Scaffold hybridization approach targeting PPIs

Informazioni relative al progetto

SHAPPI

ID dell’accordo di sovvenzione: 749012

DOI

10.3030/749012

Progetto chiuso

Data della firma CE 3 Marzo 2017

Data di avvio 1 Maggio 2017

Data di completamento 30 Aprile 2019

Finanziato da

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Costo totale

€ 183 454,80

Contributo UE

€ 183 454,80

Coordinato da

UNIVERSITY OF LEEDS
United Kingdom

Periodic Reporting for period 1 - SHAPPI (Scaffold hybridization approach targeting PPIs)

Periodo di rendicontazione: 2017-05-01 al 2019-04-30

Sintesi del contesto e degli obiettivi generali del progetto

Protein-protein interactions (PPIs) control all cellular processes relevant to health and disease, hence an outstanding challenge in chemical biology is to develop generic approaches for PPI inhibition. Such inhibitors represent unique tools to understand biological processes and starting points for drug discovery, for treatment of multiple illnesses for which effective treatments don’t currently exist e.g. cancer. Proteins recognize each other through a large, shallow, less well-defined interaction interface. Key amino acids (termed hot-spots) are responsible for the majority of the binding energy, however these tend to be similar for different PPIs, thus selective PPI inhibition particularly in related protein families is challenging. Small molecules would be ideal therapeutics, but tend to be unsuccessful against larger, more challenging interfaces.
An alternative approach to target these interfaces is the use of peptidomimetics or proteomimetics. These structures consist of non-natural building blocks capable of mimicking the 3D conformation required for bioactivity or reproducing the spatial projection of the functionality required for protein recognition. These scaffolds are generally suitable to target helix mediated interactions but targeting β-sheet mediated PPIs is still a significant challenge. Several therapeutically relevant β-sheets are known, however conventional drug design tends to be unsuccessful, thus new methodologies are needed. We hypothesized that using peptide-small molecule hybrids, where the peptide could act as an anchor on the protein surface directing the small molecule fragment toward a specific binding site could result in improved ligands for such interactions.
The fellowship focussed on inhibiting challenging PPIs using different peptidomimetic, proteomimetic, and peptide-small molecule hybrid structures (Figure 1). The objectives were to design and synthesize peptidomimetic/proteomimetic and hybrid structures and characterize their binding and structural features in order to develop generic approaches suitable for PPI inhibition.

Lavoro eseguito dall’inizio del progetto fino alla fine del periodo coperto dalla relazione e principali risultati finora ottenuti

Small molecule-peptide hybrids targeting PPIs
The fellowship focussed on developing a method that could help to identify small-molecule binding motifs targeting challenging PPI interfaces. For this we chose the -sheet mediated Shank1 PDZ/GKAP interaction, which plays a role in neurodegenerative diseases. Our goal was to replace the natural amino acid residues with more drug-like small molecule fragments thus creating hybrid structures, which could result in improved properties. Truncated peptides were designed that can serve as an anchor on the protein surface guiding the attached small molecule to a specific binding site. The peptides were synthesized equipped with a functional group (hydrazide) that can react with a library of small molecule fragments (aldehydes), which were tested against the target using an optimized biophysical assay. The most promising hit compounds were synthesized in larger quantities, their binding parameters were measured, and the key structural features required for inhibiting the interaction were determined. To shed light on the exact molecular nature of the interaction crystal structures were solved for the hybrids in complex with the target protein.
With this approach we were able to identify fragment containing ligands that have comparable affinity to the wild-type sequence, thus developed a methodology that can tackle more challenging β-sheet mediated interfaces. We anticipate that libraries of hybrid scaffolds will allow rapid identification of potent PPI inhibitors facilitating the discovery of small molecule binding motifs for challenging interactions and will have a large impact on the scientific community.
These results were presented at national and international conferences and a manuscript is nearing completion. Future research will involve the application of this method to other therapeutically relevant protein targets and preparation of a larger proposal for the application of hybrids targeting challenging PPIs.

Rationally designed ligands targeting helix mediated interactions
The HIF-1α/p300 interaction plays a key role in adaptation to hypoxia crucial for cancer cell growths, which makes this interaction an attractive target for cancer therapy. Efforts have been made to directly decouple this interaction by using small molecules, and peptidomimetics, however the lack of crystal structure of the interaction makes inhibitor design and optimization challenging. First, we sought to better understand the nature of this interaction with the goal to have high affinity and selective ligands as starting points for further inhibitor design. Based on recent literature results a hybrid structure was prepared from HIF-1α and its negative regulator CITED2 containing the overlapping p300 binding sites. The measured binding and thermodynamic parameters provided useful information about the interaction which could be advantageous for further inhibitor design.
To understand the greater capabilities of existing methods targeting alpha-helices with proteomimetics a classical model enzyme system (RnaseA) was investigated. A helical segment of a protein structure (the S-peptide from RNase S) was replaced by a foldamer that mimics an α-helix. The resultant protein-proteomimetic complex lead to restoration of catalytic function (RNA degradation). These results were published in Chemical Science as proof of concept study revealing that replacement of a significant amount of protein structure to a topographical mimic could result in a functional protein, a strategy that can be further utilized in the field of artificial protein design.
In order to aid the design of peptidomimetic ligands, an approach was investigated using stapled peptides against the ASF1/H3 interaction. Constraining the peptides into their bioactive helical conformation resulted in more stable ligands while retaining affinity toward the target protein. Exploiting this strategy may lead to more advanced tools targeting helix mediated interactions.

Progressi oltre lo stato dell’arte e potenziale impatto previsto (incluso l’impatto socioeconomico e le implicazioni sociali più ampie del progetto fino ad ora)

This fellowship provided the opportunity to raise the fellow’s research profile to represent an attractive and convincing prospect when applying for independent funding schemes. The fellowship helped the fellow to acquire diverse scientific skills and complementary training needed to address problems in the multidisciplinary field of chemical biology. The knowledge generated by this research will draw the attention of peptidomimetic scientists, synthetic chemists, cell biologists and structural biologists. Results generated so far were presented in multiple national and international conferences in the form of poster and oral presentations. Two scientific papers were published and another two are nearing completion that will be submitted to high impact scientific journals. This research topic is relevant to modern synthetic chemistry and drug design strategies targeting diseases with poor prognosis using current approaches (e.g. different cancer types), which is often in the spotlight of the general public. Communication to a broad audience focussed on relating the research objectives (targeting difficult PPIs) to current health problems, improve general understanding of targeting difficult diseases and emphasize the potential of this research to provide ground-breaking techniques and knowledge with the ultimate goal to improve health-care.

Figure 1. Schematic depicting the different scaffolds investigated in this fellowship