Chemical probes of Skp2-mediated p27 ubiquitylation and degradation

Abstract

Skp2 is a member of the F-box family of proteins that serve as substrate-specific adaptors in Skp1-CUL1-ROC1-F-box (SCF) E3 ubiquitin ligases. Skp2 (Fbxl1) directly binds to the tumor suppressor p27 in the context of the SCF^Skp2 E3 ubiquitin ligase to ubiquitylate and target-phosphorylated p27 for proteasomal degradation. As p27 is a powerful suppressor of growth in a variety of cells, and as Skp2 is also overexpressed in many human cancers, Skp2 is considered an oncogene and an intriguing drug target. However, despite 20 years of investigation, a valid chemical inhibitor of Skp2-mediated degradation of p27 has not been identified. Recently, an increasing number of compounds designed to have this bioactivity have been reported. Here, we conduct a meta-analysis of the evidence regarding bioactivity, structure, and medicinal chemistry in order to evaluate and compare these Skp2 inhibitor compounds. Despite chemically diverse compounds with a wide array of Skp2-mediated p27 ubiquitylation inhibition properties reported by several independent groups, no current chemical probe formally qualifies as a validated pharmaceutical hit compound. This finding suggests that our knowledge of the structural biochemistry of the Skp2-p27 complex remains incomplete and highlights the need for novel modes of inquiry.

Fig. 1. SCF^Skp2 complex with proposed inhibitor binding sites. ICM-Skin representation of PDB: ; 2AST. Compounds C1 and C2 target the Skp2 (cyan) and phospho-p27 peptide (green) interface. 22d targets the Skp2 and Cks1 (purple) interface preventing assembly of these subunits. Compound #25 and compound A both target the interface of Skp2 and Skp1 (orange).

Fig. 2. SCF^Skp2 druggable pockets. ICM-Skin and ribbon representation of PDB: ; 2AST. Skp1 (orange), Cks1 (purple) and phospo-p27 (green) are shown as mesh while Skp2 is represented as a yellow ribbon structure. The Skp2–Cks1–p27 interface pocket (cyan) is the target site for C1 and C2. It is not certain if linichlorin A and gentian violet also target this site. This pocket exists at an interface between Skp2 and Cks1 when phospo-p27 is removed. The Skp2–Cks1 interface pocket (red) has been previously noted to be an unusually large pocket and is the target site for 22d and NSC689857. It is also not certain if linichlorin A and gentian violet also target this site. The Skp2–Skp1 interface with no pocket is indicated with an arrow and compound #25 and compound A target this region.

Fig. 3. ICM docking scores of SCF^Skp2 inhibitors. The dotted line represents the putative threshold ICM docking score predictive of binding (≤–32). C1 and C2 were docked to their corresponding Skp2–P–p27 pocket. NSC689857 and compound A were docked to the Skp2–Skp1 interface with no originally identifiable pocket. 22d and NSC689857 were docked to the Skp2–Cks1 interface pocket. Both linichlorin A and gentian violet were docked to the Skp2–P–p27 and the Skp2–Cks1 pockets. Superscript 1 denotes docking to the Skp2–P–p27 pocket and superscript 2 denotes docking to the Skp2–Cks1 pocket.