Utilization of peptide phage display to investigate hotspots on IL-17A and what it means for drug discovery

Ting, Joey; Tung, Frances; Antonysamy, Stephen; Wasserman, S.R.; Jones, Spencer B.; Zhang, Feiyu F.; Espada, Alfonso; Broughton, Howard B.; Chalmers, Michael J.; Woodman, Michael E.; Bina, Holly A.; Dodge, Jeffrey A.; Benach, J.; Zhang, Aiping; Groshong, Christopher; Manglicmot, Danalyn; Russell, Marijane; Afshar, Sepideh

doi:10.1371/journal.pone.0190850

Cited by 18 publications

(25 citation statements)

References 27 publications

(53 reference statements)

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“…A single- to double-digit micromolar starting affinity, as reported here, is often observed for peptide identification from phage display. For example, peptides isolated to Japanese encephalitis virus, insulin-degrading enzyme, or IL17A had affinities in the 2.5–12 μ m range (42–44). Subsequent affinity maturation has been successfully employed to achieve affinities in the nanomolar range (42).…”

Section: Discussionmentioning

confidence: 99%

“…For example, peptides isolated to Japanese encephalitis virus, insulin-degrading enzyme, or IL17A had affinities in the 2.5–12 μ m range (42–44). Subsequent affinity maturation has been successfully employed to achieve affinities in the nanomolar range (42). Additional peptide phage libraries can also be constructed by randomizing the residues that are adjacent to the ones critical for the interaction (soft randomization) based on structural information and thus improve the affinity of the parental FYLIR peptide.…”

Section: Discussionmentioning

confidence: 99%

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Discovery of peptide ligands targeting a specific ubiquitin-like domain–binding site in the deubiquitinase USP11

Spiliotopoulos

Ferreras

Densham

et al. 2019

Journal of Biological Chemistry

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Ubiquitin-specific proteases (USPs) reverse ubiquitination and regulate virtually all cellular processes. Defined noncatalytic domains in USP4 and USP15 are known to interact with E3 ligases and substrate recruitment factors. No such interactions have been reported for these domains in the paralog USP11, a key regulator of DNA double-strand break repair by homologous recombination. We hypothesized that USP11 domains adjacent to its protease domain harbor unique peptide-binding sites. Here, using a next-generation phage display (NGPD) strategy, combining phage display library screening with next-generation sequencing, we discovered unique USP11-interacting peptide motifs. Isothermal titration calorimetry disclosed that the highest affinity peptides (KD of ∼10 μm) exhibit exclusive selectivity for USP11 over USP4 and USP15 in vitro. Furthermore, a crystal structure of a USP11–peptide complex revealed a previously unknown binding site in USP11's noncatalytic ubiquitin-like (UBL) region. This site interacted with a helical motif and is absent in USP4 and USP15. Reporter assays using USP11-WT versus a binding pocket–deficient double mutant disclosed that this binding site modulates USP11's function in homologous recombination–mediated DNA repair. The highest affinity USP11 peptide binder fused to a cellular delivery sequence induced significant nuclear localization and cell cycle arrest in S phase, affecting the viability of different mammalian cell lines. The USP11 peptide ligands and the paralog-specific functional site in USP11 identified here provide a framework for the development of new biochemical tools and therapeutic agents. We propose that an NGPD-based strategy for identifying interacting peptides may be applied also to other cellular targets.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Discovery of peptide ligands targeting a specific ubiquitin-like domain–binding site in the deubiquitinase USP11

Spiliotopoulos

Ferreras

Densham

et al. 2019

Journal of Biological Chemistry

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“…IL-23 bound phage was eluted by 100 mM of triethylamine and was immediately neutralized by 1 M Tris/HCl pH 6.8. After three rounds of selection, phage was screened against IL-23, IL-12, and background neutravidin-coated plates as described previously using filter-lift and phage-ELISAs [15].…”

Section: Phage Display Library Construction Selection and Screeningmentioning

confidence: 99%

“…As a result, a gradual enrichment of p19 binders was observed in the selected pool as the selection progressed. After three rounds of selection, phage pool was deconvoluted by filter-lift and single point ELISA [15]. Sequence analysis of specific p19 binders indicated that more than 40% of clones share a common motif comprised of phenylalanine-Glycine-Leucine/Threonine (FGL/T).…”

Section: Selection Of Phage Libraries Against Il-23mentioning

confidence: 99%

Integration of phage and yeast display platforms: A reliable and cost effective approach for binning of peptides as displayed on-phage

et al. 2020

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Hundreds of target specific peptides are routinely discovered by peptide display platforms. However, due to the high cost of peptide synthesis only a limited number of peptides are chemically made for further analysis. Here we describe an accurate and cost effective method to bin peptides on-phage based on binding region(s), without any requirement for peptide or protein synthesis. This approach, which integrates phage and yeast display platforms, requires display of target and its alanine variants on yeast. Flow cytometry was used to detect binding of peptides on-phage to the target on yeast. Once hits were identified, they were synthesized to confirm their binding region(s) by HDX (Hydrogen deuterium exchange) and crystallography. Moreover, we have successfully shown that this approach can be implemented as part of a panning process to deplete non-functional peptides. This technique can be applied to any target that can be successfully displayed on yeast; it narrows down the number of peptides requiring synthesis; and its utilization during selection results in enrichment of peptide population against defined binding regions on the target.

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“…At the protein–protein interface, α-helices tend to bind into the groove of their binding partner, and as a result, helix mimetics have been of great interest. A number of methods exist which aid in the discovery of new PPIs and facilitate the discovery of molecules to bind PPIs, including high throughput screening [ 20 ], phage display [ 21 ], crosslinking [ 22 ], computational studies [ 23 ] and structural based design, to name a few.…”

Section: General Structure Of Ppismentioning

confidence: 99%

Using Peptidomimetics and Constrained Peptides as Valuable Tools for Inhibiting Protein–Protein Interactions

Robertson

Spring

2018

Molecules

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Protein–protein interactions (PPIs) are tremendously important for the function of many biological processes. However, because of the structure of many protein–protein interfaces (flat, featureless and relatively large), they have largely been overlooked as potential drug targets. In this review, we highlight the current tools used to study the molecular recognition of PPIs through the use of different peptidomimetics, from small molecules and scaffolds to peptides. Then, we focus on constrained peptides, and in particular, ways to constrain α-helices through stapling using both one- and two-component techniques.

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Utilization of peptide phage display to investigate hotspots on IL-17A and what it means for drug discovery

Cited by 18 publications

References 27 publications

Discovery of peptide ligands targeting a specific ubiquitin-like domain–binding site in the deubiquitinase USP11

Discovery of peptide ligands targeting a specific ubiquitin-like domain–binding site in the deubiquitinase USP11

Integration of phage and yeast display platforms: A reliable and cost effective approach for binning of peptides as displayed on-phage

Using Peptidomimetics and Constrained Peptides as Valuable Tools for Inhibiting Protein–Protein Interactions

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