Many important diseases, including cancer, are driven by proteins that are extremely difficult to target with conventional drugs. Transcription factors are a major example. These proteins control gene expression and can sit at the top of disease-causing biological pathways, but they often work through large, flat and dynamic interaction surfaces that do not contain the binding pockets normally required for small-molecule medicines.

Researchers at the University of Bath are developing new technologies to address this challenge by discovering peptide-based molecules that can enter cells and block transcription factor function. These molecules are designed to target protein surfaces that have historically been considered “undruggable”, opening new possibilities for future therapeutic discovery.

Our Approach

This BBSRC-funded project focuses on developing an intracellular helix-constrained peptide library screening platform. Peptides can be powerful tools for blocking protein-protein interactions because they can cover larger binding surfaces than many conventional drugs. However, short peptides often lose their natural shape when removed from the proteins they come from, which can reduce their binding strength, stability and biological activity.

To overcome this, the project uses chemical constraints that act like molecular “staples”, holding peptides in an alpha-helical shape. Rather than adding these constraints after screening in a slow trial-and-error process, the Bath team developed a strategy to chemically constrain entire peptide libraries inside living cells during the screening process.

This approach is combined with the team’s Transcription Block Survival assay, a powerful live-cell screening system that selects peptides based on their ability to restore cell growth by blocking transcription factor-DNA binding. This means that the screen does not simply identify molecules that bind a target, but enriches for molecules that functionally inhibit transcription factor activity.

Aims and objectives

The project aims to:

develop intracellular methods for chemically constraining peptide libraries during live-cell screening
discover helix-constrained peptide antagonists of disease-relevant transcription factors
identify peptide sequences and constraint positions that improve binding, stability and functional activity
reduce the need for slow and costly trial-and-error optimisation of constrained peptides
generate design rules and computational tools that help researchers discover constrained peptide inhibitors for a wider range of protein-protein interactions

Methods

The team uses genetically encoded peptide libraries expressed inside bacterial cells. These libraries are designed so that selected cysteine residues can be chemically linked by cell-permeable cross-linking reagents, producing constrained peptide structures inside the cell.

The constrained libraries are screened using the Transcription Block Survival assay. In this system, a transcription factor blocks expression of an essential gene. Bacterial cells only grow when a peptide successfully inhibits the transcription factor and restores gene expression. The fastest-growing cells therefore reveal the most effective functional peptide antagonists, and the peptide sequences can be identified by DNA sequencing.

Promising peptides are then characterised using biochemical, biophysical and cell-based methods, including circular dichroism, binding assays, DNA-binding assays, reporter assays, serum stability tests and cellular studies. The project also integrates computational peptide-design methods to help define where constraints are most likely to improve peptide function.

Outcomes and impact

A major outcome of this project has been the development of intracellular-cyclisation Transcription Block Survival, or icTBS. This platform enables chemical peptide cyclisation to be carried out directly inside living cells during functional selection.

Using this approach, the team discovered constrained peptide antagonists of the oncogenic transcription factor CREB1. The resulting peptides showed high-affinity target binding, improved functional activity and, after optimisation for uptake, activity in melanoma and colorectal cancer cell models.

The technology provides a general strategy for discovering constrained peptide molecules against transcription factors and other intracellular targets that have been difficult to address using traditional drug-discovery approaches.

In the longer term, this work could support the development of new chemical biology tools and future therapeutic discovery programmes for cancers and other diseases driven by challenging protein-protein interactions.