Inhibition of Notch signalling using fucose analogs

Abstract

Notch is an evolutionarily conserved cell surface receptor for short range cell‐cell communication. Notch signalling has critical roles in many cell‐fate related events, such as cell differentiation, proliferation and survival. Dysfunction of Notch signalling is associated with various congenital disorders and a wide range of human malignancies. Notch is an important drug target for cancer therapy. However, the initial efforts to inhibit Notch activity with γ‐secretase inhibitors (GSIs) have failed because of severe gastrointestinal toxicity. Novel Notch‐targeting strategies are urgently needed. One unique characteristic of the Notch receptor is the heavy O‐glycosylation of its extracellular domain (ECD). The O‐linked glycans, especially O‐fucose added by POFUT1 (Protein O‐Fucosyltransferase 1), are necessary for proper Notch function, such as protein stability, trafficking and interaction with ligands. We hypothesize that disrupting Notch O‐fucosylation can be a potential approach for Notch inhibition. We have demonstrated that L‐fucose analogs with carbon 6 modifications are potent Notch inhibitors, especially 6‐alkynyl and 6‐alkenyl fucose. The analogs are efficiently incorporated to Notch EGF repeats by POFUT1, and inhibit Notch signalling at sub‐micromolar concentrations. Interestingly, these analogs inhibit Delta but not Jagged‐mediated Notch activity, suggesting they are the first small molecule, ligand‐specific Notch inhibitors, and can be a powerful tool for Notch study. Notch controls the early differentiation of T cells in thymus. Blocking Notch signalling with the analogs showed a complete inhibition of T cell differentiation with Delta ligands in an in vitro OP9 cell culture system. We have preliminary results showing the analogs also showed inhibitory effects in vivo using fetal thymus organ culture (FTOC). Many cancers have mutations in Notch pathway components that make Notch hyperactive. We found the analogs strongly inhibit the constitutively active signalling of NOTCH1 mutants identified from acute lymphoblastic leukemia (T‐ALL) patients. We are now examining the anti‐tumor ability of the analogs in various cancer cell lines where Notch or Notch pathway players contribute to tumorigenesis. The 6‐alkynyl fucose analog can also act as a probe for EGF repeat O‐fucosylation, and help to discover novel POFUT1 substrates. Additional fucose analogs are being synthesized and evaluated to find more effective inhibitors or analogs with distinct activities (e.g. Notch activation, Jagged‐specific or ligand‐nonspecific inhibition).Support or Funding InformationThis work was supported by NIH grant GM061126.