Direct identification of ligand-receptor interactions on living cells and tissues.

Many cellular responses are triggered by proteins, drugs or pathogens binding to cell-surface receptors, but it can be challenging to identify which receptors are bound by a given ligand. Here we describe TRICEPS, a chemoproteomic reagent with three moieties--one that binds ligands containing an amino group, a second that binds glycosylated receptors on living cells and a biotin tag for purifying the receptor peptides for identification by quantitative mass spectrometry. We validated this ligand-based, receptor-capture (LRC) technology using insulin, transferrin, apelin, epidermal growth factor, the therapeutic antibody trastuzumab and two DARPins targeting ErbB2. In some cases, we could also determine the approximate ligand-binding sites on the receptors. Using TRICEPS to label intact mature vaccinia viruses, we identified the cell surface proteins AXL, M6PR, DAG1, CSPG4 and CDH13 as binding factors on human cells. This technology enables the identification of receptors for many types of ligands under near-physiological conditions and without the need for genetic manipulations.

Differential interaction of an AmB analogue and ergosterol in enantiomeric membranes.

An amphotericin B derivative exhibits differential interaction with vesicles formed from either natural (R)-or unnatural (S)-POPC and ergosterol. This implicates a close association between the polyene macrolide and the phospholipid bilayer and may account for its increased antifungal activity.

Novel guanidine-containing molecular transporters based on lactose scaffolds: lipophilicity effect on the intracellular organellar selectivity.

We have synthesized two lactose-based molecular transporters, each containing seven guanidine residues attached to the lactose scaffold through omega-aminocarboxylate linker chains of two different lengths, and have examined their cellular uptakes and intracellular and organellar localizations in HeLa cells, as well as their tissue distributions in mice. Both molecular transporters showed higher cellular uptake efficiencies than Arg8, and wide tissue distributions including the brain. Mitochondrial localization is of special interest because of its potential relevance to "mitochondrial diseases". Interestingly, it has been found that the intracellular localization sites of the G7 molecular transporters-namely either mitochondria or lysosomes and endocytic vesicles-are largely determined by the linker chain lengths, or their associated lipophilicities.

Design, synthesis, and delivery properties of novel guanidine-containing molecular transporters built on dimeric inositol scaffolds.

We have developed a novel class of synthetic molecular transporters that contain eight residues of guanidine with an inositol dimer as the scaffold. The dimers were prepared by connecting two units of myo- or scyllo-inositol via a carbonate or amide linkage, and the multiple units of the guanidine functionality were constructed on the inositol scaffold by means of peracylation with omega-aminocarboxylate derivatives of varying length. Bioassays based on confocal laser scanning microscopy and fluorescence-activated cell sorter analyses indicated that these transporters display a varying degree of membrane translocating ability, and the intracellular localization and mouse-tissue distribution studies strongly suggested that these transporters undergo substantially different mechanistic processes from those of peptide transporters reported to date. It was also shown that doxorubicin, an anticancer antibiotic, can be efficiently delivered into mouse brain by aid of this type of transporter.