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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - PROFOLIG (Covalent-ligation-assisted elucidation of protein-aromatic foldamer interactions)

Teaser

Summary

Protein-protein interactions (PPIs) play crucial roles in many biological processes and diseases, and represent potential targets to develop new therapeutic approaches. However, PPI inhibition often requires ligands that can cover large areas of protein surfaces, such as other proteins (eg antibodies). With their medium size, the stability and predictability of their helical folded structures, aromatic foldamers constitute candidates for protein surface recognition.

The main objectives of this project consisted in the development of aromatic foldamers to interact specifically with the protein surfaces by anchoring approaches in aqueous media towards the development of specific binding ligands in the absence of anchor.

The devised strategy consisted in an iterative design process that exploits the structural information of folded synthetic oligomer sequences interacting with protein surfaces to implement subsequent refinements. Starting with a well-studied protein i.e. human carbonic anhydrase II (HCA), we used an inhibitor strategy as first-principles design to obtain structural information of the interactions between the anchored foldamer and HCA protein surface that considers features such as helical length, proteinogenic side chains and hydrogen bonding ability. This anchoring approach was then extended to a covalent strategy in the case of therapeutically interesting proteins with large contact surfaces involved in protein-protein interactions (PPI) such as cyclophilin A (CypA), interleukin-4 (IL4) and ubiquitin (Ub). We finally envisioned to remove the covalent linker in the case of protein/foldamer complexes with stronger affinity.

The methodology achieved the following objectives: a) synthesis of a library of foldamer sequences of different length containing various proteinogenic side chains, and anchoring them to protein surfaces; b) screening of the protein-foldamer interactions by induced circular dichroism (ICD); c) assessing the structural information either by solid state structures (X-ray crystallography) or by solution NMR studies; d) new design optimization based on the structural information.

Work performed

A series of foldamer libraries were designed and synthesized in such a way that these have linkers at N-terminus to anchor to the protein surface and include proteinogenic side chains on quinoline units to interact with the protein surface. These complexes/adducts were prepared by a convergent procedure.

The interactions of the foldamers with the protein surfaces were assessed in solution by induced circular dichroism (ICD), LCMS analysis, NMR spectroscopy and in the solid state by X-ray crystal structure analysis.

The results showed that two long foldamer sequences bearing proteinogenic side chains could cover up to 800 Ã…Â² of the human carbonic anhydrase II (HCA) surface. These foldamers are composed of amino-quinolinecarboxylic acids units bearing proteinogenic side chains and of more flexible aminomethyl-pyridinecarboxylic acids units that enhance helix handedness dynamics. Crystal structures of those HCA-foldamer complexes were obtained with a 9-mer and a 14-mer both showing extensive protein-foldamer hydrophobic contacts. In addition, foldamer-foldamer interactions seem to be prevalent in the crystal packing, leading to the peculiar formation of an HCA superhelix wound around a rod of stacked foldamers. Same complexes were also studied in solution by using NMR.

The concept was extended to therapeutically relevant proteins such as cyclophilin (CypA), interleukin-4 (IL4) and human ubiquitin (Ub) involved in PPIs. Since these proteins do not possess active sites or known small molecule ligands, tethering consisted of covalently ligating foldamers at a cysteine residue introduced via site-directed mutagenesis using either short or long linkers. We have used appended active disulfides at the N- terminus of aromatic oligoamide foldamers on solid phase, optimized conditions under which foldamer-protein disulfide bridges form, and developed methods to purify these adducts and characterize them by NMR and mass spectrometry. ICD and solution NMR results indicating selective foldamer-protein interactions. In the case of CypA, the experiments were also carried without any tether. Altogether, our results constitute an important milestone toward the identification of aromatic foldamer-protein interactions and toward the structure elucidation of foldamer-protein interfaces in the contexts of IL4, CypA and Ub.

One paper has been published in Eur. J. Org. Chem. Describing the results obtained in WPs 1 and 2. Recently another paper has been submitted describing the results obtained in WPs 1, 2 and 3. During his stay, the researcher has had a chance to present the preliminary work on several occasions.

Final results

The work developed during the reporting period went beyond the state of the art of the foldamer field by expanding the scope of protein surface recognition by aromatic helical foldamers: a) the discovery that the aromatic oligoamide foldamers selectively binds to protein surfaces via either complex formation with HCA or via disulphide covalent link in the case of cyclophilin A, IL4 and ubiquitin. In addition, it was found that long foldamers (having around 3kDa) were interacting with large contact surface of HCA as observed during their structural elucidation. b) the introduction of long aromatic oligoamide foldamers onto the protein surface for the purpose of enhancing interactions and covering large surface area was achieved for the first time in the foldamer field. Several cases of selective foldamer binding were found, mediated either by inhibitor strategy in the case of HCA, or by covalent ligation strategy or non-covalent/complex strategy in the case of CypA, IL4 and ubiquitin. The structures of two of HCA-foldamer complexes could be elucidated by X-ray crystallographic analysis. These are the first examples of crystal structures of long foldamers (up to 14-units) with HCA surface and constitute important milestones in the challenging area of protein surface recognition by synthetic foldamers, a field that had until recently suffered from a complete lack of solid state evidence with long foldamers; c) the studies performed successfuly provided proof of concept that other proteinogenic side chains can be incorporated onto monomers in foldamer sequences and serve as reporter of the binding event.

Overall, the project gave an important contribution to the challenging field of protein-protein interactions. Advancements in this field are poised to have a tremendous impact in the development of foldamer-based ligands that recognize large areas of protein surfaces. They can also be candidates as protein markers for imaging and diagnostic or as inhibitors of protein-protein interactions which lie at the core of most cellular processes and thus constitute targets for the development of pharmacological tools or therapeutic agents. Such developments would introduce â€œmedium-sizedâ€ molecules into pharmaceutical development which would represent a paradigm change aside of the prevalence of small molecules and protein-based therapeutics. All these potential applications are well in line with the Horizon 2020 societal challenge of â€œHealth, demographic change and wellbeingâ€ and will contribute to the strengthening of the European Unionâ€™s excellence and attractiveness in research and innovation and its economic and industrial competitiveness.