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

Abstract

Multivalent binding allows high selectivity and affinity in a ligandprotein 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.N-recognin ͉ protein degradation ͉ ubiquitin ͉ cardiovascular cardiomyocyte

Mechanisms for Rescue of Correctable Folding Defects in CFTRΔF508

“…However, cells often tolerate the loss of ER quality control factors well (Wilhovsky et al, 2000;Dai et al, 2003). Thus, the development of approaches to inactivate specific E3 ubiquitin ligases (Burger and Seth, 2004;Lee et al, 2008) have the potential to permit foldable pools of CFTR⌬F508 or other disease-causing mutants to accumulate and may serve as a means to treat CF.…”

Pharmacological Modulation of the N-End Rule Pathway and Its Therapeutic Implications

“…This conjecture was experimentally tested using RF-C11, a synthetic heterovalent compound that has both an N-terminal Arg (type 1) and an N-terminal Phe (type 2) chemically linked by two C11 hydrocarbon chains (Figure 3B) [30, 100]. Compared with homovalent controls such as RR-C11 (with two N-terminal Arg) and FF-C11 (with two N-terminal Phe) and monovalent dipeptides such as Arg-Ala and Phe-Ala, RF-C11 showed significantly higher inhibitory efficacy for in vitro ubiquitination and degradation of model N-end rule substrates.…”

Characterization of Arginylation Branch of N-end Rule Pathway in G-protein-mediated Proliferation and Signaling of Cardiomyocytes

“…Primary Cardiomyocytes and Explanted Hearts-Primary cardiomyocytes from mouse embryonic hearts were isolated as described with some modifications (30). Briefly, dissected hearts at E13.5 were digested in Hanks' balanced salt solution containing 0.2% collagenase II, 0.005% trypsin, and 0.1% chicken serum for 15 min at 37°C.…”

“…Immunostaining of paraffin sections and whole-mount immunohistochemical staining of embryos were performed as described (30). For the in vivo proliferation assay, pregnant mice were intraperitoneally injected (150 mg/g) with BrdU in 250 l of saline.…”