Re-designing proteins using Foldamer chemistry : Synthesis of artificial zinc fingers, folding and nucleic acid binding properties – FOLDART

The choice to use urea-based foldamers as a-helix mimics was based on prior work by Partner 1 demonstrating (1) a strong structural similarity between the canonical oligourea helix and the a-helix, (2) the persistence of oligourea helical folding in aqueous solution and (3) the high resistance of the oligourea backbone to the action of proteases. The knowledge of high resolution of oligourea helices combined with that of atomic structures of zinc finger motifs provided a guideline for the design of target composite sequences. The synthetic elaboration of composite zinc fingers involved (1) the synthesis of urea building blocks with the side chains of amino acids corresponding to the sequence to be mimicked, and in particular the key imidazole side chains involved in zinc coordination, (2) and the use of effective solid-phase synthesis methods. With the target sequences in hand, the project focused on studying the binding properties of these artificial zinc fingers to metal ions (zinc in particular), the folding and the tertiary structure as well as recognition of the composite motif by nucleic acids.

The FoldArt project has led to significant advances in the synthesis and characterization of small composite proteins. Notable advances include (1) the creation of a composite zinc finger mimic with potent zinc coordination properties akin to the native zinc finger, (2) the resolution of the three-dimensional structure of the composite zinc finger showing a well-defined tertiary structure. Prior to these design of composite zinc fingers, the investigation of model chimeric systems combining peptide and foldamer backbones has also proved very successful with the discovery of that peptide and oligourea helices can actually communicate within a single strand to create a unique helical structure, suggesting possible applications in the therapeutic field for mimicking bioactive a-peptide helices

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The results obtained during this project led to the publication of several articles in peer-reviewed international journals (3 articles published in 2015 and 2016 and 2 articles to be submitted shortly + one book chapter in 2017). This work has been presented at several international conferences in the form of a poster and oral communications. The discovery of the unique folding properties of peptide/foldamer chimeric helices has led to one patent application and initiation of a collaboration with a start-up company (UREkA Sarl) at IECB.

A significant number of interactions mediated by proteins involve a-helical domains. Hence, synthetic a-helices and their mimetics have attracted considerable attention as scaffolds to target biomacromolecules (e.g. nucleic acids (NA) and proteins). In this context, synthetic oligomers with predictable helical patterns also referred to as helical foldamers have gained increasing interest to mimic isolated helical peptide fragments. Advances in foldamer chemistry together with the finding that oligomeric backbones may retain folding in water bode well for the use of foldamers in biologically relevant situations. However and despite some recent successes, the elaboration of more sophisticated folded molecular architectures, such as tertiary and quaternary folds resembling proteins in terms of shape and functions remains challenging. The FoldArt project is aimed at studying the chemical synthesis, folding and functions of composite proteins created by substituting non-peptide helical segments for natural alpha-helices. We have selected Cys2His2-type zinc fingers, a family of NA-binding metalloproteins containing well defined tertiary folds, as target proteins. A hurdle when using foldamers as protein secondary structure mimetics is to faithfully reproduce the spatial arrangement of the side chains found at a protein surface. Depending on the arrangement of functional groups at the biomimetic helix surface, corresponding zinc finger foldamer (ZFF) motifs may either unfold or maintain protein topology and function. Compared to other foldamer backbones reported in the literature, aliphatic urea-based foldamers developed in the Guichard group (Partner #1) possess several features that make them well suited for the purpose of this study: (1) the canonical 2.5-helix of oligoureas and the a-helix superimpose quite well; (2) helical folding is maintained in aqueous environment, though helix stability is diminished compared to organic solvents and (3) Urea oligomers are highly resistant to the action of proteases. The project also benefits from synthetic access to required monomers bearing natural amino acid side chains including the recently developed histidine-derived monomer crucial for elaborating ZFFs (Nelli et al. Tetrahedron, 2011, DOI:10.1016/j.tet.2011.11.066). Together with partner #2 (Mergny group brings expertise in unusual nucleic acid structures, recognition by ligands and structural characterization), extensive efforts will be dedicated to precisely analyze metal-binding properties, folding patterns and NA recognition of newly generated chimeric peptide/oligourea zinc fingers. We expect the project to bring significant leap forward in the design of synthetic foldamer proteins and their application for the replication/ modulation of protein functions, and toward possible biomedical applications.