Non-competitive cyclic peptides for targeting enzyme–substrate complexes

McAllister, Tom E.; Yeh, Tzu‐Lan; Abboud, Martine I.; Leung, Ivanhoe K. H.; Hookway, Edward S.; King, Oliver N.; Bhushan, Bhaskar; Williams, Sophie; Hopkinson, Richard J.; Münzel, Martin; Loik, Nikita D.; Chowdhury, Rasheduzzaman; Oppermann, U.; Claridge, Tim D. W.; Goto, Yuki; Suga, Hiroaki; Schofield, Christopher J.; Kawamura, Akane

doi:10.1039/c8sc00286j

Cited by 27 publications

(31 citation statements)

References 48 publications

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“…4 A ), in which we maintained the four acidic residues N-terminal to Tyr 21 and also Cys at position 30. Because cyclization is known to improve conformational stability, a cyclic version BK1.2 was designed, with Cys 30 cyclized to a N-terminal Tyr residue that was introduced ( 29 , 30 ). As a control, we also created a noncyclized version of this peptide, BK1.3.…”

Section: Resultsmentioning

confidence: 99%

Engineered anti-inflammatory peptides inspired by mapping an evasin–chemokine interaction

Darlot

Eaton

Geis-Asteggiante

et al. 2020

Journal of Biological Chemistry

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Chemokines mediate leucocyte migration and homeostasis, and are key targets in inflammatory diseases including atherosclerosis, cytokine storm and chronic auto-immune disease. Chemokine redundancy and ensuing network robustness has frustrated therapeutic development. Salivary evasins from ticks bind multiple chemokines overcoming redundancy, and are effective in several pre-clinical disease models. Their clinical development has not progressed due to concerns regarding potential immunogenicity, parenteral delivery and cost. Peptides mimicking protein activity can overcome the perceived limitations of therapeutic proteins. Here we show that peptides possessing multiple-chemokine-binding and anti-inflammatory activities can be developed from the chemokine-binding site of an evasin. We used hydrogen–deuterium exchange mass spectrometry to map the binding interface of the evasin P672 that physically interacts with C-C motif chemokine ligand 8 (CCL8) and synthesized a 16-mer peptide (BK1.1) based on this interface region in evasin P672. Fluorescent polarization and native mass spectrometry approaches showed that BK1.1 binds CCL8, CCL7 and CCL18, and disrupts CCL8 homodimerization. We show that a BK1.1 derivative, BK1.3, has substantially improved ability to disrupt P672 binding to CCL8, CCL2 and CCL3 in an AlphaScreen assay. Using isothermal titration calorimetry, we show that BK1.3 directly binds CCL8. BK1.3 also has substantially improved ability to inhibit CCL8, CCL7, CCL2 and CCL3 chemotactic function in vitro. We show that local as well as systemic administration of BK1.3 potently blocks inflammation in vivo. Identification and characterization of the chemokine-binding interface of evasins could thus inspire the development of novel anti-inflammatory peptides that therapeutically target the chemokine network in inflammatory diseases.

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Section: Resultsmentioning

confidence: 99%

Engineered anti-inflammatory peptides inspired by mapping an evasin–chemokine interaction

Darlot

Eaton

Geis-Asteggiante

et al. 2020

Journal of Biological Chemistry

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“…Although the development of substrate‐selective inhibitors could be challenging, our results imply that pursuing them (either for HIFα isoforms and/or ODDs) might be viable. Recent work has revealed high‐affinity peptides that do not bind to the PHD active site . The development of these and other allosteric compounds could be the starting point for the development of substrate‐selective inhibitors that work by modulating the induced‐fit processes involved in ODD binding .…”

Section: Resultsmentioning

confidence: 99%

Studies on the Substrate Selectivity of the Hypoxia‐Inducible Factor Prolyl Hydroxylase 2 Catalytic Domain

et al. 2018

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In animals, the response to chronic hypoxia is mediated by upregulation of the α,β-heterodimeric hypoxia-inducible factors (HIFs). Levels of HIFα isoforms, but not HIFβ, are regulated by their post-translational modification as catalysed by prolyl hydroxylase domain enzymes (PHDs). Different roles for the human HIF-1/2α isoforms and their two oxygen-dependent degradation domains (ODDs) are proposed. We report kinetic and NMR analyses of the ODD selectivity of the catalytic domain of wild-type PHD2 (which is conserved in nearly all animals) and clinically observed variants. Studies using Ala scanning and "hybrid" ODD peptides imply that the relatively rigid conformation of the (hydroxylated) proline plays an important role in ODD binding. They also reveal differential roles in binding for the residues on the N- and C-terminal sides of the substrate proline. The overall results indicate how the PHDs achieve selectivity for HIFα ODDs and might be of use in identifying substrate-selective PHD inhibitors.

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“…The low-micromolar affinity of the lysozyme binding peptides was anticipated based on the affinity of previously identified cyclic peptides that are similar in size and amino acid composition 20 . Other groups, using display-based technologies, have identified cyclic peptides with low-nanomolar affinities 21 ; these cyclic peptides, however, feature a larger ring structure (up to 14 randomized amino acid positions), and therefore benefit from a higher enthalpic contribution to binding free energy. In other instances, the peptide macrocycle is constrained via multifunctional crosslinkers to increase both the enthalpic and entropic contributions of binding free energy 22 .…”

Section: Binding Affinity Analysis Of Lysozyme Cyclic Peptidesmentioning

confidence: 99%

Isolation of Chemically Cyclized Peptide Binders Using Yeast Surface Display

Bacon

Blain

Burroughs

et al. 2020

Preprint

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Cyclic peptides with engineered protein-binding activity have gained increasing attention for use in therapeutic and biotechnology applications. We describe the efficient isolation and characterization of cyclic peptide binders from genetically encoded combinatorial libraries using yeast surface display. Here, peptide cyclization is achieved by disuccinimidyl glutarate-mediated crosslinking of amine groups within a linear peptide sequence that is expressed as a yeast cell surface fusion. Using this approach, we first screened a library of cyclic heptapeptides by magnetic selection and fluorescence activated cell sorting (FACS), to isolate binders for a model target (lysozyme) with low micromolar binding affinity (K D ~ 1.2 -3.7 µM). The isolated peptides bound lysozyme selectively, and only when cyclized. Importantly, we showed that yeast surface displayed cyclic peptides could be used to efficiently obtain quantitative estimates of binding affinity, without chemical synthesis of the selected peptides. Subsequently, to demonstrate broader applicability of our approach, we isolated cyclic heptapeptides that bind human interleukin-17 (IL-17) using yeast-displayed IL-17 as a target for magnetic selection, followed by FACS using recombinant IL-17. Molecular docking simulations and follow-up experimental analyses identified a candidate cyclic peptide that binds IL-17 in its receptor binding region with moderate affinity (K D ~ 300 nM). Taken together, our results show that yeast surface display can be used to efficiently isolate and characterize cyclic peptides generated by chemical modification from combinatorial libraries.

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Non-competitive cyclic peptides for targeting enzyme–substrate complexes

Abstract: Tight, non-active site binding cyclic peptides are promising affinity reagents for studying proteins and their interactions.

Cited by 27 publications

References 48 publications

Engineered anti-inflammatory peptides inspired by mapping an evasin–chemokine interaction

Engineered anti-inflammatory peptides inspired by mapping an evasin–chemokine interaction

Studies on the Substrate Selectivity of the Hypoxia‐Inducible Factor Prolyl Hydroxylase 2 Catalytic Domain

Isolation of Chemically Cyclized Peptide Binders Using Yeast Surface Display

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