Mammalian display screening of diverse cystine-dense peptides for difficult to drug targets

Zachary R Crook,James M Olson,David Baker,Roland K Strong,Philip Bradley,Mi-Youn Brusniak,Andrew J Mhyre,Mesfin Gewe,Della Friend,Ashok D Bandaranayake,Gregory P Sevilla,Midori Clarke

doi:10.1038/s41467-017-02098-8

Zachary R Crook, James M Olson + Show 10 more

Open Access

https://doi.org/10.1038/s41467-017-02098-8

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Abstract

Protein:protein interactions are among the most difficult to treat molecular mechanisms of disease pathology. Cystine-dense peptides have the potential to disrupt such interactions, and are used in drug-like roles by every clade of life, but their study has been hampered by a reputation for being difficult to produce, owing to their complex disulfide connectivity. Here we describe a platform for identifying target-binding cystine-dense peptides using mammalian surface display, capable of interrogating high quality and diverse scaffold libraries with verifiable folding and stability. We demonstrate the platform’s capabilities by identifying a cystine-dense peptide capable of inhibiting the YAP:TEAD interaction at the heart of the oncogenic Hippo pathway, and possessing the potency and stability necessary for consideration as a drug development candidate. This platform provides the opportunity to screen cystine-dense peptides with drug-like qualities against targets that are implicated for the treatment of diseases, but are poorly suited for conventional approaches.

Highlights

Protein:protein interactions are among the most difficult to treat molecular mechanisms of disease pathology
TEAD is at the core of the oncogenic Hippo pathway, which plays a critical role in wound repair and contact inhibition[5], and is commonly dysregulated in many human cancers, including liver, breast, colon, lung, prostate, and brain[6,7,8,9,10,11]
We developed a mammalian surface display platform optimized for the folding of cystine-dense peptides (CDPs), validating it on a highly diverse library of thousands of native CDPs by using both highthroughput mammalian display screening and HPLC to evaluate their expression and stability

Summary

Results

E. coli and S. cerevisiae are routinely used for surface display screens to find target-binding peptides (yeast have the advantage of the eukaryotic secretory pathway’s oxidative environment to aid disulfide formation)[32,33], yet the variety of CDP scaffolds being reliably surface displayed or secreted is limited[27] Both species natively secrete fewer than 50 proteins with cysteine-rich domains, compared to the human secretome, of which over 1400 genes (~20%) contain such domains (Supplementary Table 1). A similar technique using yeast display was recently validated for designed, cysteine-free peptides[37], but such an analysis for CDPs cannot be performed in conventional yeast display, as the Aga1/2 scaffold is held together with disulfide bonds This high-throughput, quantitative protein content assay allows us to identify well-folded CDPs by those that confer strong surface staining to cells (high content) and/or retain their staining after protease treatment (protease resistant). Analyzing the four sorted populations of varying fluorescence ranges

1–2 Peaks by HPLC

Discussion

Methods