Synthetic heterovalent inhibitors targeting recognition E3 components of the N-end rule pathway.
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.
Lee
Min Jae
MJ
Center for Pharmacogenetics, Department of Pharmaceutical Sciences, School of Pharmacy, University of Pittsburgh, Pittsburgh, PA 15261, USA.
Pal
Krishnendu
K
Tasaki
Takafumi
T
Roy
Sayantani
S
Jiang
Yonghua
Y
An
Jee Young
JY
Banerjee
Rajkumar
R
Kwon
Yong Tae
YT
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*
D004791:Enzyme Inhibitors; D009363:Neoplasm Proteins; D025801:Ubiquitin; C021081:recognins; D044767:Ubiquitin-Protein Ligases