title: Inhibition of SCF ubiquitin ligases by engineered ubiquitin variants that target the Cul1 binding site on the Skp1-F-box interface.
abstract: Skp1-Cul1-F-box (SCF) E3 ligases play key roles in multiple cellular processes through ubiquitination and subsequent degradation of substrate proteins. Although Skp1 and Cul1 are invariant components of all SCF complexes, the 69 different human F-box proteins are variable substrate binding modules that determine specificity. SCF E3 ligases are activated in many cancers and inhibitors could have therapeutic potential. Here, we used phage display to develop specific ubiquitin-based inhibitors against two F-box proteins, Fbw7 and Fbw11. Unexpectedly, the ubiquitin variants bind at the interface of Skp1 and F-box proteins and inhibit ligase activity by preventing Cul1 binding to the same surface. Using structure-based design and phage display, we modified the initial inhibitors to generate broad-spectrum inhibitors that targeted many SCF ligases, or conversely, a highly specific inhibitor that discriminated between even the close homologs Fbw11 and Fbw1. We propose that most F-box proteins can be targeted by this approach for basic research and for potential cancer therapies.
authors.affiliation: Banting and Best Department of Medical Research, Terrence Donnelly Center for Cellular and Biomolecular Research, University of Toronto, Toronto, ON, Canada M5S 3E1; Department of Molecular Genetics, Terrence Donnelly Center for Cellular and Biomolecular Research, University of Toronto, Toronto, ON, Canada M5S 3E1;
authors.forename: Maryna
authors.initials: M
authors.lastname: Gorelik
authors.affiliation: Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON, Canada M5G 1X5;
authors.forename: Stephen
authors.initials: S
authors.lastname: Orlicky
authors.affiliation: Banting and Best Department of Medical Research, Terrence Donnelly Center for Cellular and Biomolecular Research, University of Toronto, Toronto, ON, Canada M5S 3E1; Department of Molecular Genetics, Terrence Donnelly Center for Cellular and Biomolecular Research, University of Toronto, Toronto, ON, Canada M5S 3E1;
authors.forename: Maria A
authors.initials: MA
authors.lastname: Sartori
authors.affiliation: Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON, Canada M5G 1X5;
authors.forename: Xiaojing
authors.initials: X
authors.lastname: Tang
authors.affiliation: Banting and Best Department of Medical Research, Terrence Donnelly Center for Cellular and Biomolecular Research, University of Toronto, Toronto, ON, Canada M5S 3E1; Department of Molecular Genetics, Terrence Donnelly Center for Cellular and Biomolecular Research, University of Toronto, Toronto, ON, Canada M5S 3E1;
authors.forename: Edyta
authors.initials: E
authors.lastname: Marcon
authors.affiliation: Department of Chemistry and Chemical Biology, Cornell University, Argonne, IL 60439;
authors.forename: Igor
authors.initials: I
authors.lastname: Kurinov
authors.affiliation: Banting and Best Department of Medical Research, Terrence Donnelly Center for Cellular and Biomolecular Research, University of Toronto, Toronto, ON, Canada M5S 3E1; Department of Molecular Genetics, Terrence Donnelly Center for Cellular and Biomolecular Research, University of Toronto, Toronto, ON, Canada M5S 3E1;
authors.forename: Jack F
authors.initials: JF
authors.lastname: Greenblatt
authors.affiliation: Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON, Canada M5G 1X5; Institut de Recherche en Immunologie et Cancérologie, Université de Montréal, Montreal, QC, Canada H3C 3J7;
authors.forename: Mike
authors.initials: M
authors.lastname: Tyers
authors.affiliation: Banting and Best Department of Medical Research, Terrence Donnelly Center for Cellular and Biomolecular Research, University of Toronto, Toronto, ON, Canada M5S 3E1; Department of Molecular Genetics, Terrence Donnelly Center for Cellular and Biomolecular Research, University of Toronto, Toronto, ON, Canada M5S 3E1;
authors.forename: Jason
authors.initials: J
authors.lastname: Moffat
authors.affiliation: Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON, Canada M5G 1X5; Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada M5S 3E1.
authors.forename: Frank
authors.initials: F
authors.lastname: Sicheri
authors.affiliation: Banting and Best Department of Medical Research, Terrence Donnelly Center for Cellular and Biomolecular Research, University of Toronto, Toronto, ON, Canada M5S 3E1; Department of Molecular Genetics, Terrence Donnelly Center for Cellular and Biomolecular Research, University of Toronto, Toronto, ON, Canada M5S 3E1; sachdev.sidhu@utoronto.ca.
authors.forename: Sachdev S
authors.initials: SS
authors.lastname: Sidhu
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D018797:Cell Cycle Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics; D044842:Cullin Proteins / Q000737:chemistry / Q000378:metabolism*; D015195:Drug Design; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology; D044783:F-Box Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics; D000073979:F-Box-WD Repeat-Containing Protein 7; D014644:Genetic Variation; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019151:Peptide Library; D015202:Protein Engineering; D054730:Protein Interaction Domains and Motifs; D044843:SKP Cullin F-Box Protein Ligases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D044767:Ubiquitin-Protein Ligases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics; D014452:Ubiquitins / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology*; D044844:beta-Transducin Repeat-Containing Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics
chemical_list: D018797:Cell Cycle Proteins; C100318:Cullin 1; D044842:Cullin Proteins; D004791:Enzyme Inhibitors; D044783:F-Box Proteins; D000073979:F-Box-WD Repeat-Containing Protein 7; C471645:FBXW11 protein, human; C513273:FBXW7 protein, human; D019151:Peptide Library; D014452:Ubiquitins; D044844:beta-Transducin Repeat-Containing Proteins; D044843:SKP Cullin F-Box Protein Ligases; D044767:Ubiquitin-Protein Ligases
keywords: Cul1 affinity; Fbxw11; Fbxw7; SCF inhibitors; β-Trcp