Lipid Analogues Research Articles

Modification of bipolar lipid conformation at the air/water interface by a single stereochemical variation

This article describes the design and the synthesis of two analogues of archaeal bipolar lipids that differ only by the configuration of a single stereogenic centre on a glyceryl moiety. The corresponding comparative physicochemical study by tensiometry/ellipsometry and atomic force microscopy provided a set of data that points out the effect of this single stereochemical variation. The two epimers revealed different conformations at the air/water interface resulting in a more or less tendency for a bent or a stretched conformation. It strengthens the importance of the stereochemistry in such bipolar lipid packing.

Stereoselective synthesis of perdeuterated phytanic acid, its phospholipid derivatives and their formation into lipid model membranes for neutron reflectivity studies

We describe a straightforward method, for synthesis of large scale (gram quantities) of highly deuterated phytanic acid from commercially available phytol while preserving the stereochemistry around the chiral centres. The subsequent synthesis of tail-deuterated analogues of the archeabacterial membrane lipids 1,2-di(3RS,7R,11R-phytanyl)-sn-glycero-3-phosphocholine (DPEPC) and 1,2-di(3RS,7R,11R-phytanoyl)-sn-glycero-3-phosphocholine (DPhyPC) from perdeuterated phytanic acid is also described. Both lipids were employed in construction of two different model membranes, namely Langmuir monolayers and a tethered bilayer membrane (TBM) on a solid substrate, characterised by pressure area isotherm and neutron reflectometry techniques. At 10mN/m pressure the head-group thickness of both monolayers was similar while the thickness of the tail region was significantly larger for tail-deuterated DPhyPC, which was evident from a smaller area per molecule. At 20mN/m the thickness of the head and tail regions in both lipids was comparable, yet the area per molecule of tail-deuterated DPhyPC was 10% smaller than tail-deuterated DPEPC. In the TBM bilayer model membrane, the thickness of the lipid tails in both inner and outer leaflets was 8.2Å, giving a total of 16.4Å. Deuteration enabled unambiguous determination of the relative proportion of the hydrogenous tether, phospholipid and subphase.

In Vitro Intestinal Co-Culture Cell Model to Evaluate Intestinal Absorption of Edelfosine Lipid Nanoparticles

Nanotechnology is providing a new therapeutic paradigm by enhancing drug efficacy and preventing side-effects. Edelfosine is a synthetic ether lipid analogue of platelet activating factor with high antitumor activity. The encapsulation of this potent antitumor drug in lipid nanoparticles increases its oral bioavailability; moreover, it prevents the hemolytic and gastrointestinal side-effects of the free drug. The literature points towards lymphatic absorption of lipid nanoparticles after oral administration, and previous in vitro and in vivo studies stress the protection against toxicity that these nanosystems provide. The present study is intended to assess the permeability of lipid nanoparticles across the intestinal barrier. Caco-2 monoculture and Caco-2/Raji co-culture were used as in vitro models of enterocytes and Microfold cells respectively. Results showed that free drug is internalized and possibly metabolized in enterocytes. These results do not correlate with those observed in vivo when edelfosine-lipid nanoparticles were administered orally in mice, which suggests that the microfold model is not a good model to study the absorption of edelfosine-lipid nanoparticles across the intestinal barrier in vitro.

Lipid analogues as potential drugs for the regulation of mitochondrial cell death

The mitochondrion plays an important role in the production of energy as ATP, the regulation of cell viability and apoptosis, and the biosynthesis of major structural and regulatory molecules, such as lipids. During ATP production, reactive oxygen species are generated that alter the intracellular redox state and activate apoptosis. Mitochondrial dysfunction is a well-recognized component of the pathogenesis of diseases such as cancer. Understanding mitochondrial function, and how this is dysregulated in disease, offers the opportunity for the development of drug molecules to specifically target such defects. Altered energy metabolism in cancer, in which ATP production occurs largely by glycolysis, rather than by oxidative phosphorylation, is attributable in part to the up-regulation of cell survival signalling cascades. These pathways also regulate the balance between pro- and anti-apoptotic factors that may determine the rate of cell death and proliferation. A number of anti-cancer drugs have been developed that target these factors and one of the most promising groups of agents in this regard are the lipid-based molecules that act directly or indirectly at the mitochondrion. These molecules have emerged in part from an understanding of the mitochondrial actions of naturally occurring fatty acids. Some of these agents have already entered clinical trials because they specifically target known mitochondrial defects in the cancer cell.

A divergent approach to the synthesis of iGb3 sugar and lipid analogues via a lactosyl 2-azido-sphingosine intermediate

Isoglobotrihexosylceramide (iGb3, 1) is an immunomodulatory glycolipid that binds to CD1d and is presented to the T-cell receptor (TCR) of invariant natural killer T (iNKT) cells. To investigate how modifications to the lipid tail or terminal sugar residue of iGb3 influence iNKT cell activity, we developed an efficient and divergent synthetic route that provided access to both sugar and lipid iGb3 analogues which utilised a lactosyl 2-azido-sphingosine derivative as a common intermediate. In this way, iGb3 (1) and the unprecedented analogues 6'''-deoxy-iGb3-sphingosine 2, 6'''-deoxy-iGb3-sphinganine 3, C12 N-acyl iGb3 4 and C20:2 N-acyl iGb3 5 were prepared so that key structure-activity relationships can be explored.

Chemo-enzymatic synthesis of rac 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine and its analogues.

The synthesis of rac 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholines (6a-c), blood platelet activating ether lipid analogues has been achieved in a four-step sequence from epichlorohydrin (1). Etherification of epichlorohydrin with different alcohols namely tetradecyl (2a), hexadecyl (2b) and octadecyl (2c) alcohols gave glycidyl ethers (3a-c) with 78-80% yields. The second step involved opening of the epoxide by acetic anhydride to give acetylated products (4a-c, 76-78% yield), which were subsequently hydrolyzed selectively, a key step of the method employing a 1,3 specific lipase to obtain rac 1-O-alkyl-2- acetylglycerol (5a-c) with 45-50% yields. The hydrolyzed products (5a-c) were phosphorylated to obtain rac 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholines (6a-c) in 80-85% yields.

Evaluation of the effect of miltefosine on Trichomonas vaginalis

Trichomonas vaginalis causes trichomoniasis in humans, a sexually transmitted disease commonly treated with metronidazole (MTZ). MTZ is known to cause undesirable side effects, and MTZ-resistant parasites have been reported. Thus, the development of an alternative treatment is desirable. Miltefosine (MLT) is an alkylphosphocholine synthetic lipid analogue that displays antiparasitic activity against Leishmania, Trypanosoma cruzi, Entamoeba histolytica, Acanthamoeba spp., Giardia lamblia, T. vaginalis and some fungi. Moreover, it has been used for oral treatment of visceral leishmaniosis in several countries. Here, we analysed the MLT-induced antiproliferative effect on T. vaginalis as well its effect on the fine structure and viability of the parasite. We observed a dose-dependent effect with an IC50 of 14.5 and 20 μM after 24 and 48 h, respectively. Furthermore, reversibility assays demonstrated that new incubations were necessary in order to maintain the antiproliferative effect. Ultrastructural analyses demonstrated that MLT induced several alterations, including the appearance of wrinkled and rounded cells, membrane blebbing, intense vacuolization and nuclear condensation, all indicative of cell death by apoptosis. In addition, the quantitative analyses of the viability assays using combined markers of live and dead cells demonstrated that treatment with the IC50 concentration of MLT significantly reduced the number of viable parasites compared with untreated cells. Taken together, these observations suggest that MLT is a promising compound for the treatment of trichomoniasis.

High‐Fidelity Protein Targeting into Membrane Lipid Microdomains in Living Cells

Lipid analogues carrying three nitrilotriacetic acid (tris-NTA) head groups were developed for the selective targeting of His-tagged proteins into liquid ordered (lo ) or liquid disordered (ld ) lipid phases. Strong partitioning into the lo phase of His-tagged proteins bound to tris-NTA conjugated to saturated alkyl chains (tris-NTA DODA) was achieved, while tris-NTA conjugated to an unsaturated alkyl chain (tris-NTA SOA) predominantly resided in the ld phase. Interestingly, His-tag-mediated lipid crosslinking turned out to be required for efficient targeting into the lo phase by tris-NTA DODA. Robust partitioning into lo phases was confirmed by using viral lipid mixtures and giant plasma membrane vesicles. Moreover, efficient protein targeting into lo and ld domains within the plasma membrane of living cells was demonstrated by single-molecule tracking, thus establishing a highly generic approach for exploring lipid microdomains in situ.

High-Fidelity Protein Targeting into Membrane Lipid Microdomains in Living Cells

Lipid analogues carrying three nitrilotriacetic acid (tris-NTA) head groups were developed for the selective targeting of His-tagged proteins into liquid ordered (lo) or liquid disordered (ld) lipid phases. Strong partitioning into the lo phase of His-tagged proteins bound to tris-NTA conjugated to saturated alkyl chains (tris-NTA DODA) was achieved, while tris-NTA conjugated to an unsaturated alkyl chain (tris-NTA SOA) predominantly resided in the ld phase. Interestingly, His-tag-mediated lipid crosslinking turned out to be required for efficient targeting into the lo phase by tris-NTA DODA. Robust partitioning into lo phases was confirmed by using viral lipid mixtures and giant plasma membrane vesicles. Moreover, efficient protein targeting into lo and ld domains within the plasma membrane of living cells was demonstrated by single-molecule tracking, thus establishing a highly generic approach for exploring lipid microdomains in situ.

The Immunobiology of Toll-Like Receptor 4 Agonists

Lipopolysaccharide (LPS, endotoxin) is a structural component of the gram-negative outer membrane. The lipid A moiety of LPS binds to the LPS receptor complex expressed by leukocytes, endothelial cells, and parenchymal cells and is the primary component of gram-negative bacteria that is recognized by the immune system. Activation of the LPS receptor complex by native lipid A induces robust cytokine production, leukocyte activation, and inflammation, which is beneficial for clearing bacterial infections at the local level but can cause severe systemic inflammation and shock at higher challenge doses. Interestingly, prior exposure to LPS renders the host resistant to shock caused by subsequent LPS challenge, a phenomenon known as endotoxin tolerance. Treatment with lipid A has also been shown to augment the host response to infection and to serve as a potent vaccine adjuvant. However, the adverse effects associated with the pronounced inflammatory response limit the use of native lipid A as a clinical immunomodulator. More recently, analogs of lipid A have been developed that possess attenuated proinflammatory activity but retain attractive immunomodulatory properties. The lipid A analog monophosphoryl lipid A exhibits approximately 1/1,000th of the toxicity of native lipid A but retains potent immunoadjuvant activity. As such, monophosphoryl lipid A is currently used as an adjuvant in several human vaccine preparations. Because of the potency of lipid A analogs as immunoadjuvants, numerous laboratories are actively working to identify and develop new lipid A mimetics and to optimize their efficacy and safety. Based on those characteristics, lipid A analogs represent an attractive family of immunomodulators.

Synthesis of a Comprehensive Polyprenol Library for the Evaluation of Bacterial Enzyme Lipid Substrate Specificity

Polyprenols, a type of universal glycan lipid carrier, play important roles for glycan bio-assembly in wide variety of living systems. Chemical synthesis of natural polyisoprenols such as undecaprenol and dolichols, but especially their homologs, could serves as a powerful molecular tool to dissect and define the functions of enzymes involved in glycan biosynthesis. In this paper, we report an efficient and reliable method to construct this type of hydrophoic molecule through a base-mediated iterative coupling approach using a key bifunctional (Z, Z)-diisoprenyl building block. The ligation with N-acetyl-D-glactosamine (GalNAc) with a set of the synthesized lipid analogs forming polyprenol pyrophosphate linked GalNAc (GalNAc-PP-lipid) conjugates is also demonstrated.

The supramolecular structure is decisive for the immunostimulatory properties of synthetic analogues of a mycobacterial lipid in vitro

Identification of new vaccine adjuvants with immunopotentiating properties commonly involves in vitro evaluations of candidate compounds for their ability to stimulate cells of the immune system. Subsequent elaborate experiments are then performed on only the positive candidates. Here we show how this strategy may miss good candidates due to context-dependent supramolecular characteristics of the candidate compounds, since both a specific molecular structure and the correct presentation of specific parts of the compounds are required for successful stimulation of the cells. Nevertheless, the supramolecular structure is rarely evaluated although changes in this structure may have a drastic impact on the presentation of the compounds to the cells. Synthetic analogues of the mycobacterial cell wall lipid monomycoloyl glycerol (MMG) possess immunopotentiating properties, but their biophysical characteristics are largely unresolved and the structural features determining their immunoactivating properties have been poorly explored. In the present study, we demonstrate that the immunostimulatory activity in vitro correlates with the supramolecular characteristics of the self-assembled MMG nanostructures. Thus, a series of MMG analogues displaying different stereochemistry in the hydrophobic moiety and the polar headgroup were designed and synthesized with different alkyl chain lengths. Stimulation of human monocyte-derived dendritic cells in vitro was clearly dependent on the stereochemistry of the hydrophobic part and on the alkyl chain length but not on the stereochemistry of the hydrophilic glycerol moiety. Small-angle X-ray scattering (SAXS) analysis showed that the immunoactivating analogues self-assembled into lamellar phases whereas the biologically inert analogues adopted inverse hexagonal phases. Langmuir monolayers confirmed that analogues with opposite lipid acid configurations displayed different packing modes. These data demonstrate that the biophysical properties and the lipid molecular structure are major determinants for the ability of the MMG analogues to activate antigen-presenting cells. Our findings emphasize the importance of investigating the biophysical and structural properties when assessing the effect of adjuvants in vitro.

Esterification Catalysis by Pyridinium p-Toluenesulfonate Revisited—Modification with a Lipid Chain for Improved Activities and Selectivities

The lipid analogs of pyridinium p-toluenesulfonate (PPTS) were examined for catalysis of the condensation of an equimolar mixture of carboxylic acids and alcohols under mild conditions without removal of water. Although PPTS is a poor catalyst, the introduction of a lipid chain and nitro group significantly improved the activity of PPTS and led to selectivity at suppressing the elimination side reactions of alcohols. 2-Oleamido-5-nitro-pyridinium p-toluenesulfonate (6) is a lead catalyst that promoted various esterification reactions with yields up to 99%. [Supplementary materials are available for this article. Go to the publisher's online edition of Synthetic Communications® for the following free supplemental resource: Full experimental and spectral details.]

Translational Molecular Imaging of Prostate Cancer.

Prostate cancer is a heterogeneous disease, and its management is now evolving to become more personalized and to incorporate new targeted therapies. With these new changes comes a demand for molecular imaging techniques that can not only detect disease but also assess biology and treatment response. This review article summarizes current molecular imaging approaches in prostate cancer (e.g. 99mTc bone scintigraphy and 18F-fluorodeoxyglucose positron emission tomography) and highlights emerging clinical and preclinical imaging agents, with an emphasis on mechanism and clinical application. Emerging agents at various stages of clinical translation include radiolabeled analogs of lipid, amino acid, and nucleoside metabolism, as well as agents more specifically targeting prostate cancer biomarkers including androgen receptor, prostate-specific membrane antigen and others. We also highlight new techniques and targeted contrast agents for magnetic resonance imaging and spectroscopy. For all these imaging techniques, a growing and important unmet need is for well-designed prospective clinical trials to establish clear indications with clinical benefit in prostate cancer.

Long and Short Lipid Molecules Experience the Same Interleaflet Drag in Lipid Bilayers

Membrane interleaflet viscosity ηe affects tether formation, phase separation into domains, cell shape changes, and budding. Contrary to the expected contribution to interleaflet coupling from interdigitation, the slide of lipid patches in opposing monolayers conferred the same value ηe≈3×10(9) J s m-4 for the friction experienced by the ends of both short and long chain fluorescent lipid analogues. Consistent with the weak dependence of the translational diffusion coefficient on lipid length, the in-layer viscosity was, albeit length dependent, much smaller than ηe.

Defined lipid analogues induce transient channels to facilitate drug-membrane traversal and circumvent cancer therapy resistance

Design and efficacy of bioactive drugs is restricted by their (in)ability to traverse cellular membranes. Therapy resistance, a major cause of ineffective cancer treatment, is frequently due to suboptimal intracellular accumulation of the drug. We report a molecular mechanism that promotes trans-membrane movement of a stereotypical, widely used anti-cancer agent to counteract resistance. Well-defined lipid analogues adapt to the amphiphilic drug doxorubicin, when co-inserted into the cell membrane, and assemble a transient channel that rapidly facilitates the translocation of the drug onto the intracellular membrane leaflet. Molecular dynamic simulations unveiled the structure and dynamics of membrane channel assembly. We demonstrate that this principle successfully addresses multi-drug resistance of genetically engineered mouse breast cancer models. Our results illuminate the role of the plasma membrane in restricting the efficacy of established therapies and drug resistance - and provide a mechanism to overcome ineffectiveness of existing and candidate drugs.

Membrane curvature and cholesterol effects on lipids packing and spin-labelled lipids conformational distributions

Nitroxide spin-labelled lipid analogues are often used to study model membrane properties using EPR spectroscopy. Whereas in liquid phase membranes the spin label assumes, on average, its putative location, in gel phases and frozen membrane, depending on its position along the acyl chain, it may exhibit a different average location. Here we used 2H three-pulse Electron Spin Echo Envelope Modulation (ESEEM) of phospholipid spin probes, combined with various deuteration schemes to detect the effect of the model membrane curvature and cholesterol on vertical migrations of the spin label. We compared large and small unilamellar 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) vesicles with and without cholesterol (10%). The vertical displacement of the spin label was manifested as an apparently flat trans-membrane profile of water concentration and of label proximity to the head group choline. The spin-label propensity to migrate was found to increase with vesicle curvature and decrease in the presence of cholesterol. This in turn reflects the effect of packing and ordering of the membrane lipids. The results show that in curved vesicles lacking cholesterol, the label attached to carbon 16 may travel as far high along the membrane normal as the location of the label on carbon 5, due to the presence of U-shaped lipid conformations. This phenomenon must be taken into account when using spin-labelled lipids as membrane depth markers or to trace trans-membrane profiles.

Characterization of the immunostimulatory activity and safety of a vaccine adjuvant de-O-acylated lipooligosaccharide (P4520)

Abstract Lipopolysaccharide (LPS), a constituent of the outer membrane of Gram-negative bacteria elicits strong immunopathological reactions observed during bacterial infection, and the lipid A moiety of LPS is responsible for the immunostimulatory activity. Lipid A exerts its biological activity by sending signals via toll-like receptor 4 present on immune cells. Many natural and synthetic analogues of lipid A have been developed as vaccine adjuvant. In this study, we evaluated the immunostimulatory activities of de-O-acylated lipooligosaccharide (deacyl-LOS) on murine and human immune cells in comparison with monophosphoryl lipid A. The results indicated that deacyl-LOS is more potent than MPL in inducing cytokine secretion and activation of monocytes and dendritic cells, which was confirmed to be mediated by TLR4. It also induced allogeneic T cell responses and activated B cell proliferation. The acute toxicity test showed that deacyl-LOS did not cause any pathological effects or death at 0.25, 0.5 or 1 mg per kg body weight in mice. These data suggest a potential of deacyl-LOS as a safe and effective vaccine adjuvant.

Abstract 3382: Formation of transient membrane channels targets doxorubicin resistance.

Abstract Poor accumulation of anti-cancer drugs in tumor cells is a major limitation in clinical cancer therapy. The main barrier for a drug to traverse through the body and reach its intracellular target is the plasma membrane. A typical (candidate) drug is therefore small, amphiphilic and of limited charge. Still, membrane traversal remains a highly inefficient and impeding process. In clinical oncology in particular there is an urgent need for new ways to improve drug efficacy and reduce toxicity. We have elucidated a mechanism that effectively accelerates membrane translocation of the stereotypical and widely used amphiphilic drug, doxorubicin. Well-defined short-chain sphingolipids, when co-inserted into the membrane, diminish the barrier for doxorubicin translocation. In vicinity of doxorubicin, the lipid analogues rapidly self-assemble at nanosecond timescale, and form a small, transient membrane channel. As a result, the doxorubicin drug readily translocates the membrane, thereby reducing the energetic barrier significantly by two-fold. Monte-Carlo based full-atom simulations revealed the structure and dynamics of channel formation at molecular detail. By means of a high-throughput screening approach of classical and targeted anti-cancer agent libraries, we identified various anti-cancer drugs in addition to doxorubicin, which share critical molecular characteristics, like defined amphiphilicity. Short-chain sphingolipids enhance cellular accumulation of these anti-cancer compounds similar to doxorubicin. Guided by these mechanistic insights, we applied the concept of facilitated doxorubicin traversal in genetically engineered WAPcre;EcadF/F;p53F/F mouse models for breast cancer. These tumors are multi-drug resistant to conventional and targeted anti-cancer agents. Co-administration of the sphingolipid analogue GC with liposomal doxorubicin effectively overcame drug resistance. While toxicity and normal tissue exposure reduced, GC caused elevated levels of intracellular doxorubicin in the tumor, improved tumor growth inhibition and significantly prolonged survival. Thus, a strategy of GC-mediated doxorubicin translocation was the only therapeutic approach that generated a sustained doxorubicin anti-tumor response. Notably, enhanced doxorubicin translocation was strongest over membranes of the tumor, a spectacular observation confirmed in vitro. We demonstrate that composition and local organization of the plasma membrane determine the efficiency of membrane channel formation. In conclusion, transient membrane channels target the tumor cell membrane to overcome multi-drug resistance. Our results illuminate a critical role of the (tumor) plasma membrane in restricting the efficacy of anti-cancer drugs and its contribution to multi-drug resistance. Moreover, our findings present a mechanism to address these limitations of (candidate) drugs in a clinically applicable way. Citation Format: Albert J. van Hell, Manuel Melo, Wim van Blitterswijk, Tim Dijkema, Lilia Pedrosa, Gerben Koning, Siewert Jan Marrink, Jos Jonkers, Marcel Verheij. Formation of transient membrane channels targets doxorubicin resistance. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3382. doi:10.1158/1538-7445.AM2013-3382

Phase I study of OM-174, a lipid A analogue, with assessment of immunological response, in patients with refractory solid tumors

BackgroundLipids A, the lipophilic partial structure of lipopolysaccharides, induce regression of several tumor types in animal models. Rather than exerting direct cytotoxic effect, these compounds trigger the immune system which in turn stimulates secretion of cytokines, and activates the inducible nitric oxide synthase, as well as immune cell infiltration of tumors. OM-174 is an analogue of lipid A with dual action on Toll-like receptors 2 and 4. In an experimental model of peritoneal carcinomatosis induced in BDIX rats by intraperitoneal injection of syngeneic PROb colon cancer cells, it induced a complete regression of tumors. The present phase I trial was conducted to determine the maximum tolerated dose, the recommended phase II dose and biological response associated with OM-174 administered as intravenous infusion.MethodsPatients received OM-174 twice weekly for a total of 5, 10 or 15 injections of either 600, 800 or 1000 μg/m2. Blood samples for pharmacokinetic analysis and cytokine dosages were collected. NK cells activity and Toll-like receptors 4 polymorphism analysis were also performed.ResultsSeventeen patients were included. The highest dose administered was 1000 μg/m2 repeated in 15 injections. The most common toxicities were a chills, fever, nausea/vomiting, diarrhea, fatigue and headache. No patient experienced haematological side effects. As no dose limiting toxicity was observed, despite a grade 3 respiratory complication, the maximal tolerated dose and recommended dose were not established. Three patients exhibited disease stabilization with a mean duration of 4 months. Pharmacokinetic profile of OM-174 was characterized by a low distribution volume and clearance. Analysis of TLR 4 polymorphysm showed that most (16/17) patients carried the wild type alleles. A progressive increase in NK cell number and activity was observed only in patients receiving 1000 μg/m2 of OM-174. A peak of IL-8 and IL-10 concentrations were observed after each OM-174 injection. Peaks of TNF-alpha and IL-6 concentrations were detected after the first infusion and decreased progressively suggesting tolerance.ConclusionOM-174 therapy was well tolerated at biologically active concentrations. Whereas the recommended dose was not determined, further studies are planned in combination with chemotherapy as animal models suggest a strong synergistic antitumor effect.Trial registrationNCT01800812 (ClinicalTrials.gov Identifier).