De Novo Engineering of Pd-Metalloproteins and Their Use as Intracellular Catalysts.

The development of transition metal-based catalytic platforms that promote bioorthogonal reactions inside living cells remains a major challenge in chemical biology. This is particularly true for palladium-based catalysts, which are very powerful in organic synthesis but perform poorly in the cellular environment, mainly due to their rapid deactivation. We now demonstrate that grafting Pd(II) complexes into engineered ß-sheets of a model WW domain results in cell-compatible palladominiproteins that effectively catalyze depropargylation reactions inside HeLa cells. The concave shape of the WW domain ß-sheet proved particularly suitable for accommodating the metal center and protecting it from rapid deactivation in the cellular environment. A thorough NMR and computational study confirmed the formation of the metal-stapled peptides and allowed us to propose a three-dimensional structure for this novel metalloprotein motif.

A copper(ii) peptide helicate selectively cleaves DNA replication foci in mammalian cells.

The use of copper-based artificial nucleases as potential anticancer agents has been hampered by their poor selectivity in the oxidative DNA cleavage process. An alternative strategy to solve this problem is to design systems capable of selectively damaging noncanonical DNA structures that play crucial roles in the cell cycle. We designed an oligocationic CuII peptide helicate that selectively binds and cleaves DNA three-way junctions (3WJs) and induces oxidative DNA damage via a ROS-mediated pathway both in vitro and in cellulo, specifically at DNA replication foci of the cell nucleus, where this DNA structure is transiently generated. To our knowledge, this is the first example of a targeted chemical nuclease that can discriminate with high selectivity 3WJs from other forms of DNA both in vitro and in mammalian cells. Since the DNA replication process is deregulated in cancer cells, this approach may pave the way for the development of a new class of anticancer agents based on copper-based artificial nucleases.