Lipid Analogues Research Articles

In Situ Synthesis of Lipid Analogues Leading to Artificial Cell Growth and Division

AbstractThe bottom‐up construction of an artificial cell model system starting from inanimate components helps people better understand the working mechanism of cells. Phospholipids are natural components of the cell membrane system, which are widely used to construct artificial cells. However, it is difficult to synthesize natural phospholipids in situ. Chemically synthesized lipid analogues are similar in structure to natural phospholipids, so they can be used to mimic phospholipids to build vesicles. Here, four methods of the chemical synthesis of lipid analogues (including enzyme reactions, click chemistry reactions, native chemical ligation reactions, imine condensation and decomposition reactions) are described. The construction of artificial cells based on the lipid analogues, and their growth and division are also summarized. Finally, the challenges and future directions in the field of construction and division of artificial cells based on lipid analogues are proposed.

Molecular‐scale structure of a high‐curvature membrane

Cells utilize molecular machines to form and remodel their membrane‐defined compartments’ compositions, shapes, and connections. The regulated activity of these membrane remodeling machines drives processes like vesicular traffic and organelle homeostasis. Although molecular patterning within membranes is essential to cellular life, characterizing the composition and structure of realistic biological membranes on the molecular length scale remains a challenge, particularly during membrane shape transformations. Here, we employed an ESCRT‐III protein coating model system to investigate how membrane‐binding proteins bind to and alter the structural patterns within lipid bilayers. We observe leaflet‐level and localized lipid structures within a constricted and thinned membrane nanotube. To map the fine structure of these membranes, we compared simulated bilayer nanotubes with experimental cryo‐EM reconstructions of native membranes and membranes containing halogenated lipid analogs. Halogenated lipids scatter electrons more strongly, and analysis of their surplus scattering enabled us to estimate the concentrations of lipids within each leaflet and to estimate lipid shape and sorting changes induced by high curvature and lipid‐protein interactions. Specifically, we found that cholesterol enriched within the inner leaflet due to its spontaneous curvature, while acidic lipids enriched in the outer leaflet due to electrostatic interactions with the protein coat. The docosahexaenoyl (DHA) polyunsaturated chain‐containing lipid SDPC enriched strongly at membrane‐protein contact sites. Simulations and imaging of brominated SDPC showed how a pair of phenylalanine residues opens a hydrophobic defect in the outer leaflet and how DHA tails stabilize the defect and “snorkel” up to the membrane surface to interact with these side chains. This highly curved nanotube differs markedly from protein‐free, flat bilayers in leaflet thickness, lipid diffusion, and other structural asymmetries with implications for our understanding of membrane mechanics.

Plasmalogenic Lipid Analogs as Platelet-Activating Factor Antagonists: A Potential Novel Class of Anti-inflammatory Compounds.

Plasmalogens and Platelet-Activating Factor (PAF) are both bioactive ether phospholipids. Whereas plasmalogens are recognized for their important antioxidant function and modulatory role in cell membrane structure and dynamics, PAF is a potent pro-inflammatory lipid mediator known to have messenger functions in cell signaling and inflammatory response. The relationship between these two types of lipids has been rarely studied in terms of their metabolic interconversion and reciprocal modulation of the pro-inflammation/anti-inflammation balance. The vinyl-ether bonded plasmalogen lipid can be the lipid sources for the precursor of the biosynthesis of ether-bonded PAF. In this opinion paper, we suggest a potential role of plasmalogenic analogs of PAF as modulators and PAF antagonists (anti-PAF). We discuss that the metabolic interconversion of these two lipid kinds may be explored towards the development of efficient preventive and relief strategies against PAF-mediated pro-inflammation. We propose that plasmalogen analogs, acting as anti-PAF, may be considered as a new class of bioactive anti-inflammatory drugs. Despite of the scarcity of available experimental data, the competition between PAF and its natural plasmalogenic analogs for binding to the PAF receptor (PAF-R) can be proposed as a mechanistic model and potential therapeutic perspective against multiple inflammatory diseases (e.g., cardiovascular and neurodegenerative disorders, diabetes, cancers, and various manifestations in coronavirus infections such as COVID-19).

Multifunctional Role of Lipids in Modulating the Tumorigenic Properties of 4T1 Breast Cancer Cells.

Tumor growth and progression are linked to an altered lipid metabolism in the tumor microenvironment (TME), including tumor cells and tumor-associated macrophages (TAMs). A growing number of lipid metabolism targeting drugs have shown efficacy in anti-tumor therapy. In addition, exogenously applied lipids and lipid analogues have demonstrated anti-tumor activities in several cancers, including breast cancer. In this study, we investigated the anti-tumor efficacies of the natural lipids palmitic acid (PA), sphingomyelin (SM), ceramide (Cer) and docosahexaenoic acid (DHA) on breast cancer cells. All tested lipids reduced the malignancy of breast cancer cells in vitro by impairing cell proliferation, migration and invasiveness. PA showed superior anti-tumor properties, as it additionally impaired cancer cell viability by inducing apoptosis, without affecting healthy cells. Co-culture experiments further demonstrated that Cer and PA reduced the immunosuppressive phenotype of M2 macrophages and the M2 macrophage-promoted the epithelial–mesenchymal transition (EMT) and migration of breast cancer cells. At the molecular level, this coincided with the up-regulation of E-cadherin. Our results highlight a powerful role for exogenously applied PA and Cer in reducing breast cancer tumorigenicity by simultaneously targeting cancer cells and M2 macrophages. Our findings support the notion that lipids represent alternative biocompatible therapeutic agents for breast cancer.

Contribution to development of remedies for COVID-19: focusing on Eritoran

Eritoran (E5564) is Eisai's in-house discovered and developed investigational Toll-Like Receptor 4 (TLR4) antagonist created with natural product organic synthesis technology. It is a structural analogue of Lipid A, which is an activator of endotoxins of bacteria. It has been previously observed to be safe in 14 clinical studies including a large Phase 3 randomized trial in severe sepsis. In order to evaluate therapeutic efficacy by eritoran, we are participating in the international network REMAP-CAP-COVID (Randomized, Embedded, Multi-factorial, Adaptive Platform-Community Acquired Pneumonia COVID) which aims for novel coronavirus medicine development through drug repurposing, and began an international collaborative clinical trial in October 2020 which is designated for confirmed novel coronavirus patients who are hospitalized and are in a progressing disease state. It is hoped that through suppressing the most upstream TLR4 activity which controls production of multiple cytokines by eritoran, the cytokine storm in patients can be suppressed and pneumonia can thus be prevented from becoming severe. On the other hand, E6011 is the only humanized anti-fractalkine (FKN) monoclonal antibody in the world created by KAN Research Institute. E6011 inhibits the tight binding of CD16-positive monocytes (a cell population that highly expresses the FKN receptor CX3CR1) to vascular endothelial cells, which are important for the local inflammatory response. This is expected to suppress the formation and exacerbation of vasculopathy in COVID-19.

Versatile synthesis of pathogen specific bacterial cell wall building blocks.

Full details on the design, strategies and tactics for development of a novel synthetic sequence to farnesyl lipid I and II analogs is reported. The modular route was based on a three coupling strategy involving an efficient solid phase synthesis of the elaborate peptide fragment, which proceeded with excellent yield and stereoselectivity and was efficiently applied for the convergent synthesis of 3-lipid I and II. Furthermore, the generality of this route was demonstrated by synthesis of 3-lipid I congeners that are characteristic for S. aureus and E. faecalis. All 3-lipid I and II building blocks were obtained in high purity revealing high spectroscopic resolution.

Lipid Flip-Flop-Inducing Antimicrobial Phytochemicals from Gymnema sylvestre are Bacterial Membrane Permeability Enhancers.

An amphiphilic phytochemical fraction isolated from methanol extract of Gymnema sylvestre leaf powder contained six terpenoids, two flavonoids, and one alkaloid that induced rapid flip-flop of fluorescent phospholipid analog in the phosphatidyl choline bilayer. Lipid-flipping activity of the methanol-extracted fraction of G. sylvestre (MEFGS) was dose-dependent and time-dependent with a rate constant k = (12.09 ± 0.94) mg–1 min–1 that was saturable at (40 ± 1) % flipping of the fluorescent lipid analogue. Interactions of MEFGS phytochemicals with large unilamelar vesicles led to time-dependent change in their rounded morphology into irregular shapes, indicating their membrane-destabilizing activity. MEFGS exhibited antibacterial activity on Escherichia coli (MTCC-118), Staphylococcus aureus (MTCC-212), and Pseudomonas aeruginosa (MTCC-1035) with IC50 values 0.5, 0.35, and 0.1 mg/mL, respectively. Phytochemicals in MEFGS increased membrane permeabilization in all three bacteria, as indicated by 23, 17, and 17% increase in the uptake of crystal violet, respectively. MEFGS enhanced membrane damage, resulting in a 3–5 fold increase in leakage of cytosolic ions, 0.5–2 fold increase in leakage of PO4–, and 15–20% increase in loss of cellular proteins. MEFGS synergistically increased the efficacy of curcumin, amoxillin, ampicillin, and cefotaxime on S. aureus probably by enhancing their permeability into the bacterium. For the first time, our study reveals that phytochemicals from G. sylvestre enhance the permeability of the bacterial plasma membrane by facilitating flip-flop of membrane lipids. Lipid-flipping phytochemicals from G. sylvestre can be used as adjuvant therapeutics to enhance the efficacy of antibacterials by increasing their bioavailability in the target bacteria.

α‐Tocopherol‐Conjugated, Open Chain Sugar‐Mimicking Cationic Lipids: Design, Synthesis and In–Vitro Gene Transfection Properties

AbstractSugar based cationic lipids are promising carriers of nucleic acids into various cells. Particularly, abundant hydroxyl functional groups in the sugar‐like moieties are believed to enhance target oriented uptake as well as biocompatibility. Towards this direction, we have synthesized three new cationic lipid analogues with or without sugar‐like multi‐hydroxyl functional head groups. Liposomal formulations of these lipids were developed and thoroughly characterized by means of DNA binding ability, size and zeta potential. Optimal lipid‐DNA complexes were determined by their transfection efficiency in HEK‐293 (Human embryonic kidney) cells. 1,4 gluconic γ‐lactone derived lipid (TS1) and ascorbic acid derived lipid (TS2), have exhibited 25‐fold and 12‐fold increase in transfected HEPG2 (human liver cancer) cells, respectively, when compared with a control lipid without sugar‐like moiety (TA). However, this property was reversed in the case of U87 (human primary glioblastoma) cell line. The results point out that the transfection efficiencies of sugar alcohol‐based cationic lipids are cell‐dependant. Cytotoxicity of the lipid complexes revealed that the sugar‐mimicking cationic lipids are non‐toxic as ≈80 % viable cells were observed in the treated cell lines. In conclusion, the current paper adds knowledge on the sugar based cationic lipids and reiterates that these lipids could yield potential applications towards liver targeted gene delivery.

Corrections in the CHARMM36 Parametrization of Chloride Interactions with Proteins, Lipids, and Alkali Cations, and Extension to Other Halide Anions.

The nonpolarizable CHARMM force field is one of the most widely used energy functions for all-atom biomolecular simulations. Chloride is the only halide ion included in the latest version, CHARMM36m, and is used widely in simulation studies, often as an electrolyte ion but also as the biological substrate of transport proteins and enzymes. Here, we find that existing parameters systematically underestimate the interaction of Cl- with proteins and lipids. Accordingly, when examined in solution, little to no Cl-association can be observed with most components of the protein, including backbone, polar side chains and aromatic rings. The strength of the interaction with cationic side chains and with alkali ions is also incongruent with experimental measurements, specifically osmotic coefficients of concentrated solutions. Consistent with these findings, a 4-μs trajectory of the Cl--specific transport protein CLC-ec1 shows irreversible Cl- dissociation from the so-called Scen binding site, even in a 150 mM NaCl buffer. To correct for these deficiencies, we formulate a series of pair-specific Lennard-Jones parameters that override those resulting from the conventional Lorentz-Berthelot combination rules. These parameters, referred to as NBFIX, are systematically calibrated against available experimental data as well as ab initio geometry optimizations and energy evaluations, for a wide set of binary and ternary Cl- complexes with protein and lipid analogs and alkali cations. Analogously, we also formulate parameter sets for the other three biological halide ions, namely, fluoride, bromide, and iodide. The resulting parameters are used to calculate the potential of mean force defining the interaction of each anion and each of the protein and lipid analogues in bulk water, revealing association free energies in the range of -0.3 to -3.3 kcal/mol, with the F- complexes being the least stable. The NBFIX corrections also preserve the Cl- occupancy of CLC-ec1 in a second 4-μs trajectory. We posit that these optimized molecular-mechanics models provide a more realistic foundation for all-atom simulation studies of processes entailing changes in hydration, recognition, or transport of halide anions.

Synthetic Glycolipids as Molecular Vaccine Adjuvants: Mechanism of Action in Human Cells and In Vivo Activity.

Modern adjuvants for vaccine formulations are immunostimulating agents whose action is based on the activation of pattern recognition receptors (PRRs) by well-defined ligands to boost innate and adaptive immune responses. Monophosphoryl lipid A (MPLA), a detoxified analogue of lipid A, is a clinically approved adjuvant that stimulates toll-like receptor 4 (TLR4). The synthesis of MPLA poses manufacturing and quality assessment challenges. Bridging this gap, we report here the development and preclinical testing of chemically simplified TLR4 agonists that could sustainably be produced in high purity and on a large scale. Underpinned by computational and biological experiments, we show that synthetic monosaccharide-based molecules (FP compounds) bind to the TLR4/MD-2 dimer with submicromolar affinities stabilizing the active receptor conformation. This results in the activation of MyD88- and TRIF-dependent TLR4 signaling and the NLRP3 inflammasome. FP compounds lack in vivo toxicity and exhibit adjuvant activity by stimulating antibody responses with a potency comparable to MPLA.

Measuring Membrane Lipid Turnover with the pH-sensitive Fluorescent Lipid Analog ND6.

Exo-/endocytosis is a common process mediating the exchange of biomolecules between cells and their environment and among different cells. Specialized cells use this process to execute vital body functions such as insulin secretion from β cells and neurotransmitter release from chemical synapses. Owing to its physiological significance, exo-/endocytosis has been one of the most studied topics in cell biology. Many tools have been developed to study this process at the gene and protein level, because of which much is known about the protein machinery participating in this process. However, very few methods have been developed to measure membrane lipid turnover, which is the physical basis of exo-/endocytosis. This paper introduces a class of new fluorescent lipid analogs exhibiting pH-dependent fluorescence and demonstrates their use to trace lipid recycling between the plasma membrane and the secretory vesicles. Aided by simple pH manipulations, those analogs also allow the quantification of lipid distribution across the surface and the intracellular membrane compartments, as well as the measurement of lipid turnover rate during exo-/endocytosis. These novel lipid reporters will be of great interest to various biological research fields such as cell biology and neuroscience.

Rethinking Remdesivir: Synthesis, Antiviral Activity, and Pharmacokinetics of Oral Lipid Prodrugs.

ABSTRACTRemdesivir (RDV; GS-5734) is currently the only FDA-approved antiviral drug for the treatment of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. The drug is approved for use in adults or children 12 years or older who are hospitalized for the treatment of COVID-19 on the basis of an acceleration of clinical recovery for inpatients with this disease. Unfortunately, the drug must be administered intravenously, restricting its use to those requiring hospitalization for relatively advanced disease. RDV is also unstable in plasma and has a complex activation pathway which may contribute to its highly variable antiviral efficacy in SARS-CoV-2-infected cells. Potent orally bioavailable antiviral drugs for early treatment of SARS-CoV-2 infection are urgently needed, and several, including molnupiravir and PF-07321332, are currently in clinical development. We focused on making simple, orally bioavailable lipid analogs of remdesivir nucleoside (RVn; GS-441524) that are processed to RVn monophosphate, the precursor of the active RVn triphosphate, by a single-step intracellular cleavage. In addition to high oral bioavailability, stability in plasma, and simpler metabolic activation, new oral lipid prodrugs of RVn had submicromolar anti-SARS-CoV-2 activity in a variety of cell types, including Vero E6, Calu-3, Caco-2, human pluripotent stem cell (PSC)-derived lung cells, and Huh7.5 cells. In Syrian hamsters, oral treatment with 1-O-octadecyl-2-O-benzyl-glycero-3-phosphate RVn (ODBG-P-RVn) was well tolerated and achieved therapeutic levels in plasma above the 90% effective concentration (EC90) for SARS-CoV-2. The results suggest further evaluation as an early oral treatment for SARS-CoV-2 infection to minimize severe disease and reduce hospitalizations.

Non-Polar Lipids as Regulators of Membrane Properties in Archaeal Lipid Bilayer Mimics.

The modification of archaeal lipid bilayer properties by the insertion of apolar molecules in the lipid bilayer midplane has been proposed to support cell membrane adaptation to extreme environmental conditions of temperature and hydrostatic pressure. In this work, we characterize the insertion effects of the apolar polyisoprenoid squalane on the permeability and fluidity of archaeal model membrane bilayers, composed of lipid analogues. We have monitored large molecule and proton permeability and Laurdan generalized polarization from lipid vesicles as a function of temperature and hydrostatic pressure. Even at low concentration, squalane (1 mol%) is able to enhance solute permeation by increasing membrane fluidity, but at the same time, to decrease proton permeability of the lipid bilayer. The squalane physicochemical impact on membrane properties are congruent with a possible role of apolar intercalants on the adaptation of Archaea to extreme conditions. In addition, such intercalant might be used to cheaply create or modify chemically resistant liposomes (archeaosomes) for drug delivery.

Mitochondrial Targeting Involving Cholesterol-Rich Lipid Rafts in the Mechanism of Action of the Antitumor Ether Lipid and Alkylphospholipid Analog Edelfosine.

The ether lipid edelfosine induces apoptosis selectively in tumor cells and is the prototypic molecule of a family of synthetic antitumor compounds collectively known as alkylphospholipid analogs. Cumulative evidence shows that edelfosine interacts with cholesterol-rich lipid rafts, endoplasmic reticulum (ER) and mitochondria. Edelfosine induces apoptosis in a number of hematological cancer cells by recruiting death receptors and downstream apoptotic signaling into lipid rafts, whereas it promotes apoptosis in solid tumor cells through an ER stress response. Edelfosine-induced apoptosis, mediated by lipid rafts and/or ER, requires the involvement of a mitochondrial-dependent step to eventually elicit cell death, leading to the loss of mitochondrial membrane potential, cytochrome c release and the triggering of cell death. The overexpression of Bcl-2 or Bcl-xL blocks edelfosine-induced apoptosis. Edelfosine induces the redistribution of lipid rafts from the plasma membrane to the mitochondria. The pro-apoptotic action of edelfosine on cancer cells is associated with the recruitment of F1FO–ATP synthase into cholesterol-rich lipid rafts. Specific inhibition of the FO sector of the F1FO–ATP synthase, which contains the membrane-embedded c-subunit ring that constitutes the mitochondrial permeability transcription pore, hinders edelfosine-induced cell death. Taking together, the evidence shown here suggests that the ether lipid edelfosine could modulate cell death in cancer cells by direct interaction with mitochondria, and the reorganization of raft-located mitochondrial proteins that critically modulate cell death or survival. Here, we summarize and discuss the involvement of mitochondria in the antitumor action of the ether lipid edelfosine, pointing out the mitochondrial targeting of this drug as a major therapeutic approach, which can be extrapolated to other alkylphospholipid analogs. We also discuss the involvement of cholesterol transport and cholesterol-rich lipid rafts in the interactions between the organelles as well as in the role of mitochondria in the regulation of apoptosis in cancer cells and cancer therapy.

Biomembrane mimetic electrochemical sensors

Biomimetic approaches to the assembling of regular layers of electrochemical sensors make it possible to extend their performance because of the tuning shape and charge of the lipid analogs and implementation of artificial receptors in the lipid layers. The role of artificial components in modification of the properties of the surface layers and application of the nanopore devices are of particular interest. In review, recent trends in assembling modified lipid membranes and their artificial analogs with nanopores have been considered with particular emphasis on their use in electrochemical sensors. • Following nature offers great opportunities in assembling selective sensors. • Modification of biomembranes improves their stability and sensing properties. • Introduction of synthetic components is governed by electrostatic and hydrophobic interactions. • Nanopores offer single-molecule detection of species selected by shape, size, and charge. • Biomimetic membranes can clarify membrane processes in nature.

Breakdown in membrane asymmetry regulation leads to monocyte recognition of P. falciparum-infected red blood cells.

The human malaria parasite Plasmodium falciparum relies on lipids to survive; this makes its lipid metabolism an attractive drug target. The lipid phosphatidylserine (PS) is usually confined to the inner leaflet of the red blood cell membrane (RBC) bilayer; however, some studies suggest that infection with the intracellular parasite results in the presence of this lipid in the RBC membrane outer leaflet, where it could act as a recognition signal to phagocytes. Here, we used fluorescent lipid analogues and probes to investigate the enzymatic reactions responsible for maintaining asymmetry between membrane leaflets, and found that in parasitised RBCs the maintenance of membrane asymmetry was partly disrupted, and PS was increased in the outer leaflet. We examined the underlying causes for the differences between uninfected and infected RBCs using fluorescent dyes and probes, and found that calcium levels increased in the infected RBC cytoplasm, whereas membrane cholesterol was depleted from the erythrocyte plasma membrane. We explored the resulting effect of PS exposure on enhanced phagocytosis by monocytes, and show that infected RBCs must expend energy to limit phagocyte recognition, and provide experimental evidence that PS exposure contributes to phagocytic recognition of P. falciparum-infected RBCs. Together, these findings underscore the pivotal role for PS exposure on the surface of Plasmodium falciparum-infected erythrocytes for in vivo interactions with the host immune system, and provide a rationale for targeted antimalarial drug design.

N-palmitoyl-D-glucosamine, a Natural Monosaccharide-Based Glycolipid, Inhibits TLR4 and Prevents LPS-Induced Inflammation and Neuropathic Pain in Mice.

Toll-like receptors (TLRs) are key receptors through which infectious and non-infectious challenges act with consequent activation of the inflammatory cascade that plays a critical function in various acute and chronic diseases, behaving as amplification and chronicization factors of the inflammatory response. Previous studies have shown that synthetic analogues of lipid A based on glucosamine with few chains of unsaturated and saturated fatty acids, bind MD-2 and inhibit TLR4 receptors. These synthetic compounds showed antagonistic activity against TLR4 activation in vitro by LPS, but little or no activity in vivo. This study aimed to show the potential use of N-palmitoyl-D-glucosamine (PGA), a bacterial molecule with structural similarity to the lipid A component of LPS, which could be useful for preventing LPS-induced tissue damage or even peripheral neuropathies. Molecular docking and molecular dynamics simulations showed that PGA stably binds MD-2 with a MD-2/(PGA)3 stoichiometry. Treatment with PGA resulted in the following effects: (i) it prevented the NF-kB activation in LPS stimulated RAW264.7 cells; (ii) it decreased LPS-induced keratitis and corneal pro-inflammatory cytokines, whilst increasing anti-inflammatory cytokines; (iii) it normalized LPS-induced miR-20a-5p and miR-106a-5p upregulation and increased miR-27a-3p levels in the inflamed corneas; (iv) it decreased allodynia in peripheral neuropathy induced by oxaliplatin or formalin, but not following spared nerve injury of the sciatic nerve (SNI); (v) it prevented the formalin- or oxaliplatin-induced myelino-axonal degeneration of sciatic nerve. SIGNIFICANCE STATEMENT We report that PGA acts as a TLR4 antagonist and this may be the basis of its potent anti-inflammatory activity. Being unique because of its potency and stability, as compared to other similar congeners, PGA can represent a tool for the optimization of new TLR4 modulating drugs directed against the cytokine storm and the chronization of inflammation.

Combinatorial liposomal peptide vaccine induces IgA and confers protection against influenza virus and bacterial super-infection.

ObjectivesThe upper respiratory tract is the major entry site for Streptococcus pyogenes and influenza virus. Vaccine strategies that activate mucosal immunity could significantly reduce morbidity and mortality because of these pathogens. The severity of influenza is significantly greater if a streptococcal infection occurs during the viraemic period and generally viral infections complicated by a subsequent bacterial infection are known as super‐infections. We describe an innovative vaccine strategy against influenza virus:S. pyogenes super‐infection. Moreover, we provide the first description of a liposomal multi‐pathogen‐based platform that enables the incorporation of both viral and bacterial antigens into a vaccine and constitutes a transformative development.MethodsSpecifically, we have explored a vaccination strategy with biocompatible liposomes that express conserved streptococcal and influenza A virus B‐cell epitopes on their surface and contain encapsulated diphtheria toxoid as a source of T‐cell help. The vaccine is adjuvanted by inclusion of the synthetic analogue of monophosphoryl lipid A, 3D‐PHAD.ResultsWe observe that this vaccine construct induces an Immunoglobulin A (IgA) response in both mice and ferrets. Vaccination reduces viral load in ferrets from influenza challenge and protects mice from both pathogens. Notably, vaccination significantly reduces both mortality and morbidity associated with a super‐infection.ConclusionThe vaccine design is modular and could be adapted to include B‐cell epitopes from other mucosal pathogens where an IgA response is required for protection.

Contribution to Development of Remedies for COVID-19: Focusing on Eritoran

Eritoran (E5564) is Eisai's in-house discovered and developed investigational TLR4 (Toll-Like Receptor 4) antagonist created with natural product organic synthesis technology. It is a structural analogue of Lipid A, which is an activator of endotoxins of bacteria. It has been previously observed to be safe in 14 clinical studies including a large Phase 3 randomized trial in severe sepsis. We are participating in the international network REMAP-CAP-COVID (Randomized, Embedded, Multi-factorial, Adaptive Platform-Community Acquired Pneumonia COVID) which aims for novel coronavirus medicine development through drug repurposing, and began an international collaborative clinical trial in October 2020 which is designated for confirmed novel coronavirus patients who are hospitalized and are in a progressing disease state. It is hoped that through suppressing the most upstream TLR4 activity which controls production of multiple cytokines, the cytokine storm in patients can be suppressed and pneumonia can thus be prevented from becoming severe.

Pleiotropic Properties of Amphiphilic Dihydropyridines, Dihydropyridones, and Aminovinylcarbonyl Compounds.

Three groups of synthetic lipids are chosen for studies: (1) 1,4-dihydropyridines (1,4-DHPs) containing two cationic moieties and their analogues; (2) 3,4-dihydro-2(1H)-pyridones containing a cationic moiety; and (3) acyclic, open-chain analogues, i.e., 2-amino-3-alkoxycarbonylalkylammonium derivatives. 1,4-DHPs possessing dodecyl alkyl chains in the ester groups in positions 3 and 5 and cationic nitrogen-containing groups in positions 2 and 6 have high cytotoxicity in cancer cells HT-1080 (human lung fibrosarcoma) and MH-22A (mouse hepatoma), but low cytotoxicity in the noncancerous NIH3T3 cells (mouse embryonic fibroblast). On the contrary, similar compounds having short (methyl, ethyl, or propoxyethyl) chains in the ester groups in positions 3 and 5 lack cytotoxicity in the cancer cells HT-1080 and MH-22A even at high doses. Inclusion of fluorine atoms in the alkyl chains in positions 3 and 5 of the DHP cycle decreases the cytotoxicity of the mentioned compounds. Structurally related dihydropyridones with a polar head group are substantially more toxic to normal and cancerous cells than the DHP analogues. Open-chain analogues of DHP lipids comprise the same conjugated aminovinylcarbonyl moiety and possess anticancer activity, but they also have high basal cytotoxicity. Electrochemical oxidation data demonstrate that oxidation potentials of selected compounds are in the range of 1.6–1.7 V for cationic 1,4-DHP, 2.0–2.4 V for cationic 3,4-dihydropyridones, and 1.2–1.5 V for 2-amino-3-alkoxycarbonylalkylammonium derivatives. Furthermore, the tested cationic 1,4-DHP amphiphiles possess antiradical activity. Molecular topological polar surface area values for the tested compounds were defined in accordance with the main fragments of compound structures. The determined logP values were highest for dodecyl ester groups in positions 3 and 5 of the 1,4-DHP and lowest for short alkyl chain-containing amphiphiles.