Synthetic mimics of mammalian cell surface receptors: prosthetic molecules that augment living cells

Specific receptors on the surface of mammalian cells actively internalize cell-impermeable ligands by receptor-mediated endocytosis. To mimic these internalizing receptors, my laboratory is studying artificial cell surface receptors that comprise N-alkyl derivatives of 3beta-cholesterylamine linked to motifs that bind cell-impermeable ligands. When added to living mammalian cells, these synthetic receptors insert into cellular plasma membranes, project ligand-binding small molecules or peptides from the cell surface, and enable living cells to internalize targeted proteins and other cell-impermeable compounds. These artificial receptors mimic their natural counterparts by rapidly cycling between plasma membranes and intracellular endosomes, associating with proposed cholesterol and sphingolipid-rich lipid raft membrane microdomains, and delivering ligands to late endosomes/lysosomes. This "synthetic receptor targeting" strategy is briefly reviewed here and contrasted with other related cellular delivery systems. Potential applications of artificial cell surface receptors as molecular probes, agents for cellular targeting, tools for drug delivery, and methods for ligand depletion are discussed. The construction of synthetic receptors as prosthetic molecules, designed to seamlessly augment the molecular machinery of living cells, represents an exciting new frontier in the fields of bioorganic chemistry and chemical biology.     

Synthesis of an artificial cell surface receptor that enables oligohistidine affinity tags to function as metal-dependent cell-penetrating peptides

Cell-penetrating peptides and proteins (CPPs) are important tools for the delivery of impermeable molecules into living mammalian cells. To enable these cells to internalize proteins fused to common oligohistidine affinity tags, we synthesized an artificial cell surface receptor comprising an N-alkyl derivative of 3beta-cholesterylamine linked to the metal chelator nitrilotriacetic acid (NTA). This synthetic receptor inserts into cellular plasma membranes, projects NTA headgroups from the cell surface, and rapidly cycles between the plasma membrane and intracellular endosomes. Jurkat lymphocytes treated with the synthetic receptor (10 microM) for 1 h displayed approximately 8,400,000 [corrected]NTA groups on the cell surface. Subsequent addition of the green fluorescent protein AcGFP fused to hexahistidine or decahistidine peptides (3 microM) and Ni(OAc)(2) (100 microM) enhanced the endocytosis of AcGFP by 150-fold (hexahistidine fusion protein) or 600-fold (decahistidine fusion protein) within 4 h at 37 degrees C. No adverse effects on cellular proliferation or morphology were observed under these conditions. By enabling common oligohistidine affinity tags to function as cell-penetrating peptides, this metal-chelating cell surface receptor provides a useful tool for studies of cellular biology [corrected]     

A synthetic mimic of human Fc receptors: defined chemical modification of cell surfaces enables efficient endocytic uptake of human immunoglobulin-G

Binding of ligands to macromolecular receptors on the surface of mammalian cells often results in ligand uptake through receptor-mediated endocytosis. Certain human leukocytes and epithelial cells express Fc receptors (FcRs) that bind and internalize antibodies through this mechanism. To mimic this process, we synthesized an artificial FcR comprising the membrane anchor N-alkyl-3beta-amino-5alpha-cholestane linked to a disulfide-constrained cyclic peptide, termed FcIII, known to exhibit high affinity and specificity for the Fc region of human IgG. Treatment of human Jurkat lymphocytes that lack natural FcRs with the synthetic FcR (1 microM, 1 h) installed an average of approximately 6.2 x 10(5) synthetic receptor molecules per cell surface. These treated cells gained the capacity to internalize human IgG at levels greater than human THP-1 cells that express the natural receptors FcgammaRI and FcgammaRII. By linking binding motifs for circulating ligands to membrane anchors that cycle between the cell surface and intracellular endosomes, minimalistic cell surface receptors can be used to destroy targeted ligands by endocytosis. These small mimics of macromolecular receptors may be useful for controlling the extracellular abundance of ligands involved in disease.     

Selective disruption of early/recycling endosomes: release of disulfide-linked cargo mediated by a N-alkyl-3beta-cholesterylamine-capped peptide

The use of endocytic uptake pathways to deliver poorly permeable molecules into mammalian cells is often plagued by entrapment and degradation of material in late endosomes and lysosomes. As a strategy to prevent the exposure of cargo to these highly hydrolytic membrane-sealed compartments, we synthesized derivatives of the membrane anchor N-alkyl-3beta-cholesterylamine that selectively target linked compounds to less hydrolytic early/recycling endosomes. By targeting a pH-dependent membrane-lytic dodecapeptide and a disulfide-linked fluorophore to these compartments in Chinese hamster ovary cells or Jurkat lymphocytes, membranes of early/recycling endosomes were selectively disrupted, resulting in cleavage of the disulfide and escape of the fluorophore into the cytosol and nucleus with low toxicity. The ability of appropriately designed N-alkyl-3beta-cholesterylamines to deliver cargo into and release disulfide-linked cargo from relatively nonhydrolytic early/recycling endosomes may be useful for the delivery of a variety of sensitive molecules into living mammalian cells.     

Environment-Sensitive Fluorescence of 7-Nitrobenz-2-oxa-1,3-diazol-4-yl (NBD)-Labeled Ligands for Serotonin Receptors

Serotonin is a neurotransmitter that plays a crucial role in the regulation of several behavioral and cognitive functions by binding to a number of different serotonin receptors present on the cell surface. We report here the synthesis and characterization of several novel fluorescent analogs of serotonin in which the fluorescent NBD (7-nitrobenz-2-oxa-1,3-diazol-4-yl) group is covalently attached to serotonin. The fluorescent ligands compete with the serotonin_1A receptor specific radiolabeled agonist for binding to the receptor. Interestingly, these fluorescent ligands display a high environmental sensitivity of their fluorescence. Importantly, the human serotonin_1A receptor stably expressed in CHO-K1 cells could be specifically labeled with one of the fluorescent ligands with minimal nonspecific labeling. Interestingly, we show by spectral imaging that the NBD-labeled ligand exhibits a red edge excitation shift (REES) of 29 nm when bound to the receptor, implying that it is localized in a restricted microenvironment. Taken together, our results show that NBD-labeled serotonin analogs offer an attractive fluorescent approach for elucidating the molecular environment of the serotonin binding site in serotonin receptors. In view of the multiple roles played by the serotonergic systems in the central and peripheral nervous systems, these fluorescent ligands would be useful in future studies involving serotonin receptors.     

The Pennsylvania Green Fluorophore: a hybrid of Oregon Green and Tokyo Green for the construction of hydrophobic and pH-insensitive molecular probes

[structure: see text] Fluorescent small molecules are powerful tools for exploring cellular biology. As a more hydrophobic, photostable, and less pH-sensitive alternative to fluorescein, we synthesized Pennsylvania Green, a bright, monoanionic fluorophore related to Oregon Green and Tokyo Green. Comparison of membrane probes comprising N-alkyl-3beta-cholesterylamine linked to 4-carboxy-Tokyo Green (pK(a) approximately 6.2) and 4-carboxy-Pennsylvania Green (pK(a) approximately 4.8) revealed that only Pennsylvania Green was highly fluorescent in acidic early and recycling endosomes within living mammalian cells.     

TERS Detection of αVβ3 Integrins in Intact Cell Membranes

Integrins are important membrane receptors that form focal adhesions with the extracellular matrix and are transmembrane signaling proteins. We demonstrate that nanoparticles functionalized with c‐RGDfC ligands bind to intact cell membranes and selectively enhance the amino acid signals of the integrin receptor when coupled with tip‐enhanced Raman scattering (TERS) detection. Controlling the plasmonic interaction between the functionalized nanoparticle and the TERS tip provides a clear Raman signal from α_Vβ₃ integrins in the cell membrane that matches the signal of the purified integrin receptor. Random aggregation of nanoparticles on the cell does not provide the same spectral information. Chemical characterization of membrane receptors in intact cellular membranes is important for understanding membrane signaling and drug targeting. These results provide a new method to investigate the chemical interactions associated with ligand binding to membrane receptors in cells.     

Issues of ligand accessibility and mobility in initial cell attachment

The influence of lateral ligand mobility on cell attachment and receptor clustering has previously been explored for membrane-anchored molecules involved in cell-cell adhesion. In this study, we considered instead a cell binding motif from the extracellular matrix. Even though the lateral mobility of extracellular matrix ligands in membranes does not occur in vivo, we believe it is of interest for cell engineering in vitro. As is the case for cell-cell adhesion molecules, lateral mobility of extracellular matrix ligands could influence cell attachment and, subsequently, cell behavior in cell culture. In this paper, the accessibility and functionality of extracellular matrix ligands presented at surfaces were evaluated for the conditions of laterally mobile versus non-mobile ligands by studying ligand-antibody binding events and early cell attachment as a function of ligand concentration. We compare the initial attachment of rat-derived adult hippocampal progenitor (AHP) cells on laterally mobile, supported phospholipid bilayer membranes to non-mobile, poly-L-lysine-grafted-poly(ethylene glycol) (PLL-g-PEG) polymer films functionalized with a range of laminin-derived IKVAV-containing peptide densities. To this end, synthesis of a new PLL-g-PEG/PEG-IKVAV polymer is described. The characterization of available IKVAV peptides on both surface presentations schemes was explored by studying the mass uptake of anti-IKVAV antibodies using a combination of the surface-sensitive techniques quartz crystal microbalance with dissipation monitoring, surface plasmon resonance spectroscopy, and optical waveguide lightmode spectroscopy. IKVAV-containing peptides presented on laterally mobile, supported phospholipid bilayers and non-mobile PLL-g-PEG were recognized by the anti-IKVAV antibody in a dose-dependent manner, indicating that the amount of available IKVAV ligands increases proportionally with ligand density over the concentrations tested. Attachment of AHP cells to IKVAV-functionalized PLL-g-PEG and supported phospholipid bilayers followed a sigmoidal dependence on peptide concentration, with a critical concentration of approximately 3 pmol/cm2 IKVAV ligands required to support initial AHP cell attachment for both surface modifications. There appeared to be little influence of IKVAV peptide mobility on the initial attachment of AHP cells. Although the spread in the cell attachment data was larger for the PLL-g-PEG surface modification, this was reduced when observed after 24 h, indicating that the cells might need longer times to establish attachment strengths equivalent to those observed on peptide-functionalized supported lipid bilayers. The present study is a step toward understanding the influence of extracellular-matrix-derived ligand mobility on cell fate. Further analysis should focus on the systematic tuning of lateral ligand diffusion, as well as a comparison between the response of non-spreading cells (i.e., AHPs), versus spreading cells (i.e., fibroblasts).     

Synthesis of chimeric 7alpha-substituted estradiol derivatives linked to cholesterol and cholesterylamine

We report the synthesis of 7alpha-substituted beta-estradiol derivatives bearing side chains terminated with cholesterol and 3beta-cholesterylamine. These chimeric compounds were designed to exhibit high affinity for estrogen receptors (ERs) and cellular plasma membranes to potentially enable regulated uptake of ERs by mammalian cells. Evaluation with recombinant yeast reporting compound-mediated ER dimerization revealed potencies similar to the antiestrogen ICI 182780. Compounds that efficiently deliver dominant negative ERs into cells may provide novel therapeutics against breast cancers.     

Snooker: a structure-based pharmacophore generation tool applied to class A GPCRs

G-protein coupled receptors (GPCRs) are important drug targets for various diseases and of major interest to pharmaceutical companies. The function of individual members of this protein family can be modulated by the binding of small molecules at the extracellular side of the structurally conserved transmembrane (TM) domain. Here, we present Snooker, a structure-based approach to generate pharmacophore hypotheses for compounds binding to this extracellular side of the TM domain. Snooker does not require knowledge of ligands, is therefore suitable for apo-proteins, and can be applied to all receptors of the GPCR protein family. The method comprises the construction of a homology model of the TM domains and prioritization of residues on the probability of being ligand binding. Subsequently, protein properties are converted to ligand space, and pharmacophore features are generated at positions where protein ligand interactions are likely. Using this semiautomated knowledge-driven bioinformatics approach we have created pharmacophore hypotheses for 15 different GPCRs from several different subfamilies. For the beta-2-adrenergic receptor we show that ligand poses predicted by Snooker pharmacophore hypotheses reproduce literature supported binding modes for ～75% of compounds fulfilling pharmacophore constraints. All 15 pharmacophore hypotheses represent interactions with essential residues for ligand binding as observed in mutagenesis experiments and compound selections based on these hypotheses are shown to be target specific. For 8 out of 15 targets enrichment factors above 10-fold are observed in the top 0.5% ranked compounds in a virtual screen. Additionally, prospectively predicted ligand binding poses in the human dopamine D3 receptor based on Snooker pharmacophores were ranked among the best models in the community wide GPCR dock 2010.     

Multimodal Use of New Coumarin‐Based Fluorescent Chemosensors: Towards Highly Selective Optical Sensors for Hg2+ Probing

Despite several types of fluorescent sensing molecules have been proposed and examined to signal Hg²⁺ ion binding, the development of fluorescence‐based devices for in‐field Hg²⁺ detection and screening in environmental and industrial samples is still a challenging task. Herein, we report the synthesis and characterization of three new coumarin‐based fluorescent chemosensors featuring mixed thia/aza macrocyclic framework as receptors units, that is, ligands L1 – L3 . These probes revealed an OFF–ON selective response to the presence of Hg²⁺ ions in MeCN/H₂O 4:1 (v/v), which allowed imaging of this metal ion in Cos‐7 cells in vitro. Once included in silica core–polyethylene glycol (PEG) shell nanoparticles or supported on polyvinyl chloride (PVC)‐based polymeric membranes, ligands L1 – L3 can also selectively sense Hg²⁺ ions in pure water. In particular we have developed an optical sensing array tacking advantage of the fluorescent properties of ligand L3 and based on the computer screen photo assisted technique (CSPT). In the device ligand L3 is dispersed into PVC membranes and it quantitatively responds to Hg²⁺ ions in natural water samples.     

AFM imaging of ligand binding to platelet integrin alphaIIbbeta3 receptors reconstituted into planar lipid bilayers

The platelet integrin alphaIIbbeta3 plays a key role in platelet adhesion, activation, and aggregation at the subendothelium and at protein-coated synthetic biomaterials. In this study, interactions between alphaIIbbeta3 and both protein and peptide ligands for the receptor were imaged under physiological conditions by high-resolution atomic force microscopy (AFM). To directly image the ligand-receptor interactions, alphaIIbbeta3 receptors were reconstituted into a supported lipid bilayer formed on a mica surface in the AFM fluid cell assembly and subsequently activated with Mn2+. Fibrinogen, the natural protein ligand for the integrin, as well as a nanogold-labeled peptide ligand (an RGD-containing heptamer) were infused into the AFM fluid cell, incubated with the reconstituted and activated receptors, and imaged under buffer. Height images illustrating topographical features showed the integrin reconstituted in the bilayer. Fibrinogen molecules binding to the receptors were easily observed in the height images, with fibrinogen showing its characteristic trinodular structure and occasionally bridging integrin receptors. Fibrinogen was observed to bind to integrins at the D-domain consistent with the location of the gamma-chain dodecapeptide, while fibrinogen bridging integrins bound to receptors on opposite sides of the protein consistent with a 2-fold axis of symmetry. Peptide ligands were not visible in height images; however, phase images that map the mechanical properties detected the nanogold labels and demonstrated the presence of peptide ligands bound to the receptors. The results demonstrate the ability of this high-resolution microscopy technique to directly visualize single ligand/receptor interactions in a dynamic and physiologically relevant environment, and establish a framework for future fundamental studies of single protein/receptor interactions during normal pathological processes as well as biomaterial surface-induced thrombosis.     

Efficient delivery of streptavidin to mammalian cells: clathrin-mediated endocytosis regulated by a synthetic ligand

The efficient delivery of macromolecules to living cells presents a formidable challenge to the development of effective macromolecular therapeutics and cellular probes. We describe herein a novel synthetic ligand termed "Streptaphage" that enables efficient cellular uptake of the bacterial protein streptavidin by promoting noncovalent interactions with cholesterol and sphingolipid-rich lipid raft subdomains of cellular plasma membranes. The Streptaphage ligand comprises an N-alkyl derivative of 3 beta-cholesterylamine linked to the carboxylate of biotin through an 11-atom tether. Molecular recognition between streptavidin and this membrane-bound ligand promotes clathrin-mediated endocytosis, which renders streptavidin partially intracellular within 10 min and completely internalized within 4 h of protein addition. Analysis of protein uptake in Jurkat lymphocytes by epifluorescence microscopy and flow cytometry revealed intracellular fluorescence enhancements of over 300-fold (10 microM ligand) with >99% efficiency and low toxicity. Other mammalian cell lines including THP-1 macrophages, MCF-7 breast cancer cells, and CHO cells were similarly affected. Structurally related ligands bearing a shorter linker or substituting the protonated steroidal amine with an isosteric amide were ineffective molecular transporters. Confocal fluorescence microscopy revealed that Streptaphage-induced uptake of streptavidin functionally mimics the initial cellular penetration steps of Cholera toxin, which undergoes clathrin-mediated endocytosis upon binding to the lipid raft-associated natural product ganglioside GM1. The synthetic ligand described herein represents a designed cell surface receptor capable of targeting streptavidin conjugates into diverse mammalian cells by hijacking the molecular machinery used to organize cellular membranes. This technology has potential applications in DNA delivery, tumor therapy, and stimulation of immune responses.     

Evaluation of N-alkyl derivatives of radioiodinated spiperone as radioligands for in vivo dopamine D2 receptor studies: effects of lipophilicity and receptor affinity on the in vivo biodistribution.

A series of radioiodinated spiperone (2'-ISP) derivatives bearing amide N-alkyl substituents (N-methyl-2'-ISP, N-ethyl-2'-ISP, and N-propyl-2'-ISP) were synthesized and evaluated as potential singlet photon emission computed tomographic radiopharmaceuticals for visualizing dopaminergic receptors. The lipophilicity of these ligands (i.e., the partition coefficient for octanol-phosphate buffer) increased as the chain length increased. Investigation of blood-brain barrier permeability in rats showed a parabolic relationship between the brain uptake index and the partition coefficient. In vitro competitive binding studies showed that the relative affinity for the dopamine D2 receptor was in the order of N-propyl-2'-ISP greater than 2'-ISP greater than N-methyl-2'-ISP approximately N-ethyl-2'-ISP. In vivo biodistribution studies showed that the initial brain uptake correlated fairly well with the brain uptake index and that the kinetics of the radioactivity specifically bound to the striatum were strongly influenced by the dopamine receptor binding affinity of the compounds. Thus, the in vivo behavior of these N-alkylated 2'-ISP derivatives involved a complex interplay between receptor affinity, lipophilicity, and blood-brain barrier permeability.     

Affinity chromatography in purification of A1 adenosine receptors.

Purification of A1 adenosine receptor of rat brain membranes was performed using a newly developed affinity gel employing xanthine amine congener (XAC) as an immobilized ligand. The A1 adenosine receptor was solubilized with digitonin-cholate from brain membranes and then purified by a sequential use of affinity chromatography on XAC-agarose, hydroxyapatite chromatography and reaffinity chromatography on XAC-agarose. The A1 adenosine receptor was purified ca. 45,000-fold with a yield of 5%. The final receptor preparation gave a single broad band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a Mr approximately 34,000. This band was also shown to be specifically labelled with an affinity labelling reagent for A1 adenosine receptors. This purification method was also applicable for the complete purification of A1 adenosine receptors from rat testis and human brain membranes.     

Fluorescent iminodiacetamide derivatives as potential ionophores for optical zinc ion-selective sensors.

Fluorescent sensor molecules were synthesized by conjugation of iminodiacetamide derivatives with fluorescent moieties of different structures and their UV-visible and fluorescent properties were characterized in acetonitrile solvent. The fluorescent measurements revealed that the N-(2-naphthyl) and N-phenyl derivatives exhibit a distinct zinc ion-selectivity over alkali and alkaline earth metal ions, while N-(anthrylmethyl) and N-(3-methoxyphenyl) derivatives do not possess any ion-selectivities. In contrast to the fluorescent measurements, all ligands show Zn(2+) selectivity over Ca(2+) and Mg(2+) ions in plasticized PVC membranes using potentiometric signal transduction. This observation found for N-(anthrylmethyl) and N-(3-methoxyphenyl) derivatives can be ascribed to the more hindered interaction between the signalling group of the ionophore and the central metal ion in PVC membranes than in acetonitrile solution upon complexation. From the fluorescent measurements it can also be concluded that the ligands with metal ions form complexes mainly with 2:1 stoichiometry (L(2)M). On complex formation a considerable decrease in the fluorescent intensity was observed for all ligands except the N-(anthrylmethyl) derivative, where a 25 - 30 fold fluorescence enhancement was found, which is explained by the photoinduced electron transfer (PET) mechanism. All ionophores exhibited serious hydrogen ion interference, therefore complexation-induced spectral changes were measured in aprotic acetonitrile solution.     

Different temperature sensitivity of endosomes involved in transport to lysosomes and transcytosis in rat hepatocytes: analysis by free-flow electrophoresis

Endocytosis at reduced temperature has been used to define and characterize endosome subpopulations. Thus, the temperature sensitivity of endosome subpopulations involved in transport to lysosomes and transcytosis in rat hepatocytes was analyzed applying endosome labeling in the isolated perfused rat liver with route-specific ligands in combination with temperature shift protocols. Free-flow electrophoresis (FFE) that separates membranes and organelles based on their surface charge was then applied to isolate functional endosomes. Using asialoorosomucoid (ASOR) and polymeric immunoglobulin A (pIgA) as specific ligands of the lysosomal and transcytotic route, respectively, two distinct endosome subpopulations along either pathway were separated by FFE. Upon a short (1-3 min) internalization at 37 degrees C, 125I-ASOR and fluorescein isothiocyanate (FITC)-pIgA were colocalized in common early endosomes. Following a 5-10 min chase of the ligands at 37 degrees C endosomes labeled with 125I-ASOR were separated from endosomes labeled with FITC-pIgA, indicative of two distinct late compartments along the lysosomal and transcytotic route. Internalization at 16 degrees C resulted in accumulation of both ligands in common early endosomes and, consequently, in inhibition of transport to lysosomes and transcytosis. When 125I-ASOR or 125I-pIgA were first chased into late compartments at 37 degrees C and the temperature was subsequently lowered to 16 degrees C, biliary secretion of 125I-ASOR-derived counts was arrested, while biliary output of 125I-pIgA continued. In summary, ASOR en route to lysosomes can be blocked in early as well as in late endosomes at 16 degrees C, while biliary secretion of pIgA cannot be prevented by temperature reduction once the ligand had been transferred from early to late compartments.     

Disclosing Interfaces of ZnO Nanocrystals Using Dynamic Nuclear Polarization: Sol‐Gel versus Organometallic Approach

The unambiguous characterization of the coordination chemistry of nanocrystal surfaces produced by wet‐chemical synthesis presently remains highly challenging. Here, zinc oxide nanocrystals (ZnO NCs) coated by monoanionic diphenyl phosphate (DPP) ligands were derived by a sol‐gel process and a one‐pot self‐supporting organometallic (OSSOM) procedure. Atomic‐scale characterization through dynamic nuclear polarization (DNP‐)enhanced solid‐state NMR (ssNMR) spectroscopy has notably enabled resolving their vastly different surface‐ligand interfaces. For the OSSOM‐derived NCs, DPP moieties form stable and strongly‐anchored μ₂‐ and μ₃‐bridging‐ligand pairs that are resistant to competitive ligand exchange. The sol‐gel‐derived NCs contain a wide variety of coordination modes of DPP ligands and a ligand exchange process takes place between DPP and glycerol molecules. This highlights the power of DNP‐enhanced ssNMR for detailed NC surface analysis and of the OSSOM approach for the preparation of ZnO NCs.     

Biomimetic droplets for artificial engagement of living cell surface receptors: the specific case of the T-cell

Liquid colloids, in the form of droplets grafted with specific biomolecules, are emerging as potential biomimetic systems. Here we show for the first time the possibility of forming hybrid conjugates between an advanced living cell model, the T-cell of the Jurkat cell line, and a specifically grafted droplet. Using T-cells expressing a fluorescent chimeric protein associated with the TCR/CD3 complex and fluorescent ligand-grafted droplets, we demonstrate formation of an interfacial contact concentrated in linking molecules, the morphology and dynamics of which strongly depend on the targeted receptor. The sequence of events ranges from the initial concentration of molecules following an unbound molecule gradient to active actin-driven spreading and fragmentation of the contact, ending with droplet internalization. We observed synchronized colocalization of receptors and ligands driven by cell dynamics and closely mirrored by the droplet interface. Using intracellular calcium probe Fura-2, we also showed that the cell/droplet interaction can trigger the T-cell signaling cascade. By examining molecular dynamics using FRAP measurements, we observed a nearly frozen cell droplet joining interface. Taken together, our results point to liquid colloids as promising new tools both for probing cell surface interactions and receptor dynamics and for manipulating biological cell functions.     

Identification of synthetic phosphatidylserine translocases from a combinatorial library prepared by directed split-and-pool synthesis

Simple sulfonamide and amide derivatives of tris(2-aminoethyl)amine (Tren) are known to promote the translocation or flip-flop of phosphatidylcholine, but not phosphatidylserine, across bilayer membranes. This paper describes the synthesis of a 300-member, spatially encoded library of Tren derivatives with appended peptide--sulfonamide and peptide--urea arms. The library was synthesized using the Encore method with SynPhase lanterns as the solid support. A high-throughput assay was developed to screen individual members of the library for an ability to translocate a fluorescent NBD derivative of phosphatidylserine across vesicle membranes. Several lead compounds were identified, and one was synthesized independently to confirm its high phosphatidylserine translocation activity.