Combined computational and intracellular peptide library screening: towards a potent and selective Fra1 inhibitor

To date, most research into the inhibition of oncogenic transcriptional regulator, Activator Protein 1 (AP-1), has focused on heterodimers of cJun and cFos. However, the Fra1 homologue remains an important cancer target. Here we describe library design coupled with computational and intracellular screening as an effective methodology to derive an antagonist that is selective for Fra1 relative to Jun counterparts. To do so the isCAN computational tool was used to rapidly screen >75 million peptide library members, narrowing the library size by >99.8% to one accessible to intracellular PCA selection. The resulting 131 072-member library was predicted to contain high quality binders with both a high likelihood of target engagement, while simultaneously avoiding homodimerization and off-target interaction with Jun homologues. PCA screening was next performed to enrich those members that meet these criteria. In particular, optimization was achieved via inclusion of options designed to generate the potential for compromised intermolecular contacts in both desired and non-desired species. This is an often-overlooked prerequisite in the conflicting design requirement of libraries that must be selective for their target in the context of a range of alternative potential interactions. Here we demonstrate that specificity is achieved via a combination of both hydrophobic and electrostatic contacts as exhibited by the selected peptide (Fra1W). In vitro analysis of the desired Fra1–Fra1W interaction further validates high Fra1 affinity (917 nM) yet selective binding relative to Fra1W homodimers or affinity for cJun. The isCAN → PCA based multidisciplinary approach provides a robust screening pipeline in generating target-specific hits, as well as new insight into rational peptide design in the search for novel bZIP family inhibitors.