Exploiting Overlapping Advantages of In Vitro and In Cellulo Selection Systems to Isolate a Novel High-Affinity cJun Antagonist

Abstract

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.

The Role of AP-1 Transcription Factors in Plasma Cell Biology and Multiple Myeloma Pathophysiology

“…In addition, many efforts have been made to search for peptides which exhibit high affinity for the leucine zipper dimerization domains of c-Jun or c-Fos and to inhibit these bZIP proteins by preventing the formation of functional c-Jun homodimers and c-Jun: c-Fos heterodimers [ 71 , 72 , 73 , 74 , 75 , 76 , 89 ]. Excitingly, an anti-Jun and anti-Fos superzipper has been demonstrated to bind to both the c-Jun and c-Fos leucine zipper peptides [ 77 ].…”

Selective antagonism of cJun for cancer therapy

“…Another library screening methodology called CIS display has also been utilised in tandem with PCA to allow for larger libraries to be screened in vitro before further in cell optimisation by PCA to produce a peptide named CPW. This has been shown to bind to the cJun LZ with a K d of 750 nM [147]. A wide range of peptide library screening techniques exists beyond CIS and PCA but these have not yet been used to screen for cJun antagonists.…”

Coupling Computational and Intracellular Screening and Selection Toward Co-compatible cJun and cFos Antagonists

“…The thermal stability of FosU PCA −cJun is comparable to previous peptides for which extensive biophysical data is available. 11,13,22,28 In particular, the PCA derived FosW−cJun exhibited a T m value of 63 °C and a K d value of 39 nM, whereas 4hFosW−cJun exhibited a T m of 49 °C and a K d value of 480 nM. Since the latter exhibited thermal stability within 3 °C of both FosU PCA −cJun and JunW CANDI − cFos, it can be estimated that the interaction K d for both of these complexes is also within the nanomolar range.…”