Natural ligand-nonmimetic inhibitors of the lipid-transfer protein CERT

• Published in: Communications chemistry Vol. 2; no. 1
• Main Authors: Nakao, Naoki, Ueno, Masaharu, Sakai, Shota, Egawa, Daichi, Hanzawa, Hiroyuki, Kawasaki, Shohei, Kumagai, Keigo, Suzuki, Makoto, Kobayashi, Shu, Hanada, Kentaro
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 20.02.2019; Nature Publishing Group
• Subjects: 631/92/287/1192; 639/638/309; 639/638/45/612/1222; 82; Affinity; Chemical synthesis; Chemistry; Chemistry and Materials Science; Chemistry/Food Science; Crystallography; Domains; Eukaryotes; Hydrogen bonding; In vivo methods and tests; Inhibitors; Ligands; Lipids; Membranes; Mimetic compounds; Proteins; Screening; Surface plasmon resonance; Transport
• ISSN: 2399-3669; 2399-3669
• DOI: 10.1038/s42004-019-0118-3

Lipid transfer proteins mediate inter-organelle transport of membrane lipids at organelle contact sites in cells, playing fundamental roles in the lipidome and membrane biogenesis in eukaryotes. We previously developed a ceramide-mimetic compound as a potent inhibitor of the ceramide transport protein CERT. Here we develop CERT inhibitors with structures unrelated to ceramide. To this aim, we identify a seed compound with no ceramide-like structure but with the capability of forming a hydrogen-bonding network in the ceramide-binding START domain, by virtual screening of ~3 × 10 6 compounds. We also establish a surface plasmon resonance-based system to directly determine the affinity of compounds for the START domain. Then, we subject the seed compound to a series of in silico docking simulations, efficient chemical synthesis, affinity analysis, protein-ligand co-crystallography, and various in vivo assays. This strategy allows us to obtain ceramide-unrelated compounds that potently inhibited the function of CERT in human cultured cells. The ceramide transport protein CERT is of potential therapeutic interest but is typically targeted using ceramide-derived ligands. Here the authors use virtual screening and a quantitative surface plasmon resonance assay to identify ceramide-nonmimetic inhibitors of a CERT subdomain and test their activity in live cells.