Dynamic assembly of aromatic oligoamide foldamers for stabilizing protein-protein interactions – FOLDynamic

The tethering method (covalent anchoring) used in this project relies on the screening of a library of disulfide-containing foldamers under partially reducing conditions to promote rapid thiol exchange and enable the selection of the highest affinity complex which can be then identified by Mass Spectrometry. Concurrent studies by crystallography and NMR will provide atomic level data on the protein-foldamer covalent adduct to facilitate the optimization of foldamer-protein interactions, ultimately allowing to remove the tether. The methodology relies on iterative improvements via feedback loops : once a first heterodimer of proteins mediated by hybridized double helical foldamers is obtained and characterized by biogysical methods (Solution NMR, X-ray crystallography), optimization of foldamer sequences to get a stable non covalent quaternary complex will proceed via molecular modelling by in silico side chain replacement and subsequent energy minimization. The affinity and kinetics of the complexes will be determined by NMR titration and SPR studies.

The completion of the first part of the FOLDynamic project enabled the following achievements:

- The controlled synthesis of hybrid foldamers of the QnQXm type on solid support (solid-phase synthesis, SPS), with good yields and high purity. (Q: delta-amino acid monomer based on 2,8-quinoline, functionalized at position 4 with a proteinogenic side chain; QX: amino acid monomer based on 2,7-quinoline, substituted at position 8 with a group such as chloro, fluoro, methoxy, ethoxy, etc., and functionalized at position 4 with a proteinogenic side chain.)

- The validation of ligand attachment to a side chain of a monomer directly on solid support (SPS) after the oligomer sequence had been completed.

- The determination of the optimal number (n and m) and nature (QX) of monomers required for foldamer sequences that enable complete hybridization into double helices in aqueous media.

In the second phase, the production of cysteine mutants of the proteins CypA and CnB, targeted for this study, was successfully validated. However, solubility issues with the foldamers prevented successful complexation studies. As an alternative, new protein targets were explored in order to establish a dimerization study using model proteins that are easy to produce and manipulate: human carbonic anhydrase II (HCAII) and ubiquitin (Ub).

Anchoring to HCAII was achieved using a sulfonamide-type ligand with strong affinity for the enzyme’s active site, while anchoring to Ub was carried out via a disulfide bridge as described in the project.

Covalent attachment of these anchors to a foldamer side chain was validated through SPS synthesis of the foldamers.

In the third phase, these foldamers were anchored to HCAII on one hand, and Ub on the other, in order to form protein/foldamer homodimers, which were successfully characterized by mass spectrometry.

Finally, mixing these homodimers resulted in the preferential formation of heterodimers, thanks to the complementarity of foldamer strands bearing complementary chemical functions that stabilize hetero-double-helix formation (X = OMe and F).

These heterodimers were characterized by mass spectrometry, size-exclusion chromatography, and electrophoresis gel analysis.

The core concept of the project has been successfully validated using model proteins. The next step is to apply this approach to proteins of therapeutic interest, with the goal of using it to understand and regulate protein–protein interactions (PPIs) involved in pathological processes.

To achieve this, the side chains decorating the foldamers will need to be optimized to enhance interactions with the selected protein surfaces. Biophysical studies (NMR, X-ray crystallography) will enable atomic-scale characterization of these interactions, and with the support of molecular modeling, it will be possible to design specific and high-affinity sequences to stabilize the targeted protein dimers.

These developments could also be leveraged for shorter-term applications, such as the development of pharmacological tools for imaging, PPI visualization, or diagnostic purposes.

no results published yet

Protein-protein interactions (PPIs) play crucial roles in many biological and pathological processes and represent potential targets for developing new therapeutic approaches. Most targeted PPIs require the development of inhibitors to prevent the association of the two protein partners and abolish the resulting chain of biological events. However, the converse approach remains largely unexplored for therapeutic targets, and would modulate PPIs via stabilizers that reinforce their interactions. An example system is the case of cyclosporin A (CsA) which stabilizes the formation of the immunosuppressant complex CyclophilineA/Calcineurin (CypA/Cn). However, the small molecule CsA, commonly used in the treatment against graft rejection, has significant side effects mainly because of its lack of specificity for the Cn protein. The search for new strategies for stabilizing the CypA/Cn complex is therefore desirable.
Synthetic foldamers have undergone a rapid development in the last decade, driven by the hope that they could achieve functions that match or even go beyond those of biopolymers. Among these, aromatic amide foldamers stand out thanks to their exceptionally predictable, tunable and stable conformations in solution, relatively easy synthesis of secondary and tertiary-like objects as large as small proteins, and a high amenability to crystal growth and structural elucidation. These features all point to aromatic amide foldamers as potential scaffolds to bear proteinogenic side chains and thus allow for ligands that target large protein surface areas. An interesting property of 8-fluoro/chloro-quinoline based foldamers developed in our team, is their ability to form hybridized double helices. This feature will be exploited to build supramolecular complexes stabilizing protein heterodimers, and in particular CypA/Cn within the framework of this project.
A main question is how to rationally arrange proteinogenic side chains on a foldamer to bind to a given protein? Structure-based design is only possible if structures are available, and as yet, atomic details of foldamer-protein complexes have been challenging and are therefore scarce. Key preliminary results support the new possibility to obtain detailed structural information about interactions at a foldamer-protein interface even in the absence of strong binding, provided a type of attachment links the two molecules. The approach we propose falls within the field of dynamic combinatorial chemistry, by screening a library of foldamers equipped with disulfide terminations on the CypA and CnB proteins (Cn subunit B) for which a thiol will be introduced on the surface by mutagenesis (Xaa ? Cys). MS analysis will reveal the complexes of better affinity and will be used to guide design of the corresponding foldamer sequences. Further structural analysis will then be performed with these favourable sequences, by crystallography and by NMR in solution, using labeled proteins. The data obtained will be used to optimize foldamer-protein interactions, in particular using modeling tools. The iterative enhancement of the interactions should allow the design of double-helical hybridized foldamers, capable of dimerizing the CypA and Cn proteins in a supramolecular complex, even in the absence of covalent bonds.
The data resulting from the proposed investigation will provide an essential framework toward future foldamers that could be used to target immunosuppression via the above protein-protein interactions. However, numerous steps that fall out of the scope of the proposed research would have to be made before following a therapeutic objective (evaluation of toxicity, metabolism, bioavailability, and immunogenicity). We nevertheless anticipate a significant impact in the specific field of biomolecular recognition by foldamers, or in the more general area of understanding communication between synthetic and biological molecules.