Protein-protein interactions, and in particular Transcription Factors (TFs), remain compelling drug targets, yet are often intractable to small molecules and inaccessible to larger biologics. Peptides occupy an attractive middle ground if they can become suitable ordered for target engagement. We utilise intracellular peptide library screening approaches to identify selective peptide-based inhibitors that can functionally antagonise TFs. There are two major novelties to our approach:
i) our Transcription Block Survival (TBS) peptide-library screening platform in which TF consensus sites are placed directly into the coding region of an essential gene. Subsequent TF binding within the gene directly blocks gene transcription leading to cell death under selective conditions. Cell survival is therefore only possible if antagonists bind to the TF, but more importantly can prevent it from binding to its consensus sequence, thus shutting down TF function. TBS is an entirely tag-free genotype-to-phenotype approach, selecting desirable attributes such as high solubility, target specificity, biostability and low toxicity within the complex environment of the cell. TBS facilitates rapid library screening to accelerate identification of therapeutically valuable sequences.
ii) concomitant deployment of cell penetrating crosslinkers. These enter cells to post-translationally constrain every library member into conformations not possible via genetic encoding alone, to select only those in which crosslinking translates into improve target antagonism. Screening ultra-structured biostable peptide libraries, where entire libraries are constrained during the search is highly desirable as it prevents a slow and costly retrospective trial-and-error search for beneficial crosslinkers, positions, and sequences.
Using several different exemplars, I will discuss how library-derived constrained peptide antagonists are derived and discuss their characterisation using a range of biophysical and cancer cell-based assays.