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.

Small molecules that dimerize proteins in living cells provide powerful probes of biological processes and have potential as tools for the identification of protein targets of natural products. We synthesized 7-alpha-substituted derivatives of beta-estradiol tethered to the natural product biotin to regulate heterodimerization of estrogen receptor (ER) and streptavidin (SA) proteins expressed as components of a yeast three-hybrid system. Addition of an estradiol-biotin chimera bearing a 19-atom linker to yeast expressing DNA-bound ER-alpha or ER-beta LexA fusion proteins and wild-type SA protein fused to the B42 activation domain activated reporter gene expression by as much as 450-fold in vivo (10 muM ligand). Comparative analysis of lower affinity Y43A (biotin Kd approximately 100 pM) and W120A (biotin Kd approximately 100 nM) mutants of SA indicated that moderate affinity interactions can be readily detected with this system. Comparison of a 7-alpha-substituted estradiol-biotin chimera with a structurally similar dexamethasone-biotin chimera revealed that yeast expressing ER proteins can detect cognate ligands with up to 5-fold greater potency and 70-fold higher activity than yeast expressing analogous glucocorticoid receptor (GR) proteins. This approach may facilitate the identification of protein targets of biologically active small molecules screened against genetically encoded libraries of proteins expressed in yeast three-hybrid systems.

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We report the synthesis of 7r-substituted β-estradiol derivatives bearing side chains terminated with cholesterol and 3β-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.

Das geht der Zelle unter die Haut: Ein Fluorescein‐Amphotericin‐B‐Konjugat mit einem neuen Piperazin‐Linker (siehe Struktur) diente als Sonde für den Wirkmechanismus von Amphotericin B auf Biomembranen in vivo und in Liposomen‐Experimenten. Hefezellen lokalisieren das Amphotericin‐B‐Analogon in der Membran, während es von Säugetierzellen aufgenommen wird.