or milk-fat based Western diets. Accumulation of PL and FC was dramatically increased in PLTP0/HL0 mice, compared to PLTP0 mice, but only on the coconut oil diet. Turnover studies indicated that the coconut oil diet was associated with delayed catabolism of PL of PL/FC-rich particles. Incubation of these particles with primary hepatocytes in the presence of SRBI neutralizing antibody indicated that SRBI was primarily responsible for removal of FC and PL on the Western diet. In hepatocytes of coconut oil-fed mice, removal of FC and PL from these particles by SRBI was markedly reduced, even though SRBI protein expression levels were unchanged. These studies indicate that HL and SRBI both have major role in the clearance of PL and FC of surface remnants in PLTP0 mice. SRBI appears to be dysfunctional in coconut oil diet-fed animals, possibly related to changes in hepatocyte membrane fatty acid composition.