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

Abstract

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.

Fig. 1.

Structures of the compounds that inhibit PLTP activity.

Fig. 2.

Inhibition of human PLTP-mediated phospholipid transfer activity by the compounds. The phospholipid transfer activity of human PLTP was measured in the presence of 0 to 30 μM compound (cpd) as described under Materials and Methods. The data are expressed as percentage of inhibition.

Fig. 3.

Inhibition of human MTP-mediated triglyceride transfer activity by the compounds. Triglyceride transfer activity of human MTP was measured in the presence of 0 to 30 μM compound (Cpd) as described under Materials and Methods. The data are expressed as percentage of inhibition.

Fig. 4.

A and C, effects of the compounds on ApoB secretion in HepG2 cells (A) and Huh7 cells (C). A–C, HepG2 or Huh7 cells were treated with the compounds (Cpd) for 24 h, and medium was collected for apoB (A and C) and transferrin (B) measurement by ELISA. ApoB levels normalized to transferrin are presented. B, transferrin levels from experiments shown in A. *, P < 0.05. D, effects of compounds on triglyceride secretion in HepG2 cells. Cells were treated with compounds for 24 h, and medium was harvested to measure triglyceride levels as described under Materials and Methods. E, MTT cytotoxicity for experiment shown in C. Cells were incubated with MTT reagent, and cytotoxicity was measured as described under Materials and Methods. Data are the average of triplicates ± standard deviation.

Fig. 5.

Effect of selective PLTP and dual inhibitors on apoB secretion in mouse primary hepatocytes isolated from wild-type mice (A) and PLTP-deficient mice (B). Cells were labeled with [³⁵S]methionine/cysteine in the presence of vehicle (DMSO) or 30 μM compound (cpd) for 4 h. Medium and cells were collected to detect labeled apoB as described under Materials and Methods. ApoB levels are the average of triplicate experiments. Representative images are shown in the insets.