The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences Dissertations and Theses (Open Access)
Discovery and effects of pharmacological inhibition of the E3 ligase Skp2 by small molecule protein-protein interaction disruptors
Author ORCID Identifier
Date of Graduation
Doctor of Philosophy (PhD)
Shuxing Zhang, Ph.D.
Varsha Gandhi, Ph.D.
Scott Gilbertson, Ph.D.
Zhimin Lu, Ph.D.
Giulio Draetta, Ph.D.
Skp2 (S-phase kinase-associated protein 2), one component of the SCF E3 ubiquitin ligase complex, directly interacts with Skp1 and indirectly associates with Cullin1 and Rbx1 to bridge the E2 conjugating enzyme with its protein substrate to execute its E3 ligase activity. Skp2 is an Fbox protein (due to it containing an Fbox domain) and it is the rate-limiting component of the SCF complex. Skp2 targets several cell-cycle regulatory proteins for ubiquitination and degradation; most notable and significant for cancer are the cyclin-dependent kinase inhibitor, p27. Skp2 is an oncogene and studies have shown that over-expression of Skp2 leads to increased degradation of p27 and increased proliferation in several tumor types. Additionally, Skp2 is over-expressed in multiple human cancers. Clearly, Skp2 represents an attractive target for attenuating p27 ubiquitination and subsequent cell cycle progression. However, Skp2 does not have an easily identifiable and druggable “pocket” on which small molecules can bind; it interacts with Skp1 through the Fbox domain and binds to an accessory protein called Cks1 to bind to p27. Despite this hurdle, in this study, two selective small molecule inhibitors of the Skp2 SCF complex were discovered via an in silico screen that disrupt two places: the Skp1/Skp2 interaction site and the p27 binding site via targeting hot-spot residues. The Skp1/Skp2 inhibitor disruption resulted in restoring p27 levels in the nucleus and blocks cancer progression and cancer stem cell traits. Additionally, the inhibitors phenocopy the effects of genetic Skp2 deficiency. Two specific residues on Skp2 were predicted to bind to this Skp1/Skp2 inhibitor: Trp97 and Asp98. When these residues were mutated to alanine, the inhibitor lost its ability to bind to Skp2. To investigate the flexibility and understand the conformational change upon inhibitor binding and dynamics of the SCF complex, molecular dynamics simulations, homology models, and structural analysis was carried out on the complex with and without the inhibitors. These simulations showed that the contributions of the N-terminal tail region of Skp2 does not contribute directly to the binding of these inhibitors; but its conformation is important in the context of the other members of the SCF complex. Further dynamics analysis validated the mutagenesis results, showing that the two Skp2 mutants (Trp97Ala, Asp98Ala) that retained Skp1 binding but blocked inhibitor binding were stable, whereas the mutant that was unable to retain Skp1 binding (Trp127Ala) showed destabilization in the Fbox domain. Finally, active recruitment events after post-translational modifications are shown to be possible by the interaction of phosphorylated Ser256 on Skp2 with Lys104 loop region on Cul1 The model shows that this is due to the significant flexibility in the F-box domain of Skp2, making this interaction very likely. These results show that Skp2 is a promising target on which protein-protein interaction disruptors can be designed, and consideration of the dynamics of protein complexes is required to understand ligand binding.
Skp2, Hot spots, Drug Design, protein-protein interaction, E3 ligase, SCF complex, molecluar dynamics, virtual screening, homology modeling, rational drug design
Medicinal Chemistry and Pharmaceutics Commons, Pharmaceutics and Drug Design Commons, Pharmacology Commons, Structural Biology Commons, Therapeutics Commons