Flavonoid-alkylphospholipid conjugates elicit dual inhibition of cancer cell growth and lipid accumulation.

Cancer development is often associated with lipid metabolic reprogramming, including aberrant lipid accumulation. We create novel paradigms endowed with dual functions of anticancer activity and inhibition of lipid accumulation by conjugating the natural product quercetin and synthetic alkylphospholipid drugs, and harnessing the biomedical effects of both. These conjugates offer fresh perspectives in the search for anticancer candidates.

Molecular dynamics simulations and neutron reflectivity as an effective approach to characterize biological membranes and related macromolecular assemblies.

In combination with other spectroscopy, microscopy, and scattering techniques, neutron reflectivity is a powerful tool to characterize biological systems. Specular reflection of neutrons provides structural information at the nanometer and subnanometer length scales, probing the composition and organization of layered materials. Currently, analysis of neutron reflectivity data involves several simplifying assumptions about the structure of the sample under study, affecting the extraction and interpretation of information from the experimental data. Computer simulations can be used as a source of structural and dynamic data with atomic resolution. We present a novel tool to compare the structural properties determined by neutron reflectivity experiments with those obtained from molecular simulations. This tool allows benchmarking the ability of molecular dynamics simulations to reproduce experimental data, but it also promotes unbiased interpretation of experimentally determined quantities. Two application examples are presented to illustrate the capabilities of the new tool. The first example is the generation of reflectivity profiles for a 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) lipid bilayer from molecular dynamics simulations using data from both atomistic and coarse-grained models, and comparison with experimentally measured data. The second example is the calculation of lipid volume changes with temperature and composition from all atoms simulations of single and mixed 1,2-di-palmitoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dihexadecanoyl-sn-glycero-3-phosphocholine (DPPC) bilayers.

Synthesis of enantiopure reversed structured ether lipids of the 1-O-alkyl-sn-2,3-diacylglycerol type.

This report describes the synthesis of reversed structured 1-O-alkyl-2,3-diacyl-sn-glycerols (DAGEs) possessing a pure saturated even number fatty acid (C6:0-C16:0) at the sn-2 position along with a pure EPA or DHA located at the terminal sn-3 position of the glycerol backbone of chimyl, batyl and selachyl alcohols. These adducts were synthesized by a highly efficient two-step chemoenzymatic process involving an immobilized Candida antarctica lipase to introduce pure EPA and DHA activated as oxime esters exclusively to the sn-3 terminal position of enantiopure chimyl, batyl and selachyl alcohols in excellent yields. The saturated fatty acids were subsequently incorporated to the remaining sn-2 position of the resulting 3-monoacylglyceryl ethers (3-MAGEs) using EDAC coupling agent in the presence of DMAP in very high to excellent yields (85%-98%). No losses of enantiomeric composition were observed during these processes. The multiple utilities of the resulting focused library of reversed structured DAGEs are discussed including how such compounds may possibly be utilized within the pharmaceutical area.

The effect of lipocisplatin on cisplatin efficacy and nephrotoxicity in malignant breast cancer treatment.

A lipid-cisplatin conjugate was synthesized for super-molecular assembly with lipids to form a new generation of liposomal cisplatin formulation, lipocisplatin. In vitro, lipocisplatin has higher efficacy in human ovarian cancer A2780 and human breast cancer MCF-7 with the murine breast cancer cell line 4T1 which is currently an established model for stage IV breast cancer as the most sensitive strain. Moreover, lipocisplatin demonstrated a greater MTD value and relatively longer blood circulation as compared to cisplatin. Lipocisplatin preferentially accumulate drugs to the tumor site, resulting in a better tumor inhibition efficacy. Moreover, lipocisplatin exceeds the size cutoff for kidney clearance, hence it bypasses the nephrotoxicity of cisplatin which is a major curse of one of the most efficient anticancer drugs nowadays in clinic. The results here indicated lipocisplatin may be translated into a new generation of liposomal based cisplatin drug in clinic.

Design and synthesis of new adamantyl-substituted antileishmanial ether phospholipids.

A series of new 2-[3-(2-alkyloxy-ethyl)-adamantan-1-yl]-ethoxy substituted ether phospholipids was synthesized and their antileishmanial activity was evaluated against Leishmania infantum amastigotes. The majority of the new analogues were significantly less cytotoxic than miltefosine while, antiparasitic activity depended on the length of the 2-alkyloxy substituent. The most potent compounds were {2-[[[3-(2-hexyloxy-ethyl)-adamant-1-yl]-ethoxy]hydroxyphosphinyloxy]ethyl}-Nu,Nu,Nu-trimethyl-ammonium inner salt (5b) and {2-[[[3-(2-octyloxy-ethyl)-adamant-1-yl]-ethoxy]hydroxyphosphinyloxy]ethyl}-Nu,Nu,Nu-trimethyl-ammonium inner salt (5c).

Synthesis and biophysical characterization of chlorambucil anticancer ether lipid prodrugs.

The synthesis and biophysical characterization of four prodrug ether phospholipid conjugates are described. The lipids are prepared from the anticancer drug chlorambucil and have C16 and C18 ether chains with phosphatidylcholine or phosphatidylglycerol headgroups. All four prodrugs have the ability to form unilamellar liposomes (86-125 nm) and are hydrolyzed by phospholipase A(2), resulting in chlorambucil release. Liposomal formulations of prodrug lipids displayed cytotoxicity toward HT-29, MT-3, and ES-2 cancer cell lines in the presence of phospholipase A(2), with IC(50) values in the 8-36 microM range.

[Synthesis and cytotoxicity of novel phosphorusless analogues of edelfosine].

Modified series of phosphorusless edelfosine analogues bearing the polar heads of aliphatic bases, N,N-dimethylethanolamine and N,N,N(1),N(1)-tetramethylethylenediamine, were synthesized, with the length of the spacer varying from three to four methylene units. The cytotoxic characteristics of the compounds synthesized were studied.

Edelfosine and perifosine induce selective apoptosis in multiple myeloma by recruitment of death receptors and downstream signaling molecules into lipid rafts.

Multiple myeloma (MM) is an incurable B-cell malignancy, requiring new therapeutic strategies. We have found that synthetic alkyl-lysophospholipids (ALPs) edelfosine and perifosine induced apoptosis in MM cell lines and patient MM cells, whereas normal B and T lymphocytes were spared. ALPs induced recruitment of Fas/CD95 death receptor, Fas-associated death domain-containing protein, and procaspase-8 into lipid rafts, leading to the formation of the death-inducing signaling complex (DISC) and apoptosis. TNF-related apoptosis-inducing ligand receptor-1/death receptor 4 (TRAIL-R1/DR4) and TRAIL-R2/DR5, as well as Bid, were also recruited into lipid rafts, linking death receptor and mitochondrial signaling pathways. ALPs induced mitochondrial cytochrome c release. Bcl-X(L) overexpression prevented cytochrome c release and apoptosis. A Fas/CD95-deficient MM subline expressing DR4 and DR5 was resistant to edelfosine. Fas/CD95 retrovirus transduction bestowed edelfosine sensitivity in these cells. A Fas/CD95 mutant lacking part of the intracellular domain was ineffective. Lipid raft disruption prevented ALP-induced Fas/CD95 clustering, DISC formation, and apoptosis. ALP-induced apoptosis was Fas/CD95 ligand (FasL/CD95L) independent. ALP-induced recruitment of death receptors in lipid rafts potentiated MM cell killing by FasL/CD95L and TRAIL. These data uncover a novel lipid raft-mediated therapy in MM involving concentration of death receptors in membrane rafts, with Fas/CD95 playing a major role in ALP-mediated apoptosis.

3D-Quantitative structure-activity relationships of synthetic antileishmanial ring-substituted ether phospholipids.

The application of 2D-NMR spectroscopy and Molecular Modeling in determining the active conformation of flexible molecules in 3D-QSAR was demonstrated in the present study. In particular, a series of 33 flexible synthetic phospholipids, either 2-(4-alkylidene-cyclohexyloxy)ethyl- or omega-cycloalkylidene-substituted ether phospholipids were systematically evaluated for their in vitro antileishmanial activity against the promastigote forms of Leishmania infantum and Leishmania donovani by CoMFA and CoMSIA 3D-QSAR studies. Steric and hydrophobic properties of the phospholipids under study appear to govern their antileishmanial activity against both strains, while the electrostatic properties have no significant contribution. The acknowledgment of these important properties of the pharmacophore will aid in the rational design of new analogues with higher activity.

Import and fate of fluorescent analogs of oxidized phospholipids in vascular smooth muscle cells.

Lipid oxidation is now thought to be an initiating and sustaining event in atherogenesis. Oxidatively fragmented phospholipids, namely 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC) and 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC), present in minimally modified LDL and atherosclerotic lesions, have been reported to elicit a wide range of pathophysiological responses in the cells of the vascular wall. Nevertheless, the question of their potential sites of action and their primary molecular targets remains open. To address this issue, a series of fluorescently labeled analogs, which differ with regard to structure and binding site of the fluorophore, were synthesized and used as tools for studying the uptake, intracellular stability, and distribution of PGPC and POVPC in vascular smooth muscle cells (VSMCs). We demonstrate that in accordance with their lysophospholipid-like structure, these highly similar molecules transferred rapidly either from aqueous phospholipid dispersions or preloaded native LDL into VSMCs, producing disparate fluorescence patterns irrespective of the attached fluorophore. PGPC derivatives were translocated to the lysosomes. In sharp contrast, POVPC analogs were initially captured in the plasma membrane, most likely in consequence of the formation of covalent adducts with free amino and sulfhydryl groups of proteins and phospholipids. LDL internalization is not required for cellular lipid uptake. Collectively, our data provide evidence that oxidized phospholipids, owing to their high exchangeability between lipoproteins and cell membranes, may act within a short time on different cellular sites in VSMCs and affect various lipid and protein components through physical or chemical interactions, which might then serve as starting points for intracellular signaling.

[Synthesis of ethriolophospholipids of acetal type].

New analogues of acetal-type phospholipids were obtained on the basis of ethriol (2-hydroxymethyl-2-ethyl-1,3-propanediol). The starting triol originally was condensed with decanal or dodecanal to form acetals, which were then phosphorylated with tetraethyldiamidophosphorous acid chloride. The amidophosphites were further oxidized with iodosobenzene or sulfurized to the corresponding acetal-type phospholipids and their thio analogues.

Synthesis and structure-activity relationship effects on the tumor avidity of radioiodinated phospholipid ether analogues.

Radioiodinated phospholipid ether analogues have shown a remarkable ability to selectively accumulate in a variety of human and animal tumors in xenograft and spontaneous tumor rodent models. It is believed that this tumor avidity arises as a consequence of metabolic differences between tumor and corresponding normal tissues. The results of this study indicate that one factor in the tumor retention of these compounds in tumors is the length of the alkyl chain that determines their hydrophobic properties. Decreasing the chain length from C12 to C7 resulted in little or no tumor accumulation and rapid clearance of the compound in tumor-bearing rats within 24 h of administration. Increasing the chain length had the opposite effect, with the C15 and C18 analogues displaying delayed plasma clearance and enhanced tumor uptake and retention in tumor-bearing rats. Tumor uptake displayed by propanediol analogues NM-412 and NM-413 was accompanied by high levels of liver and abdominal radioactivity 24 h postinjection to tumor-bearing rats. Addition of a 2-O-methyl moiety to the propanediol backbone also retarded tumor uptake significantly. A direct comparison between NM-404 and its predecessor, NM-324, in human PC-3 tumor bearing immune-compromised mice revealed a dramatic enhancement in both tumor uptake and total body elimination of NM-404 relative to NM-324. On the basis of imaging and tissue distribution studies in several rodent tumor models, the C18 analogue, NM-404, was chosen for follow-up evaluation in human lung cancer patients. Preliminary results have been extremely promising in that selective uptake and retention of the agent in tumors is accompanied by rapid clearance of background radioactivity from normal tissues, especially those in the abdomen. These results strongly suggest that extension of the human trials to include other cancers is warranted, especially when NM-404 is radiolabeled with iodine-124, a new commercially available positron-emitting isotope. The relatively long physical half-life of 4 days afforded by this isotope appears well-suited to the pharmacodynamic profile of NM-404.

Synthesis and biological evaluation of novel steroid-modified ether phospholipids.

Platelet activating factor is one of the most potent inflammatory ether phospholipid mediators known and structurally modified analogues are of considerable interest as potential therapeutic preparations. Inspired by the proposed structure for a novel endogenous hydroxy-PAF analogue isolated recently from gingival crevicular fluid, we designed and prepared two novel steroid-modified ether phospholipids. These two novel compounds exhibit marked chemical and biological similarities to their endogenous prototype and they antagonize it being less active in inducing washed platelet aggregation through PAF receptors.

Fluorescent phenylpolyene analogues of the ether phospholipid edelfosine for the selective labeling of cancer cells.

Edelfosine (ET-18-OCH3), a synthetic antitumor ether lipid, is taken up by malignant but not by normal cells, triggering apoptosis in a large variety of human tumor cells. The synthesis of the first fluorescent edelfosine analogue (6), with apoptotic activity comparable to that of the parent drug, is described. Fluorescence microscopy experiments show that 6 selectively labels human cancer cells, accumulating into specific domains of the plasma membrane.

Enzymatic release of antitumor ether lipids by specific phospholipase A2 activation of liposome-forming prodrugs.

An enzymatically activated liposome-based drug-delivery concept involving masked antitumor ether lipids (AELs) has been investigated. This concept takes advantage of the cytotoxic properties of AEL drugs as well as the membrane permeability enhancing properties of these molecules, which can lead to enhanced drug diffusion into cells. Three prodrugs of AELs (proAELs) have been synthesized and four liposome systems, consisting of these proAELs, were investigated for enzymatic degradation by secretory phospholipase A(2) (sPLA(2)), resulting in the release of AELs. The three synthesized proAELs were (R)-1-O-hexadecyl-2-palmitoyl-sn-glycero-3-phosphocholine (1-O-DPPC), (R)-1-O-hexadecyl-2-palmitoyl-sn-glycero-3-phosphoethanolamine poly(ethylene glycol)(350) (1-O-DPPE-PEG(350)), and 1-O-DPPE-PEG(2000) of which 1-O-DPPC was the main liposome component. All three phospholipids were synthesized from the versatile starting material (R)-O-benzyl glycidol. A phosphorylation method, employing methyl dichlorophosphate, was developed and applied in the synthesis of two analogues of (R)-1-O-hexadecyl-2-palmitoyl-sn-glycero-3-phosphoethanolamine poly(ethylene glycol). Differential scanning calorimetry has been used to investigate the phase behavior of the lipid bilayers. A release study, employing calcein encapsulated in non-hydrolyzable 1,2-bis-O-octadecyl-sn-glycero-3-phosphocholine (D-O-SPC) liposomes, showed that proAELs, activated by sPLA(2), perturb membranes because of the detergent-like properties of the released hydrolysis products. A hemolysis investigation was conducted on human red blood cells, and the results demonstrate that proAEL liposomes display a very low hemotoxicity, which has been a major obstacle for using AELs in cancer therapy. The results suggest a possible way of combining a drug-delivery and prodrug concept in a single liposome system. Our investigation of the permeability-enhancing properties of the AEL molecules imply that by encapsulating conventional chemotherapeutic drugs, such as doxorubicin, in liposomes consisting of proAELs, an increased effect of the encapsulated drug might be achievable due to an enhanced transmembrane drug diffusion.

1-O-Hexadecyl-2-desoxy-2-amino-sn-glycerol, a substrate for human sphingosine kinase.

The substrate specificity of human sphingosine kinase was investigated using a bacterially expressed poly(His)-tagged protein. Only the D-erythro isomer of the sphingoid bases, sphinganine and sphingenine, was effectively phosphorylated. Long chain 1-alkanols, alkane-1,2-diols, 2-amino-1-alkanol or 1-amino-2-alkanol and short chain 2-amino-1,3-alkanediols were very poor substrates, indicating that the kinase is recognizing the chain length and the position of the amino and secondary hydroxy group. A free hydroxy group at carbon 3 is not a prerequisite, however, since 1-O-hexadecyl-2-desoxy-2-amino-sn-glycerol was an efficient substrate with an apparent K(m) value of 3.8 microM (versus 15.7 microM for sphingenine). This finding opens new perspectives to design sphingosine kinase inhibitors. It also calls for some caution since it cannot be excluded that this ether lipid analogue is formed from precursors that are frequently used in research on platelet activating factor or from phospholipid analogues which are less prone to degradation.

Supramolecular self-assembling properties of membrane-spanning archaeal tetraether glycolipid analogues.

The self-assembling properties of a new series of archaeal tetraether glycolipid analogues 1-6 that are characterized by a bipolar architecture with two similar or different glycosidic and/or phosphate polar heads and a lipid core possessing a cyclopentane unit and/or branched chains were studied by means of differential scanning calorimetry, optical microscopy, X-ray scattering, freeze-fracture electron microscopy and dynamic light scattering. Unsymmetrical phosphate derivatives 1 and 2 spontaneously formed thermostable multilamellar and unilamellar vesicles in which most of the bipolar lipids adopted a trans-membrane conformation, as revealed by freeze-fracture electron microscopy. Supramolecular aggregates of neutral glycolipids 3-6 were found to depend on both the saccharidic polar heads and the chain composition. The presence of one glycosidic residue with rather marked hydrophilic properties, such as the lactosyl moiety, was required to allow the formation of multilamellar vesicles. Surprisingly, the introduction of a cyclopentane unit in the bridging chain was able to induce an apparent two-by-two membrane association: this unusual behaviour might be the result of unsymmetrical interfacial properties of the lipid layer caused by the presence of the cyclopentane unit.

Core aldehydes of alkyl glycerophosphocholines in atheroma induce platelet aggregation and inhibit endothelium-dependent arterial relaxation.

Plaque disruption with superimposed thrombosis is considered to be responsible for precipitating acute coronary syndrome. We identified sn-1-alkyl- and sn-1-acyl-type glycerophosphocholine (GroPCho) core aldehydes from human atheromas and demonstrated their activities on platelets and arteries. The naturally occurring core aldehydes were identified and quantified in relation to synthetic standards by high performance liquid chromatography with on-line electrospray mass spectrometry. 1-O-Hexadecyl-2-(5-oxovaleroyl)-sn-GroPCho (C(5) alkyl GroPCho core aldehyde), occurring in atheroma at less than 0.1% of total phosphatide, induced aggregation of washed rabbit platelets (50% effective dose was approximately 50 nM). Aggregations induced by C(5) alkyl GroPCho core aldehydes were completely inhibited by two different platelet-activating factor receptor antagonists. 1-Palmitoyl-2-(5-oxovaleroyl)-sn-GroPCho (C(5) acyl GroPCho core aldehyde) induced platelet shape change, but not aggregation. By contrast, 10 microM C(5) alkyl and C(5) acyl GroPCho core aldehydes both inhibited endothelium-dependent relaxation of rabbit artery by 50% (endothelium-independent relaxation was not affected). The present demonstration of platelet aggregation by physiologically relevant concentrations of alkyl GroPCho core aldehydes suggests that alkyl GroPCho core aldehyde generated in atheroma could be involved in precipitating acute coronary events, in which thrombus formation following lipid-rich plaque disruption plays an important role.

Preparation of optically pure 4-(hydroxymethyl)-2-pentadecyl-1,3-dioxolanes and their corresponding phosphodiester ether lipid derivatives.

Semi-preparative HPLC on a chiral stationary phase (Chiracel OD) was utilized in the course of this synthesis to separate the four possible diastereomers [cis-(2R,4S)-2a, trans-(2S,4S)-2b, cis-(2S,4R)-2a', and trans-(2R,4R)-2b'] of a 2,4-disubstituted-1,3-dioxolane into optically pure forms (100% de, 100% ee). The syntheses of phosphodiester head group derivatives from each of these four conformationally constrained diastereomeric dioxolanes gave phospholipids which are monocyclic ether lipid analogs. First, the series of four [[(2-pentadecyl-1,3-dioxolan-4-yl)methyl]oxy]phosphocholines 5 were synthesized to give optically pure conformationally constrained analogues of ET-16-OCH(3). A head group variation was also demonstrated by the syntheses of the four diastereomeric [[(2-pentadecyl-1,3-dioxolan-4-yl)-methyl]oxy]phospho-beta-(N-methylmorpholino)ethanols 6.