An Intracellular Helix-constrained Peptide Library Screening Platform to Derive Functional Transcription Factor Antagonists

Project: Research council

Project Details

Description

>60% of all multi-protein complexes feature helical interfaces, with >20% participating in gene regulation. Helical interaction inhibitors therefore have enormous potential to become a useful class of transcriptional modulator. Small molecules typically fail to abrogate these interactions owing to their limited ability to extend beyond interaction hotspots. In contrast, peptide-based inhibitors can block much larger interaction areas, making them a preferred approach for protein-protein interaction (PPI) inhibition.

However, peptide sequences corresponding to binding sites within a protein can lose their structure when created in isolation. For helical peptides, this can be compensated for with the use of 'helix-inducing constraints' which can be thought of as safety pins that hold the peptide together to retain helical structure. However, the beneficial effect of a constraint can be very hard to predict, leading to considerable time and expense. We will tackle this challenging problem by screening entire libraries of peptides inside cells where each members has been locked into a helical conformation. By constraining all peptides during the library screen we will identify those in which the constraint provides improved target affinity, resistance to proteases, and potentially the potential to cross biological membranes. We will ensure that antagonists are truly functional in blocking transcription factor-DNA binding by screening the helix-constrained libraries within living cells using our Transcription Block Survival (TBS) Assay. We will fully characterise peptides to establish how they acheive their favourble properties using a range of biochemical assays.

Finally, using knowledge gained we will create updated versions of our widely used isPCA/isCAN peptide library screening software that searches vast numbers of peptides to identify those most likely to bind with high affinity and selectivity to a given target (e.g. Chen et al, Nature 2019, Aupic et al, Nat Commun 2021, Daudey et al, Chem Sci 2021). Building on these we will further identify sequences within which 'modules' can be introduced that both tolerate constraints but that more importantly promote increased helicity, binding, biostability, and potentially membrane permeability. The software is anticipated to be widely used by the community to facilitate selective inhibiton of a wide range of disease relevant PPIs in which helical interfaces present. The computational tools will be made freely available and accessible online to the scientific community. We envisage that they will be widely adopted by protein biochemists, cell biologists and synthetic biologists.
Short title628000
StatusNot started
Effective start/end date1/07/2330/06/26

Funding

  • Biotechnology and Biological Sciences Research Council

RCUK Research Areas

  • Bioengineering
  • Biomolecules and biochemistry
  • Protein engineering
  • Chemical biology

Fingerprint

Explore the research topics touched on by this project. These labels are generated based on the underlying awards/grants. Together they form a unique fingerprint.