Synthetic heterovalent inhibitors targeting recognition E3 components of the N-end rule pathway

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 105; no. 1; pp. 100 - 105
• Main Authors: Lee, Min Jae, Pal, Krishnendu, Tasaki, Takafumi, Roy, Sayantani, Jiang, Yonghua, An, Jee Young, Banerjee, Rajkumar, Kwon, Yong Tae
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 08.01.2008; National Acad Sciences
• Subjects: Animals; Binding sites; Biological Sciences; Cell Proliferation; Cellular biology; Cellular immunity; Enzyme Inhibitors - chemistry; Enzyme Inhibitors - pharmacology; Hypertrophy; Inhibitory Concentration 50; Ligands; Mice; Mice, Transgenic; Molecular interactions; Molecules; Myocardium; Myocardium - metabolism; Myocytes, Cardiac - metabolism; Neoplasm Proteins - chemistry; Protein Binding; Protein Structure, Tertiary; Protein synthesis; Proteins; Reticulocytes; RGS proteins; Rodents; Signal Transduction; Triticum - metabolism; Ubiquitin - chemistry; Ubiquitin-Protein Ligases - chemistry; Western blotting
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.0708465105

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.