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Screening and optimization of phage display cyclic peptides against the WDR5 WBM site.

ABSTRACT: Of the various WD40 family proteins, WDR5 is a particularly important multifunctional adaptor protein that can bind to several protein complexes to regulate gene activation, so it was considered as a promising epigenetic target in anti-cancer drug development. Despite many inhibitors having been discovered directing against the arginine-binding cavity in WDR5 called the WIN site, the side hydrophobic cavity called the WBM site receives rather scant attention. Herein, we aim to obtain novel WBM-targeted peptidic inhibitors with high potency and selectivity. We employed two improved biopanning approaches with a disulfide-constrained cyclic peptide phage library containing 7 randomized residues and identified several peptides with micromole binding activity by docking and binding assay. To further optimize the stability and activity, 9 thiol-reactive chemical linkers were utilized in the cyclization of the candidate peptide DH226027, which had good binding affinity. This study provides an effective method to discover potent peptides targeting protein-protein interactions and highlights a broader perspective of peptide-mimic drugs.

SUBMITTER: Song L

PROVIDER: S-EPMC10583817 | biostudies-literature | 2023 Oct

REPOSITORIES: biostudies-literature

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Screening and optimization of phage display cyclic peptides against the WDR5 WBM site.

Song Lingyu L Cao Jiawen J Chen Lin L Du Zhiyan Z Zhang Naixia N Cao Danyan D Xiong Bing B

RSC medicinal chemistry 20230817 10

Of the various WD40 family proteins, WDR5 is a particularly important multifunctional adaptor protein that can bind to several protein complexes to regulate gene activation, so it was considered as a promising epigenetic target in anti-cancer drug development. Despite many inhibitors having been discovered directing against the arginine-binding cavity in WDR5 called the WIN site, the side hydrophobic cavity called the WBM site receives rather scant attention. Herein, we aim to obtain novel WBM-t ...[more]

PMID: 37859722

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Project description:BackgroundMetal borides are a class of inorganic solids that is much less known and investigated than for example metal oxides or intermetallics. At the same time it is a highly versatile and interesting class of compounds in terms of physical and chemical properties, like semiconductivity, ferromagnetism, or catalytic activity. This makes these substances attractive for the generation of new materials. Very little is known about the interaction between organic materials and borides. To generate nanostructured and composite materials which consist of metal borides and organic modifiers it is necessary to develop new synthetic strategies. Phage peptide display libraries are commonly used to select peptides that bind specifically to metals, metal oxides, and semiconductors. Further, these binding peptides can serve as templates to control the nucleation and growth of inorganic nanoparticles. Additionally, the combination of two different binding motifs into a single bifunctional phage could be useful for the generation of new composite materials.ResultsIn this study, we have identified a unique set of sequences that bind to amorphous and crystalline nickel boride (Ni3B) nanoparticles, from a random peptide library using the phage display technique. Using this technique, strong binders were identified that are selective for nickel boride. Sequence analysis of the peptides revealed that the sequences exhibit similar, yet subtle different patterns of amino acid usage. Although a predominant binding motif was not observed, certain charged amino acids emerged as essential in specific binding to both substrates. The 7-mer peptide sequence LGFREKE, isolated on amorphous Ni3B emerged as the best binder for both substrates. Fluorescence microscopy and atomic force microscopy confirmed the specific binding affinity of LGFREKE expressing phage to amorphous and crystalline Ni3B nanoparticles.ConclusionsThis study is, to our knowledge, the first to identify peptides that bind specifically to amorphous and to crystalline Ni3B nanoparticles. We think that the identified strong binding sequences described here could potentially serve for the utilisation of M13 phage as a viable alternative to other methods to create tailor-made boride composite materials or new catalytic surfaces by a biologically driven nano-assembly synthesis and structuring.

Project description:PURPOSE:Osteosarcoma represents the most common malignant primary bone tumor in childhood; however, the survival rate has remained unchanged for the past 20 years. To improve existing diagnosis and treatment methods and broaden the spectrum of imaging agents that can be used for early detection and assessment of tumor response to therapy, we performed a phage display-based screening for peptide sequences that bind specifically to osteosarcoma cells. EXPERIMENTAL DESIGN:From the Ph.D.-12 phage display peptide library composed of 2.7 x 10(9) different displayed peptides, one peptide was enriched after four rounds of in vitro selection in 143B osteosarcoma tumor cells with 293T human embryonic kidney cells as a control. Both the peptide and the phage clone displaying the peptide were conjugated with fluorescent dyes for in vitro cell and ex vivo tumor tissue stainings. The peptide was further labeled with (18)F for positron emission tomography imaging studies. Cell uptake and efflux and ex vivo biodistribution were also done with (18)F-labeled osteosarcoma specific peptide. RESULTS:ASGALSPSRLDT was the dominant sequence isolated from biopanning and named as OSP-1. OSP-1 shares a significant homology with heparinase II/III family protein, which binds and reacts with heparan sulfate proteoglycans. The fluorescence staining showed that FITC-OSP-1-phage or Cy5.5-OSP-1 had high binding with a panel of osteosarcoma cell lines, much less binding with UM-SCC1 human head and neck squamous cell carcinoma cells, and almost no binding with 293T cells, whereas the scrambled peptide OSP-S had virtually no binding to all the cell lines. (18)F-OSP-1 had significantly higher accumulation in 143B tumor cells both in vitro and in vivo than (18)F-OSP-S. (18)F-OSP-1 also had higher uptake in 143B tumors than in UM-SCC-1 tumors. CONCLUSIONS:Our data suggest that OSP-1 peptide is osteosarcoma specific, and the binding site of OSP-1 might be related to heparan sulfate proteoglycans. Appropriately labeled OSP-1 peptide has the potential to serve as a novel probe for osteosarcoma imaging.

Project description:Coeliac disease (CD) is a common and complex disorder of the small intestine caused by intolerance to wheat gluten and related edible cereals like barley and rye. Peptides originating from incomplete gliadin digestion activate the lamina propria infiltrating T cells to release proinflammatory cytokines, which in turn cause profound tissue remodelling of the small intestinal wall. There is no cure for CD except refraining from consuming gluten-containing products.Phage from a random oligomer display library were enriched by repeated pannings against immobilised gliadin proteins. Phage from the final panning round were plated, individual plaques picked, incubated with host bacteria, amplified to a population size of 1011 to 1012 and purified. DNA was isolated from 1000 purified phage populations and the region covering the 36 bp oligonucleotide insert from which the displayed peptides were translated, was sequenced. Altogether more than 150 different peptide-encoding sequences were identified, many of which were repeatedly isolated under various experimental conditions. Amplified phage populations, each expressing a single peptide, were tested first in pools and then one by one for their ability to inhibit binding of human anti-gliadin antibodies in ELISA assays. These experiments showed that several of the different peptide-expressing phage tested inhibited the interaction between gliadin and anti-gliadin antibodies. Finally, four different peptide-encoding sequences were selected for further analysis, and the corresponding 12-mer peptides were synthesised in vitro. By ELISA assays it was demonstrated that several of the peptides inhibited the interaction between gliadin molecules and serum anti-gliadin antibodies. Moreover, ELISA competition experiments as well as dot-blot and western blot revealed that the different peptides interacted with different molecular sites of gliadin.We believe that several of the isolated and characterised gliadin-binding peptides described here could provide valuable tools for researchers in the field of CD by facilitating studies on localisation and uptake of various gliadin peptides in the small intestine. In future work, the potential of these peptides to detoxify gluten will be investigated.

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Screening and optimization of phage display cyclic peptides against the WDR5 WBM site.

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Screening and optimization of phage display cyclic peptides against the WDR5 WBM site.

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