Development of Cell-Permeable, Non-Helical Constrained Peptides to Target a Key Protein-Protein Interaction in Ovarian Cancer.

There is a lack of current treatment options for ovarian clear cell carcinoma (CCC) and the cancer is often resistant to platinum-based chemotherapy. Hence there is an urgent need for novel therapeutics. The transcription factor hepatocyte nuclear factor 1ß (HNF1ß) is ubiquitously overexpressed in CCC and is seen as an attractive therapeutic target. This was validated through shRNA-mediated knockdown of the target protein, HNF1ß, in five high- and low-HNF1ß-expressing CCC lines. To inhibit the protein function, cell-permeable, non-helical constrained proteomimetics to target the HNF1ß-importin α protein-protein interaction were designed, guided by X-ray crystallographic data and molecular dynamics simulations. In this way, we developed the first reported series of constrained peptide nuclear import inhibitors. Importantly, this general approach may be extended to other transcription factors.

Structural and calorimetric studies demonstrate that the hepatocyte nuclear factor 1ß (HNF1ß) transcription factor is imported into the nucleus via a monopartite NLS sequence.

The transcription factor hepatocyte nuclear factor 1ß (HNF1ß) is ubiquitously overexpressed in ovarian clear cell carcinoma (CCC) and is a potential therapeutic target. To explore potential approaches that block HNF1ß transcription we have identified and characterised extensively the nuclear localisation signal (NLS) for HNF1ß and its interactions with the nuclear protein import receptor, Importin-α. Pull-down assays demonstrated that the DNA binding domain of HNF1ß interacted with a spectrum of Importin-α isoforms and deletion constructs tagged with eGFP confirmed that the HNF1ß (229)KKMRRNR(235) sequence was essential for nuclear localisation. We further characterised the interaction between the NLS and Importin-α using complementary biophysical techniques and have determined the 2.4Å resolution crystal structure of the HNF1ß NLS peptide bound to Importin-α. The functional, biochemical, and structural characterisation of the nuclear localisation signal present on HNF1ß and its interaction with the nuclear import protein Importin-α provide the basis for the development of compounds targeting transcription factor HNF1ß via its nuclear import pathway.

Discovery of quorum quenchers targeting the membrane-embedded sensor domain of the Staphylococcus aureus receptor histidine kinase, AgrC.

We combined mRNA display technology with lipid-nanodisc based selections and identified high-affinity ligands targeting the integral membrane sensor domain of the histidine kinase AgrC as potent inhibitors of Staphylococcus aureus quorum sensing-modulated virulence. Our study highlights the potential of this integrated approach for identifying functional modulators of integral membrane proteins.

Toolbox of Diverse Linkers for Navigating the Cellular Efficacy Landscape of Stapled Peptides.

Stapled peptides have great potential as modulators of protein-protein interactions (PPIs). However, there is a vast landscape of chemical features that can be varied for any given peptide, and identifying a set of features that maximizes cellular uptake and subsequent target engagement remains a key challenge. Herein, we present a systematic analysis of staple functionality on the peptide bioactivity landscape in cellular assays. Through application of a "toolbox" of diversified dialkynyl linkers to the stapling of MDM2-binding peptides via a double-click approach, we conducted a study of cellular uptake and p53 activation as a function of the linker. Minor changes in the linker motif and the specific pairing of linker with peptide sequence can lead to substantial differences in bioactivity, a finding which may have important design implications for peptide-based inhibitors of other PPIs. Given the complexity of the structure-activity relationships involved, the toolbox approach represents a generalizable strategy for optimization when progressing from in vitro binding assays to cellular efficacy studies.