Development of constrained peptide inhibitors targeting an oncogenic E3 ubiquitin ligase

SCF Skp2/Cks1 is an E3 ubiquitin ligase, whose substrate specificity is determined by the oncogenic F-box protein Skp2 and the adaptor protein Cks1. A principal target of SCF Skp2/Cks1 is the cyclin-dependent kinase inhibitor p27. Elevated levels of Skp2 and reduced levels of p27 are common in a variety of cancers, and there is consequently a need to develop effective inhibitors of the Skp2-p27 interaction. However, conventional small-molecule approaches are challenging due to the extended bi-molecular interface that spans both Skp2 and Cks1, the lack of suitable binding pockets on this surface, and the intrinsically disordered nature of p27. Here, we develop macrocyclic peptides capable of binding to SCF Skp2/Cks1 with nanomolar affinities, an enhancement of almost two orders of magnitude over the natural p27 peptide. We show that these macrocyclic peptides inhibit p27 ubiquitination in vitro, restore p27 levels in a breast cancer cell line, and reduce cell proliferation.