Synthesis and Biological Evaluation of Metallocene-Tethered Peptidyl Inhibitors of CDC25

Using the pioneering concept developed by Jaouen, Brocard, and co-workers on the replacement of an organic moiety of a known drug by a ferrocenyl unit, we developed organometallic-containing peptides as inhibitors of dual-specificity phosphatases, cell division cycle 25 (CDC25). More precisely, we designed and prepared the first organometallic-containing CDC25 inhibitors based on the lead structure of an organic pentapeptide inhibitor. Extensive (bio)chemical studies were then undertaken. We first determined that the ferrocene- and CpMn(CO)3-containing compounds were unstable in aqueous media, while the ruthenocene and CpRe(CO)3 derivatives displayed robust stability. All peptidyl compounds were found to effectively inhibit the enzymatic activity of purified CDC25 phosphatases in a dose-dependent manner, with the ruthenocene derivative (3) being the most potent of the series. Low doses of 3 could partially prevent CDC25-mediated dephosphorylation of its physiological target CDK1 when ex vivo studies were conducted on cellular extracts containing endogenous and exogenously supplemented CDC25. Unfortunately, cellular studies to assess the overall cytotoxicity of the peptidyl inhibitors as well as specific effects on cell cycle perturbance, such as arrest at checkpoints that depend on CDC25 activity, were unsuccessful, even when compound shuttling to the cell interior was facilitated by a cell-penetrating carrier peptide. A low cellular uptake of the peptides, as determined by inductively coupled plasma mass spectrometry (ICP-MS), was found to be responsible for this result. However, taking into account the observed enzyme inhibition with our organometallic-containing peptides, this study demonstrates the validity of such an approach and paves the way for future studies in this direction.