Oxidized phospholipids as potential novel drug targets

Abstract

The interactions of three therapeutic agents, viz. the antipsychotics HPD and CPZ, and the antineoplastic anthracycline DOX, with oxidatively modified phospholipids were studied by monitoring the quenching of fluorescence of an incorporated pyrene-labeled lipid derivative. All three drugs bound avidly to the two oxidized PCs bearing either an aldehyde or carboxylic function at the end of the sn-2 nonanoyl chain, with the highest affinity measured between CPZ and the latter oxidized lipid. Subsequent dissociation of the above drugs from the oxidized lipids by DNA, acidic phospholipids, and NaCl revealed the binding of these drugs with the aldehyde lipid to be driven by hydrophobicity similarly to their binding to lysophosphatidylcholine, whereas a significant contribution of electrostatics was evident for the lipid with the carboxylic moiety. These results connect to previous experimental data, demonstrating the induction by these drugs of oxidative stress and binding to membrane phospholipids. These issues are elaborated with reference to their clinical use and side effects.

FIGURE 1

The chemical structures of the two oxidatively modified phospholipids used in this study, viz. PazePC and PoxnoPC.

FIGURE 2

Surface tension γ versus concentration of (A) PoxnoPC and (B) PazePC in 20 mM Hepes, 0.1 mM EDTA, pH 7.4. Values in Table 1 were obtained by fitting the Gibbs adsorption isotherm to the measured data. Experiments were carried out at ambient temperature of ∼23°C. The data points represent the mean of three or four measurements with the respective standard deviations.

FIGURE 3

(A) Binding of DOX (▪), CPZ (•), and HPD (▴) to PazePC as revealed by the decrease in the RFI of PPDPC as a function of [drug]. Subsequent detachment of the bound drugs by added (B) NaCl, (C) POPC/POPS = 8:2 LUVs, or (D) DNA, evident as RFQ compared to the initial value before addition of the indicated solute. Total lipid concentration in 20 mM Hepes, 0.1 mM EDTA, pH 7.4 was 25 μM. Temperature was maintained at 25°C.

FIGURE 4

(A) Binding of DOX (▪), CPZ (•), and HPD (▴) to PoxnoPC as revealed by the decrease in the RFI of PPDPC as a function of [drug]. Subsequent detachment of the bound drugs by added (B) NaCl, (C) POPC/POPS = 8:2 LUVs, or (D) DNA, evident as RFQ compared to the initial value before addition of the indicated solute. The experimental conditions were as described in the legend for Fig. 3.

FIGURE 5

(A) Binding of DOX (▪), CPZ (•), and HPD (▴) to lysoPC as revealed by the decrease in the RFI of PPDPC as a function of [drug]. (B) Subsequent detachment of the bound drugs by added NaCl evident as RFQ compared to the initial value before addition of the salt. The experimental conditions were as described in the legend for Fig. 3.

FIGURE 6

The effect of PazePC on the binding of CPZ to LUVs composed of 79:20:1 POPC/POPS/PPDPC (total lipid concentration 25 μM in 20 mM Hepes, 0.1 mM EDTA, pH 7.4). Quenching of RFI of PPDPC is illustrated in the absence (▪) and in the presence of 25 (•), 50 (▴), and 100 (▾) μM PazePC. Temperature was maintained at 25°C.

FIGURE 7

Size distributions by volume of aggregates of lysoPC and indicated binary mixtures with oxPLs measured by DLS. LysoPC (▪), lysoPC/PazePC = 8:2 (•), lysoPC/PoxnoPC = 8:2 (▴), lysoPC/PazePC = 1:1 (▾), and lysoPC/PoxnoPC = 1:1 (♦). The total lipid concentration in 20 mM Hepes, 0.1 mM EDTA, pH 7.4 was 200 μM in all measurements. Temperature was maintained at 25°C.