Interactions between naturally occurring proteins are highly specific, with protein-network imbalances associated with numerous diseases. For designed protein–protein interactions (PPIs), required specificity can be notoriously difficult to engineer. To accelerate this process, we have derived peptides that form heterospecific PPIs when combined. This is achieved using software that generates large virtual libraries of peptide sequences and searches within the resulting interactome for preferentially interacting peptides. To demonstrate feasibility, we have (i) generated 1536 peptide sequences based on the parallel dimeric coiled-coil motif and varied residues known to be important for stability and specificity, (ii) screened the 1,180,416 member interactome for predicted Tm values and (iii) used predicted Tm cutoff points to isolate eight peptides that form four heterospecific PPIs when combined. This required that all 32 hypothetical off-target interactions within the eight-peptide interactome be disfavoured and that the four desired interactions pair correctly. Lastly, we have verified the approach by characterising all 36 pairs within the interactome. In analysing the output, we hypothesised that several sequences are capable of adopting antiparallel orientations. We subsequently improved the software by removing sequences where doing so led to fully complementary electrostatic pairings. Our approach can be used to derive increasingly large and therefore complex sets of heterospecific PPIs with a wide range of potential downstream applications from disease modulation to the design of biomaterials and peptides in synthetic biology.

The oncogenic transcription factor activator protein-1 (AP-1) is a DNA-binding protein that assembles through dimerization of Fos and Jun protein subunits, their leucine-rich helical sequences entwining into a coiled-coil structure. This study reports on downsizing the proto-oncogene cFos protein (380 residues) to shorter peptides (37-25 residues) modified with helix-inducing constraints to enhance binding to Jun. A crystal structure is reported for a 37-residue Fos-derived peptide (FosW) bound to Jun. This guided iterative downsizing of FosW to shorter peptide sequences that were constrained into stable water-soluble α-helices by connecting amino acid side chains to form cyclic pentapeptide components. Structural integrity in the presence and absence of Jun was assessed by circular dichroism spectroscopy, while the thermodynamics of binding to cFos was measured by isothermal titration calorimetry. A 25-residue constrained peptide, one-third shorter yet 25% more helical than the structurally characterized 37-residue Fos-derived peptide, retained 80% of the binding free energy as a result of preorganization in a Jun-binding helix conformation, with the entropy gain (TΔS = +3.2 kcal/mol) compensating for the enthalpy loss. Attaching a cell-penetrating peptide (TAT) and a nuclear localization signal (SV40) promoted cell uptake, localization to the nucleus, and inhibition of the proliferation of two breast cancer cell lines.

In the modern age of proteomics, vast numbers of protein-protein interactions (PPIs) are being identified as causative agents in pathogenesis, and are thus attractive therapeutic targets for intervention. Although traditionally regarded unfavorably as druggable agents relative to small molecules, peptides in recent years have gained considerable attention. Their previous dismissal had been largely due to the susceptibility of unmodified peptides to the barriers and pressures exerted by the circulation, immune system, proteases, membranes and other stresses. However, recent advances in high-throughput peptide isolation techniques, as well as a huge variety of direct modification options and approaches to allow targeted delivery, mean that peptides and their mimetics can now be designed to circumvent many of these traditional barriers. As a result, an increasing number of peptide-based drugs are reaching clinical trials and patients beyond.

We have combined two peptide library-screening systems, exploiting the benefits offered by both to select novel antagonistic agents of cJun. CIS display is an in vitro cell-free system that allows very large libraries (≤10) to be interrogated. However, affinity-based screening conditions can poorly reflect those relevant to therapeutic application, particularly for difficult intracellular targets, and can lead to false positives. In contrast, an in cellulo screening system such as the Protein-fragment Complementation Assay (PCA) selects peptides with high target affinity while additionally profiling for target specificity, protease resistance, solubility, and lack of toxicity in a more relevant context. A disadvantage is the necessity to transform cells, limiting library sizes that can be screened to ≤10. However, by combining both cell-free and cell-based systems, we isolated a peptide (CPW) from a ∼10 member library, which forms a highly stable interaction with cJun (T = 63 °C, K = 750 nM, ΔG = -8.2 kcal/mol) using the oncogenic transcriptional regulator Activator Protein-1 (AP-1) as our exemplar target. In contrast, CIS display alone selected a peptide with low affinity for cJun (T = 34 °C, K = 25 μM, ΔG = -6.2 kcal/mol), highlighting the benefit of CIS → PCA. Furthermore, increased library size with CIS → PCA vs PCA alone allows the freedom to introduce noncanonical options, such as interfacial aromatics, and solvent exposed options that may allow the molecule to explore alternative structures and interact with greater affinity and efficacy with the target. CIS → PCA therefore offers significant potential as a peptide-library screening platform by synergistically combining the relative attributes of both assays to generate therapeutically interesting compounds that may otherwise not be identified.