Peptide-based immunoadsorbents: Molecular grafting of IgG–Fc-binding epitopes of Protein A onto a de novo-designed helix-loop-helix peptide

Kawabata, Kumiko; Nagai, Hirokazu; Konishi, Nao; Fujiwara, Daisuke; Sasaki, Ryo; Ichikawa, Takafumi; Fujii, Ikuo

doi:10.1016/j.bmc.2014.01.053

Cited by 5 publications

(6 citation statements)

References 19 publications

(18 reference statements)

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“…5−7 Furthermore, an IgG-Fc-binding HLH peptide was designed using a protein grafting strategy. 3,8 Here, we describe the use of yeast surface-display libraries of HLH peptides to generate HLH peptide inhibitors for the cytotoxic T lymphocyte antigen-4 (CTLA-4)−B7-1 interaction occurring at immune checkpoints that suppress immune-stimulatory activity.…”

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confidence: 99%

“…For example, we have developed an alternative binding molecule, a helix–loop–helix (HLH) peptide, containing a nonimmunoglobulin domain. , This peptide is resistant to enzyme degradation in vivo and is too small to induce an immune response. We have constructed phage-display libraries of HLH peptides and obtained ligands and inhibitors for the granulocyte-colony stimulating factor receptor (G-CSFR), ganglioside GM1, and aurora kinase A. − Furthermore, an IgG-Fc-binding HLH peptide was designed using a protein grafting strategy. , Here, we describe the use of yeast surface-display libraries of HLH peptides to generate HLH peptide inhibitors for the cytotoxic T lymphocyte antigen-4 (CTLA-4)–B7-1 interaction occurring at immune checkpoints that suppress immune-stimulatory activity.…”

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confidence: 99%

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An Immune-Stimulatory Helix–Loop–Helix Peptide: Selective Inhibition of CTLA-4–B7 Interaction

Mudiyanselage

Michigami

et al. 2019

ACS Chem. Biol.

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Molecular-targeting peptides and mini-proteins are promising alternatives to antibodies in a wide range of applications in bioscience and medicine. We have developed a helix–loop–helix (HLH) peptide as an alternative to antibodies to inhibit specific protein interactions. Cytotoxic T lymphocyte antigen-4 (CTLA-4) downregulates immune responses of cytotoxic T-cells by interaction with B7-1, a co-stimulatory molecule expressed on antigen presenting cells (APCs). To induce immune stimulatory activity, we used directed evolution methods to generate a HLH peptide that binds to CTLA-4, inhibiting the CTLA-4–B7-1 interaction and inducing immune stimulatory activity. Yeast-displayed libraries of HLH peptides were constructed and screened against CTLA-4 and identified the binding peptide Y-2, which exhibits a moderate affinity. The affinity of Y-2 was improved by in vitro affinity maturation to afford a stronger binder, ERY2-4. Peptide ERY2-4 specifically bound to CTLA-4 with a K D of 196.8 ± 2.3 nM, comparable to the affinity of the CTLA-4–B7-1 interaction. Furthermore, ERY2-4 inhibited the CTLA-4–B7-1 interaction with an IC50 of 1.1 ± 0.03 μM and blocked the interaction between CTLA-4 and dendritic cells (DCs) presenting B7 on their surface. Importantly, ERY2-4 showed no cross-reactivity against CD28, suggesting it does not suppress T-cell activation. Finally, in a mixed lymphocyte reaction assay with DCs and T cells, ERY2-4 enhanced an allogeneic lymphocyte response. Since CTLA-4 is a critical immune checkpoint for restricting the cancer immune response, this inhibitory HLH peptide represents a new class of drug candidates for immunotherapy.

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An Immune-Stimulatory Helix–Loop–Helix Peptide: Selective Inhibition of CTLA-4–B7 Interaction

Mudiyanselage

Michigami

et al. 2019

ACS Chem. Biol.

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“…Thepeptide YT1 retains its secondary structure when solvent-exposed amino acids outside the C-terminal helix are randomly substituted with av ariety of amino acids. [6] In this work, we attempted to design an intracellular PPI inhibitor for the p53-HDM2 interaction, based on the HLH scaffold. [5] Furthermore,a nI gG-Fc-binding HLH peptide was successfully designed by using aprotein grafting strategy.…”

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A Cyclized Helix‐Loop‐Helix Peptide as a Molecular Scaffold for the Design of Inhibitors of Intracellular Protein–Protein Interactions by Epitope and Arginine Grafting

et al. 2016

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The design of inhibitors of intracellular proteinprotein interactions (PPIs) remains ac hallenge in chemical biology and drug discovery.W epropose acyclized helix-loophelix (cHLH) peptide as as caffold for generating cellpermeable PPI inhibitors through bifunctional grafting:e pitope grafting to provide binding activity,and arginine grafting to endowcell-permeability.Toinhibit p53-HDM2 interactions, the p53 epitope was grafted onto the C-terminal helix and six Arg residues were grafted onto another helix. The designed peptide cHLHp53-R showed high inhibitory activity for this interaction, and computational analysis suggested ab inding mode for HDM2. Confocal microscopyo fc ells treated with fluorescently labeled cHLHp53-R revealed cell membrane penetration and cytosolic localization. The peptide inhibited the growth of HCT116 and LnCap cancer cells.This strategy of bifunctional grafting onto aw ell-structured peptide scaffold could facilitate the generation of inhibitors for intracellular PPIs.

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“…[ 4,5 ] Using phage or yeast surface display, we obtained HLH peptide ligands against ganglioside GM3, granulocyte colony‐stimulating factor receptor, the Fc domain of IgG, cytotoxic T lymphocyte antigen‐4, and vascular endothelial growth factor. [ 6–10 ] Moreover, we developed the inhibitory peptide cHLHp53‐R for p53–HDM2 interaction inside cells. [ 11 ] The binding activity of the inhibitory peptide to HDM2 was achieved by protein epitope grafting, and the cell membrane permeability was conferred to the peptide by substituting the six solvent‐exposed alanines in the N‐terminal helix to arginines.…”

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Generation of inhibitory peptides for IKKε from a kinase‐focused phage library of helix‐loop‐helix peptides

et al. 2021

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Conformationally constrained peptides are attracting attention as peptide-based molecular tools in chemical biology and drug discovery because of their desirable properties such as high selectivity for target binding and stability toward proteolytic enzymes. We previously reported a protein kinase-focused helix-loop-helix (HLH) peptide library. The library was constructed by phage display and subsequent chemical modification with adenosine that was employed as an anchor molecule binding to the ATP-binding pocket of kinase. Here, we discovered and characterized HLH peptides that bind to and inhibit the serine/threonine protein kinase IKKε, a member of the IκB kinase (IKK) family. Screening the library against IKKε identified de novo HLH peptides binding to the kinase. One of the peptides, IKK-05, showed a high α-helical content and inhibited IKKε with a mixed-inhibition mechanism with respect to ATP.The adenosine-tethering peptide Adc-IKK-05 demonstrated significantly enhanced enzyme inhibition activity, indicating a bivalency effect in binding to IKKε. In addition, Adc-IKK-05 showed the highest inhibitory activity against IKKε of the IKK family members and other families tested. The peptides obtained in this research can be developed as molecular tools to study the biological functions of IKKε and to obtain structural insights for the design of selective IKKε inhibitors.

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Peptide-based immunoadsorbents: Molecular grafting of IgG–Fc-binding epitopes of Protein A onto a de novo-designed helix-loop-helix peptide

Cited by 5 publications

References 19 publications

An Immune-Stimulatory Helix–Loop–Helix Peptide: Selective Inhibition of CTLA-4–B7 Interaction

An Immune-Stimulatory Helix–Loop–Helix Peptide: Selective Inhibition of CTLA-4–B7 Interaction

A Cyclized Helix‐Loop‐Helix Peptide as a Molecular Scaffold for the Design of Inhibitors of Intracellular Protein–Protein Interactions by Epitope and Arginine Grafting

Generation of inhibitory peptides for IKKε from a kinase‐focused phage library of helix‐loop‐helix peptides

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