P-selectin-dependent leukocyte adhesion is governed by endolysosomal two-pore channel 2.

Upon proinflammatory challenges, endothelial cell surface presentation of the leukocyte receptor P-selectin, together with the stabilizing co-factor CD63, is needed for leukocyte capture and is mediated via demand-driven exocytosis from the Weibel-Palade bodies that fuse with the plasma membrane. We report that neutrophil recruitment to activated endothelium is significantly reduced in mice deficient for the endolysosomal cation channel TPC2 and in human primary endothelial cells with pharmacological TPC2 block. We observe less CD63 signal in whole-mount stainings of proinflammatory-activated cremaster muscles from TPC2 knockout mice. We find that TPC2 is activated and needed to ensure the transfer of CD63 from endolysosomes via Weibel-Palade bodies to the plasma membrane to retain P-selectin on the cell surface of human primary endothelial cells. Our findings establish TPC2 as a key element to leukocyte interaction with the endothelium and a potential pharmacological target in the control of inflammatory leukocyte recruitment.

Combining lipid-mimicking-enabled transition metal and enzyme-mediated catalysis at the cell surface of E. coli.

Being an essential multifunctional platform and interface to the extracellular environment, the cell membrane constitutes a valuable target for the modification and manipulation of cells and cellular behavior, as well as for the implementation of artificial, new-to-nature functionality. While bacterial cell surface functionalization via expression and presentation of recombinant proteins has extensively been applied, the corresponding application of functionalizable lipid mimetics has only rarely been reported. Herein, we describe an approach to equip E. coli cells with a lipid-mimicking, readily membrane-integrating imidazolium salt and a corresponding NHC-palladium complex that allows for flexible bacterial membrane surface functionalization and enables E. coli cells to perform cleavage of propargyl ethers present in the surrounding cell medium. We show that this approach can be combined with already established on-surface functionalization, such as bacterial surface display of enzymes, i.e. laccases, leading to a new type of cascade reaction. Overall, we envision the herein presented proof-of-concept studies to lay the foundation for a multifunctional toolbox that allows flexible and broadly applicable functionalization of bacterial membranes.

Programming inactive RNA-binding small molecules into bioactive degraders.

Target occupancy is often insufficient to elicit biological activity, particularly for RNA, compounded by the longstanding challenges surrounding the molecular recognition of RNA structures by small molecules. Here we studied molecular recognition patterns between a natural-product-inspired small-molecule collection and three-dimensionally folded RNA structures. Mapping these interaction landscapes across the human transcriptome defined structure-activity relationships. Although RNA-binding compounds that bind to functional sites were expected to elicit a biological response, most identified interactions were predicted to be biologically inert as they bind elsewhere. We reasoned that, for such cases, an alternative strategy to modulate RNA biology is to cleave the target through a ribonuclease-targeting chimera, where an RNA-binding molecule is appended to a heterocycle that binds to and locally activates RNase L1. Overlay of the substrate specificity for RNase L with the binding landscape of small molecules revealed many favourable candidate binders that might be bioactive when converted into degraders. We provide a proof of concept, designing selective degraders for the precursor to the disease-associated microRNA-155 (pre-miR-155), JUN mRNA and MYC mRNA. Thus, small-molecule RNA-targeted degradation can be leveraged to convert strong, yet inactive, binding interactions into potent and specific modulators of RNA function.

A bifunctional imidazolium-based cholesterol analog for the tracking of cellular cholesterol distributions and cholesterol-protein interactions.

Cholesterol is a sterol lipid found in all higher eukaryotic organisms. It is required to consolidate the basic structural integrity and dynamic principles of cellular membranes and participates in many essential cellular processes that range from signal transduction to membrane traffic and metabolism. Moreover, a growing number of clinically highly relevant diseases such as immunological disorders or cancer has been linked to changes or misfunctions in cholesterol homeostasis. Therefore, the development of molecular tools that help to further unravel the role of cholesterol in essential cellular processes is of high relevance. Herein, we report the synthesis and proof-of-concept of a novel bifunctional imidazolium-based cholesterol analog (X-CHIM) that we envision to serve as a broadly applicable tool for the simultaneous investigation of cellular cholesterol distributions as well as cholesterol-protein interactions.

NTA-Cholesterol Analogue for the Nongenetic Liquid-Ordered Phase-Specific Functionalization of Lipid Membranes with Proteins.

The nongenetic modification of cell membranes with proteins is a straightforward way of cellular engineering. In these processes, it is important to specifically address the proteins to liquid-ordered (Lo) or liquid-disordered (Ld) domains as this can largely affect their biological functions. Herein, we report a cholesterol analogue (CHIM) with a nitrilotriacetic acid (NTA) headgroup, named CHIM-NTA. CHIM-NTA integrates into lipid membranes similar to the widely used phospholipid-derived DGS-NTA and, when loaded with Ni2+, allows for specific membrane immobilization of any polyhistidine-tagged proteins of choice. Yet, unlike DGS-NTA, it localizes to the Lo phase in phase-separated giant unilamellar vesicles (GUVs) and allows addressing His-tagged proteins to Lo domains. Furthermore, CHIM-NTA readily integrates into the membranes of live cells and thus enables the nongenetic modification of the cell surface with proteins. Overall, CHIM-NTA provides a facile and flexible way to modify biological membranes, in particular Lo domains, with His-tagged proteins and can serve as a broadly applicable molecular tool for cell surface engineering.

Lipid mimetics: A versatile toolbox for lipid biology and beyond.

Being the principal component of biological membranes lipids are essential building blocks of life. Given their huge biological importance, the investigation of lipids, their properties, interactions and metabolic pathways is of prime importance for the fundamental understanding of living cells and organisms as well as the emergence of diseases. Different strategies have been applied to investigate lipid-mediated biological processes, one of them being the use of lipid mimetics. They structurally resemble their natural counterparts but are equipped with functionality that can be used to probe or manipulate lipid-mediated biological processes and biomembranes. Lipid mimetics therefore constitute an indispensable toolbox for lipid biology and membrane research but also beyond for potential applications in medicine or synthetic biology. Herein, we highlight recent advances in the development and application of lipid-mimicking compounds.

Cationic, Steroid-Based Imidazolium Amphiphiles Show Tunable Backbone-Dependent Membrane Selectivity in Fungi.

Cationic amphiphiles have been reported to show broad antimicrobial activity. The potential for antimicrobial resistance to these molecules is low owing to their general cell membrane permeabilizing mode of action. However, their applications are often limited by toxicity resulting from their low selectivity for microbial cell membranes. Herein, we report a library of cationic, steroid-based imidazolium amphiphiles that show tunable antifungal activity in a variety of fungal pathogens of the genus Candida. We show that adoption of an ergosterol-derived backbone increases antifungal activity while modestly affecting hemolytic activity, thereby increasing overall selectivity by more than 8-fold in comparison to cholesterol-derived imidazolium salts. We hypothesize that this effect is caused by a privileged integration of the ergosterol-derived salts into fungal membranes leading to increased membrane disorder. We propose that these findings offer a useful platform for the development of improved amphiphilic fungicides.

Bipolar Imidazolium-Based Lipid Analogues for Artificial Archaeosomes.

Archaeal lipids have harvested biomedical and biotechnological interest because of their ability to form membranes with low permeability and enhanced temperature and pressure stability. Because of problems in isolating archaeal lipids, chemical synthesis appears to be a suitable means of producing model lipids that mimic the biological counterparts. Here, we introduce a new concept: we synthesized bipolar alkylated imidazolium salts of different chain lengths (BIm10-32) and studied their structure and lyotropic phase behavior. Furthermore, mixtures of the bolalipid analogues with phospholipid model biomembranes of diverse complexity were studied. DSC, fluorescence and FTIR spectroscopy, confocal fluorescence microscopy, DLS, SAXS, and TEM were used to reveal changes in lipid phase behavior, fluidity, the lipid's conformational order, and membrane morphology over a wide range of temperatures and for selected pressures. It could be shown that the long-chain BImN32 can form monolayer sheets. Integrated in phospholipid membranes, it reveals a fluidizing effect. Here, the two polar head groups, connected by a long alkyl chain, enable the integration into the bilayer. Interestingly, addition of BImN32 to fluid DPPC liposomes increased the lipid packing markedly, rendering the membrane system more stable at higher temperatures. The membrane system is also stable against compression as indicated by the high-pressure stability of the system, mimicking an archaeal lipid-like behavior. BImN32 incorporation into raft-like anionic model biomembranes led to marked changes in lateral membrane organization, topology, and fusogenicity of the membrane. Overall, it was found that long-chain imidazolium-based bolalipid analogues can help adjust membrane's biophysical properties, while the imidazolium headgroup provides the ability for crucial electrostatic interaction for vesicle fusion or selective interaction with membrane-related signaling molecules and polypeptides in a synthetically tractable manner. The results obtained may help to develop new approaches for rational design of extremophilic bolalipid-based liposomes for various applications, including delivery of drugs and vaccines.

CHIMs are versatile cholesterol analogs mimicking and visualizing cholesterol behavior in lipid bilayers and cells.

Cholesterol is an essential component of cellular membranes regulating the structural integrity and fluidity of biological bilayers and cellular processes such as signal transduction and membrane trafficking. However, tools to investigate the role and dynamics of cholesterol in live cells are still scarce and often show limited applicability. To address this, we previously developed a class of imidazolium-based cholesterol analogs, CHIMs. Here we confirm that CHIM membrane integration characteristics largely mimic those of cholesterol. Computational studies in simulated phospholipid bilayers and biophysical analyses of model membranes reveal that in biologically relevant systems CHIMs behave similarly to natural cholesterol. Importantly, the analogs can functionally replace cholesterol in membranes, can be readily labeled by click chemistry and follow trafficking pathways of cholesterol in live cells. Thus, CHIMs represent chemically versatile cholesterol analogs that can serve as a flexible toolbox to study cholesterol behavior and function in live cells and organisms.

Vancomycin-Arginine Conjugate Inhibits Growth of Carbapenem-Resistant E. coli and Targets Cell-Wall Synthesis.

The emergence of multi-drug-resistant Gram-negative bacteria, including carbapenem-resistant Enterobacteriaceae, is a major health problem that necessitates the development of new antibiotics. Vancomycin inhibits cell-wall synthesis in Gram-positive bacteria but is generally ineffective against Gram-negative bacteria and is unable to penetrate the outer membrane barrier. In an effort to determine whether vancomycin and other antibiotics effective against Gram-positive bacteria could, through modification, be rendered effective against Gram-negative bacteria, we discovered that the covalent attachment of a single arginine to vancomycin yielded conjugates with order-of-magnitude improvements in activity against Gram-negative bacteria, including pathogenic E. coli. The vancomycin-arginine conjugate (V-R) exhibited efficacy against actively growing bacteria, induced the loss of rod cellular morphology, and resulted in the intracellular accumulation of peptidoglycan precursors, all consistent with cell-wall synthesis disruption as its mechanism of action. Membrane permeabilization studies demonstrated an enhanced outer membrane permeability of V-R as compared with vancomycin. The conjugate exhibited no mammalian cell toxicity or hemolytic activity in MTT and hemolysis assays. Our study introduces a new vancomycin derivative effective against Gram-negative bacteria and underscores the broader potential of generating new antibiotics through combined mode-of-action and synthesis-informed design studies.

Intermolecular 1,4-Carboamination of Conjugated Dienes Enabled by Cp*RhIII -Catalyzed C-H Activation.

A protocol for the three-component 1,4-carboamination of dienes is described. Synthetically versatile Weinreb amides were coupled with 1,3-dienes and readily available dioxazolones as the nitrogen source using [Cp*RhCl2 ]2 -catalyzed C-H activation to deliver the 1,4-carboaminated products. This transformation proceeds under mild reaction conditions and affords the products with high levels of regio- and E-selectivity. Mechanistic investigations suggest an intermediate RhIII -allyl species is trapped by an electrophilic amidation reagent in a redox-neutral fashion.

Altered representation of expected value in the orbitofrontal cortex in mania.

OBJECTIVE: Increased responsiveness to appetitive and reduced responsiveness to aversive anticipatory cues may be associated with dysfunction of the brain reward system in mania. Here we studied neural correlates of gain and loss expectation in mania using functional magnetic resonance imaging (fMRI). METHOD: Fifteen manic patients and 26 matched healthy control individuals performed a monetary incentive delay task, during which subjects anticipated to win or lose a varying amount of money. Varying both magnitude and valence (win, loss) of anticipatory cues allowed us to isolate the effects of magnitude, valence and expected value (magnitude-by-valence interaction). RESULTS: Response times and total gain amount did not differ significantly between groups. FMRI data indicated that the ventral striatum responded according to cued incentive magnitude in both groups, and this effect did not significantly differ between groups. However, a significant group difference was observed for expected value representation in the left lateral orbitofrontal cortex (OFC; BA 11 and 47). In this region, patients showed increasing BOLD responses during expectation of increasing gain and decreasing responses during expectation of increasing loss, while healthy subjects tended to show the inverse effect. In seven patients retested after remission OFC responses adapted to the response pattern of healthy controls. CONCLUSIONS: The observed alterations are consistent with a state-related affective processing bias during the expectation of gains and losses which may contribute to clinical features of mania, such as the enhanced motivation for seeking rewards and the underestimation of risks and potential punishments.