title: Synthetic heterovalent inhibitors targeting recognition E3 components of the N-end rule pathway.
abstract: Multivalent binding allows high selectivity and affinity in a ligand-protein interaction. The N-end rule pathway is a ubiquitin (Ub)-dependent proteolytic system in which specific E3s, called N-recognins, mediate ubiquitylation through the recognition of types 1 and 2, destabilizing N-terminal residues of substrates. We recently identified a set of E3 Ub ligases (named UBR1-UBR7) containing the 70-residue UBR box, and we demonstrated that UBR1, UBR2, UBR4, and UBR5 can bind to destabilizing N-terminal residues. To explore a model of heterovalent interaction to the N-recognin family, we synthesized the small-molecule compound RF-C11, which bears two heterovalent ligands designed to target N-recognins, together with control molecules with two homovalent ligands. We demonstrate that heterovalent ligands of RF-C11 selectively and cooperatively bind cognate-binding sites of multiple N-recognins and thereby inhibit both types 1 and 2 N-end rule activities. Furthermore, the efficacy of heterovalent RF-C11 was substantially higher than homovalent inhibitors, which can target either a type 1 or type 2 site, providing the molecular basis of designing multivalent inhibitors for the control of specific intracellular pathways. In addition, RF-C11 exhibited higher efficacy and stability, compared with dipeptides bearing destabilizing N-terminal residues, which are known competitive inhibitors of the pathway. We also used the heterovalent compound to study the function of N-recognins in cardiac signaling. Using mouse and rat cardiomyocytes, we demonstrate that the N-end rule pathway has a cell-autonomous function in cardiac proliferation and hypertrophy, explaining our earlier results implicating the pathway in cardiac development and proteolysis of multiple cardiovascular regulators.
authors.affiliation: Center for Pharmacogenetics, Department of Pharmaceutical Sciences, School of Pharmacy, University of Pittsburgh, Pittsburgh, PA 15261, USA.
authors.forename: Min Jae
authors.initials: MJ
authors.lastname: Lee
authors.forename: Krishnendu
authors.initials: K
authors.lastname: Pal
authors.forename: Takafumi
authors.initials: T
authors.lastname: Tasaki
authors.forename: Sayantani
authors.initials: S
authors.lastname: Roy
authors.forename: Yonghua
authors.initials: Y
authors.lastname: Jiang
authors.forename: Jee Young
authors.initials: JY
authors.lastname: An
authors.forename: Rajkumar
authors.initials: R
authors.lastname: Banerjee
authors.forename: Yong Tae
authors.initials: YT
authors.lastname: Kwon
mesh_terms: D000818:Animals; D049109:Cell Proliferation; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D006984:Hypertrophy; D020128:Inhibitory Concentration 50; D051379:Mice; D008822:Mice, Transgenic; D009206:Myocardium / Q000378:metabolism; D032383:Myocytes, Cardiac / Q000378:metabolism*; D009363:Neoplasm Proteins / Q000737:chemistry*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D015398:Signal Transduction; D014908:Triticum / Q000378:metabolism; D025801:Ubiquitin / Q000737:chemistry*; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry*
chemical_list: D004791:Enzyme Inhibitors; D009363:Neoplasm Proteins; D025801:Ubiquitin; C021081:recognins; D044767:Ubiquitin-Protein Ligases