Selective inhibitors of a PAF biosynthetic enzyme lysophosphatidylcholine acyltransferase 2

Abstract

Platelet-activating factor (PAF) is a potent pro-inflammatory phospholipid mediator. In response to extracellular stimuli, PAF is rapidly biosynthesized by lyso-PAF acetyltransferase (lyso-PAFAT). Previously, we identified two types of lyso-PAFATs: lysophosphatidylcholine acyltransferase (LPCAT)1, mostly expressed in the lungs where it produces PAF and dipalmitoyl-phosphatidylcholine essential for respiration, and LPCAT2, which biosynthesizes PAF and phosphatidylcholine (PC) in the inflammatory cells. Under inflammatory conditions, LPCAT2, but not LPCAT1, is activated and upregulated to produce PAF. Thus, it is important to develop inhibitors specific for LPCAT2 in order to ameliorate PAF-related inflammatory diseases. Here, we report the first identification of LPCAT2-specific inhibitors, N-phenylmaleimide derivatives, selected from a 174,000-compound library using fluorescence-based high-throughput screening followed by the evaluation of the effects on LPCAT1 and LPCAT2 activities, cell viability, and cellular PAF production. Selected compounds competed with acetyl-CoA for the inhibition of LPCAT2 lyso-PAFAT activity and suppressed PAF biosynthesis in mouse peritoneal macrophages stimulated with a calcium ionophore. These compounds had low inhibitory effects on LPCAT1 activity, indicating that adverse effects on respiratory functions may be avoided. The identified compounds and their derivatives will contribute to the development of novel drugs for PAF-related diseases and facilitate the analysis of LPCAT2 functions in phospholipid metabolism in vivo.

Keywords: LPCAT2 inhibitor; N-phenylmaleimide derivatives; TSI-01; fluorescent probe; high-throughput screening; inflammation; lipid mediator; lyso-PAF acetyltransferase; lysophospholipid acyltransferase; platelet-activating factor.

Fig. 1.

Identification of LPCAT2-specific inhibitors via HTS. A: Screening cascade to identify LPCAT2-specific inhibitors. For each assay, 20 μM of each compound was used. Hit compounds were determined by the indicated criteria. CoA-SH was detected by a fluorescent probe in step 1 (HTS). PAF, DPPC, and 16:0/20:4 alkyl-PC were measured by LC-MS/MS in the following steps. Human enzymes were used in steps 1–6. Both human and mouse enzymes were used in step 7. Two compounds were identified as potent and selective inhibitors of LPCAT2 in the 9-step selection process. *The increased value of inflorescence intensity elicited by the thiol-reactive CPM. Screening steps 1–5 (n = 1), steps 6 and 7 (n = 3), steps 8 and 9 (n = 2). The details are described in the supplementary Methods. B, C: Schematic presentation of the acetyltransferase and acyltransferase activities of LPCAT2 and LPCAT1. Both enzymes have lyso-PAFAT activity [(a) and (b)] and long chain acyltransferase activity [(c) and (d)].

Fig. 2.

HTS of 174,131 compounds. A, B: Microsomal proteins of hLPCAT2-transfected CHO-S-PAFR cells, stimulated with 200 nM mcPAF, were incubated with lyso-PAF and acetyl-CoA. Thiol-containing CoA was detected by thiol-reactive fluorescent probe, CPM. The data were normalized to each positive control set at 100% activation. Before the library screening, 1,500 compounds were tested and a strong positive correlation was observed (r = 0.97) in two independent experiments (runs 1 and 2) (A). Subsequently, 174,131 compounds were analyzed (B). C: The assay performance was consistent across all plates, with robust Z′ factors (>0.5).

Fig. 3.

Enzyme expression and activity. A: Microsomal fractions obtained from CHO-S-PAFR cells stimulated or not with mcPAF were separated by SDS-PAGE or Phos-tag SDS-PAGE and analyzed by Western blotting using the anti-FLAG M2 antibody. Upper: protein expression. Lower: on the Phos-tag SDS-PAGE, band shifts (arrow heads) were observed in the stimulated fractions of LPCAT2, indicating phosphorylation upon mcPAF-stimulation. B–D: Both LPCAT acetyltransferase (B) and LPCAT2 acyltransferase (C), or LPCAT1 acyltransferase (D) activities were measured in microsomal fractions; PAF (B), 16:0/20:4 alkyl-PC (C), and DPPC (D) were detected in each assay. The experiment was performed in triplicate (mean ± SEM). Four independent experiments were performed with similar results.

Fig. 4.

Effects of compounds on RAW-mLPCAT2 cells. A, B: RAW-mLPCAT2 cells and control RAW cells were stimulated with 5 μM A23187 for 5 min and the levels of PAF (A) and lyso-PAF (B) were determined by LC-MS/MS; statistical analyses were performed using ANOVA and Tukey’s multiple comparison test. C–F: RAW-mLPCAT2 cells were treated with the indicated concentrations of TSI-01 (C), TSI-07 (D), TSI-10 (E), or TSI-11 (F) for 1 h before A23187 stimulation, and PAF and lyso-PAF levels were determined by LC-MS/MS. All compounds dose-dependently decreased PAF levels. Data are presented as percentages of the control (0.2% DMSO): 100% PAF and lyso-PAF levels were 1.2 and 5.0 (C), 2.4 and 11.8 (D), or 1.0 and 5.9 (E and F) pmol/1 × 10⁶ cells, respectively. All data are shown as the mean ± SEM of three independent experiments performed in triplicate.

Fig. 5.

Effects of TSI-01 on PAF and lyso-PAF production in mouse peritoneal macrophages. A, B: Cells were pretreated with LPS for 18 h and then treated with the indicated concentrations of TSI-01 for 1 h, then PAF (A) and lyso-PAF (B) levels were measured after 5 μM A23187 stimulation for 5 min. TSI-01 dose-dependently reduced PAF levels; in the cells treated with 60 μM TSI-01 or not stimulated with A23187, PAF levels were below the detection limit. Lyso-PAF levels were unchanged. Data are shown as the mean ± SEM of three independent experiments performed in triplicate (#, n = 2).

Fig. 6.

Effects of TSI-01 on kinetics of LPCAT2 enzymatic activity. A, C, D: Lyso-PAFAT activity was measured with 5 μM lyso-PAF and the indicated concentrations of acetyl-CoA using microsomal fractions of mcPAF-stimulated (A, D) or nonstimulated (C) CHO-S-PAFR cells transiently transfected with hLPCAT2; TSI-01 dose-dependently increased K_m values (A, C) and Dixon plot represents the inhibition constant (K_i value) (D). B: Lyso-PAFAT activity was measured with 10 mM acetyl-CoA and the indicated concentrations of lyso-PAF using microsomal fractions of mcPAF-stimulated CHO-S-PAFR cells transiently transfected with hLPCAT2; K_m values were unchanged. The data represent the mean ± SD of triplicate measurements. The experiment was repeated twice with similar results.