Conformationally restrained de novo peptides to modulate Skp1-Skp2 interaction of the SCF E3 ligase

Skp2, the substrate-recognition component of the SCF ubiquitin ligase complex, has been widely implicated in oncogenesis across diverse malignancies, positioning it as a compelling therapeutic target. The protein-protein interaction interface of Skp1-Skp2 offers an effective intervention point, as disruption of this interaction can impair the ability of Skp2 to identify and bind to its downstream substrates. However, inhibiting this interface with small-molecule inhibitors poses considerable challenge. Here, we introduce de novo peptide sequences through in silico screening of a peptide pool to target the Skp1-Skp2 core interface. The screening identified four linear precursors, which were further conformationally restricted to increase affinity and conformational stability by deploying cysteine-mediated late-stage crosslinking. Biophysical and cellular analyses of these crosslinked peptides revealed candidate peptides, P2SS and P4SS, which bind effectively to the desired interface and reduce Skp2 protein expression. Skp2 reduction further attenuates p27 ubiquitination, leading to the accumulation of p27 and subsequent G0/G1 cell-cycle arrest, ultimately resulting in cell death. Collectively, our findings establish the potential of first-in-class peptides in targeting the Skp1-Skp2 interface for the treatment of cancers driven by Skp2.