- Cell-Surface Receptor-Ligand Interaction Analysis with Homogeneous Time-Resolved FRET and Metabolic Glycan Engineering: Application to Transmembrane and GPI-Anchored Receptors
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Ligand-binding assays are the linchpin of drug discovery and medicinal chemistry. Cell-surface receptors and their ligands have traditionally been characterized by radioligand-binding assays, which have low temporal and spatial resolution and entail safety risks. Here, we report a powerful alternative (GlycoFRET), where terbium-labeled fluorescent reporters are irreversibly attached to receptors by metabolic glycan engineering. For the first time, we show time-resolved fluorescence resonance energy transfer between receptor glycans and fluorescently labeled ligands. We describe GlycoFRET for a GPI-anchored receptor, a G-protein-coupled receptor, and a heterodimeric cytokine receptor in living cells with excellent sensitivity and high signal-to-background ratios. In contrast to previously described methods, GlycoFRET does not require genetic engineering or antibodies to label receptors. Given that all cell-surface receptors are glycosylated, we expect that GlycoFRET can be generalized with applications in chemical biology and biotechnology, such as target engagement, receptor pharmacology, and high-throughput screening.
- Stockmann, Henning,Todorovic, Viktor,Richardson, Paul L.,Marin, Violeta,Scott, Victoria,Gerstein, Clare,Lake, Marc,Wang, Leyu,Sadhukhan, Ramkrishna,Vasudevan, Anil
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p. 16822 - 16829
(2017/11/28)
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- Novel hyaluronic acid-methotrexate conjugates for osteoarthritis treatment
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Hyaluronic acid (HA) provides synovial fluid viscoelasticity and has a lubricating effect. Injections of HA preparations into the knee joint are widely used as osteoarthritis therapy. The current HA products reduce pain but do not fully control inflammation. Oral methotrexate (MTX) has anti-inflammatory efficacy but is associated with severe adverse events. Based on the rationale that a conjugation of HA and MTX would combine the efficacy of the two clinically evaluated agents and avoid the risks of MTX alone, we designed HA-MTX conjugates in which the MTX connects with the HA through peptides susceptible to cleavage by lysosomal enzymes. Intra-articular injection of our HA-MTX conjugate (conjugate 4) produced a significant reduction of the knee swelling in antigen-induced arthritis rat, whereas free MTX, HA or a mixture of HA and MTX showed no or marginal effects on the model. The efficacy of conjugate 4 was almost the same as that of MTX oral treatment. Conjugate 4 has potential as a compound for the treatment of osteoarthritis.
- Homma, Akie,Sato, Haruhiko,Okamachi, Akira,Emura, Takashi,Ishizawa, Takenori,Kato, Tatsuya,Matsuura, Tetsu,Sato, Shigeo,Tamura, Tatsuya,Higuchi, Yoshinobu,Watanabe, Tomoyuki,Kitamura, Hidetomo,Asanuma, Kentaro,Yamazaki, Tadao,Ikemi, Masahisa,Kitagawa, Hironoshin,Morikawa, Tadashi,Ikeya, Hitoshi,Maeda, Kazuaki,Takahashi, Koichi,Nohmi, Kenji,Izutani, Noriyuki,Kanda, Makoto,Suzuki, Ryochi
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experimental part
p. 4647 - 4656
(2009/12/01)
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