Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - PEP-PRO-RNA (Peptide-derived bioavailable macrocycles as inhibitors of protein-RNA and protein-protein interactions)

Teaser

Over the last decade, drug discovery faced the problem of decreasing success rates which is mainly caused by the fact that numerous novel biological targets are reluctant to classic small molecule modulation. In particular, that holds true for PPIs and PRIs. Approaches that...

Summary

Over the last decade, drug discovery faced the problem of decreasing success rates which is mainly caused by the fact that numerous novel biological targets are reluctant to classic small molecule modulation. In particular, that holds true for PPIs and PRIs. Approaches that allow the modulation of these interactions provide access to therapeutic agents targeting crucial biological processes that have been considered undruggable so far. The objective of this proposal is the elucidation of general principles for the design of bioavailable peptide-derived macrocyclic compounds and their use for the development of inhibitors of protein‒protein (PPI) and protein‒RNA interactions (PRI). It is proposed to use irregularly structured peptide binding epitopes as starting point for the design of bioactive macrocycles. In a two-step process, high target affinity and bioavailability are installed: a) Peptide macrocyclization for the stabilization of the irregular bioactive secondary structure, and b) Evolution of the cyclic peptide into a bioavailable macrocyclic compound. Using a well-characterized model system developed in my lab, initial design principles will be elucidated. These principles are subsequently used and refined for the development of macrocyclic inhibitors of protein‒protein interactions (PPI) and of protein‒RNA interactions (PRI). The protein‒protein and protein‒RNA complexes selected as targets are of therapeutic interest and corresponding inhibitors hold the potential to be pursued in subsequent drug discovery campaigns.

Work performed

- The potential of novel macroyclzation strategies was explored focusing on the use of ring-closing alkyne metathesis for the stabilization of an irregular peptide secondary structure. A small library of alkyne-crosslinked peptides provided a number of derivatives with improved target affinity relative to the linear parent peptide. In addition, we report the crystal structure of the highest-affinity derivative in a complex with its protein target. It can be expected that the alkyne-based macrocyclization of irregular binding epitopes should give rise to new scaffolds suitable for targeting of currently intractable proteins. (publication: Cromm et al. ChemBioChem, 2016).
- Contribution of flexibilty of the bound ligand to target binding was investigated. Unexpected trends in the binding kinetics of two closely related macrocyclic peptides that bind their receptor protein with high affinity were observed. Isothermal titration calorimetry, 19F NMR experiments and molecular dynamics simulations reveal that increased conformational flexibility of the macrocycle–receptor complex reduces dissociation rates and contributes to complex stability. This observation has impact on macrocycle design strategies that have so far mainly focused on the stabilization of bioactive ligand conformations (Colaboration partner: Christoph Rademacher, MPI Potsdam; publicaiton: Glas et al. Chem. Eur. J. 2017).
- We investigated the potential of computational approaches for the affintiy maturation of macrocyclic peptide ligands. For that purpose a virtual screen based on molecular docking was performed that takes peptides with non-natural amino acids into consideration. These macrocycles bear large and flexible substituents which usually complicate the use of docking approaches. A virtual library containing more than 1400 structures was screened against the target focusing on docking poses with the core structure resembling a known bioactive conformation. Based on this screen, a macrocyclic peptide involving two non-natural amino acids was evolved showing increased target affinity and biological activity. Predicted binding modes were verified by X-ray crystallography. The presented workflow allows the screening of large macrocyclic peptides with diverse modifications thereby expanding the accessible chemical space and reducing synthetic efforts (Colaboration parnter: Sven Hennig, VU Amsterdam; Oliver Koch, TU Dortmund; publication: Krüger et al. J. Med. Chem. 2017).
- We developed a stapled peptide inhibitor that targets the interaction between b-catenin and T cell factor/lymphoid enhancer-binding factor transcription factors, which are crucially involved in Wnt signaling. Our integrative approach combines peptide stapling to optimize proteolytic stability, with lessons learned from
cell-penetrating peptide (CPP) design to maximize cellular uptake resulting in NLS-StAx-h, a selective, cell permeable, stapled peptide inhibitor of oncogenic Wnt signaling that efficiently inhibits b-catenin-transcription factor interactions. We expect that this type of integrative strategy that endows stapled peptides with CPP features will be generally useful for developing inhibitors of intracellular PPIs. (collaboration partners: Trevor C. Dale, Cardiff University; Stefan Heinrichs, Uniklinikum Essen; Dennis Schade, University Greifswald; Tanja Bange, MPI Dortmund; publication: Dietrich et al. Cell Chem. Biol. 2017).

Final results

Summary of major achievments so far:
- A computational tool that facilitates affinity maturation of flexible macrocyclic peptide ligands has been developed.
- Cell-permeble and selective inhibitor of the protein beta-catenin has been designed.

Expected results:
- Insights into the requirements for selective RNA binding.
- Novel PPI and PRI inhibitors.