Epitope Targeting of Tertiary Protein Structure Enables Target‐Guided Synthesis of a Potent In‐Cell Inhibitor of Botulinum Neurotoxin

Farrow, Blake; Wong, Michelle; Malette, Jacquie; Lai, Bert; Deyle, Kaycie M.; Das, Samir; Nag, Arundhati; Agnew, Heather D.; Heath, James R.

doi:10.1002/anie.201502451

Cited by 33 publications

(30 citation statements)

References 33 publications

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“…Previous efforts have demonstrated that binding affinity enhancement through cooperativity can be achieved by conjoining two binders against a target protein via a chemical linker . The length of the linker should correspond to the distance between the first and second epitopes.…”

Section: Resultsmentioning

confidence: 99%

Epitope‐Targeted Macrocyclic Peptide Ligand with Picomolar Cooperative Binding to Interleukin‐17F

Lai¹,

Wilson²,

Loredo³

et al. 2018

Chemistry A European J

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The IL-17 cytokine family is associated with multiple immune and autoimmune diseases and comprises important diagnostic and therapeutic targets. This work reports the development of epitope-targeted ligands designed for differential detection of human IL-17F and its closest homologue IL-17A. Non-overlapping and unique epitopes on IL-17F and IL-17A were identified by comparative sequence analysis of the two proteins. Synthetic variants of these epitopes were utilized as targets for in situ click screens against a comprehensive library of synthetic peptide macrocycles with 5-mer variable regions. Single generation screens yielded selective binders for IL-17F and IL-17A with low cross-reactivity. Macrocyclic peptide binders against two distinct IL-17F epitopes were coupled using variable length chemical linkers to explore the physical chemistry of cooperative binding. The optimized linker length yielded a picomolar affinity binder, while retaining high selectivity. The presented method provides a rational approach towards targeting discontinuous epitopes, similar to what is naturally achieved by many B cell receptors.

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Section: Resultsmentioning

confidence: 99%

Epitope‐Targeted Macrocyclic Peptide Ligand with Picomolar Cooperative Binding to Interleukin‐17F

Lai¹,

Wilson²,

Loredo³

et al. 2018

Chemistry A European J

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“…The results here with mAbs confirm our earlier studies with a single domain antibody that mAbs that bind to the α-exosite, or other sites within the extended substrate-binding groove can inhibit substrate cleavage. Such exosites have been targeted for inhibitor development to other toxins [ 37 , 38 ] and more recently for BoNT/A [ 52 ], and a refinement to this approach could be to string together small molecules binding at different sites to achieve avidity and better substrate inhibition [ 53 ]. Alternatively, recently described liposomal “delivery vehicles” could be used to deliver inhibitory single chain or single domain antibodies into motor neurons to inhibit BoNT and reverse paralysis [ 54 , 55 ].…”

Section: Discussionmentioning

confidence: 99%

Monoclonal Antibodies Targeting the Alpha-Exosite of Botulinum Neurotoxin Serotype/A Inhibit Catalytic Activity

et al. 2015

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The paralytic disease botulism is caused by botulinum neurotoxins (BoNT), multi-domain proteins containing a zinc endopeptidase that cleaves the cognate SNARE protein, thereby blocking acetylcholine neurotransmitter release. Antitoxins currently used to treat botulism neutralize circulating BoNT but cannot enter, bind to or neutralize BoNT that has already entered the neuron. The light chain endopeptidase domain (LC) of BoNT serotype A (BoNT/A) was targeted for generation of monoclonal antibodies (mAbs) that could reverse paralysis resulting from intoxication by BoNT/A. Single-chain variable fragment (scFv) libraries from immunized humans and mice were displayed on the surface of yeast, and 19 BoNT/A LC-specific mAbs were isolated by using fluorescence-activated cell sorting (FACS). Affinities of the mAbs for BoNT/A LC ranged from a KD value of 9.0×10−11 M to 3.53×10−8 M (mean KD 5.38×10−9 M and median KD 1.53×10−9 M), as determined by flow cytometry analysis. Eleven mAbs inhibited BoNT/A LC catalytic activity with IC50 values ranging from 8.3 ~73×10−9 M. The fine epitopes of selected mAbs were also mapped by alanine-scanning mutagenesis, revealing that the inhibitory mAbs bound the α-exosite region remote from the BoNT/A LC catalytic center. The results provide mAbs that could prove useful for intracellular reversal of paralysis post-intoxication and further define epitopes that could be targeted by small molecule inhibitors.

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“…However done,t he general ability to target specific epitopes on specific proteins is enabling.F or example,t he broad flexibility of the epitope targeting strategy is already permitting exploitation of the tertiary structure of the drug target as as caffold for developing highly potent inhibitors. [10]…”

Section: Methodsmentioning

confidence: 99%

“…Akt1 E17K (Protein kinase Bwith oncogenic point mutation) [9] Biotin-PEG 5 -PEVAIVKEGWLKKRGK Y-Pra-KTWRPRYFLLKNDG yleaf (Li)6 1nm 54 nm XII. BoNT ALC(Botulinum neurotoxin serotype Alight chain) [10] Az4-SFGHEVLNLTRN-PEG 4 -Biotin (amino acids 166-179)…”

Section: Monoclonalantibodies(mabs)raisedagainstshortpeptidementioning

confidence: 99%

A General Synthetic Approach for Designing Epitope Targeted Macrocyclic Peptide Ligands

et al. 2015

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We describe a general synthetic strategy for developing high affinity peptide binders against specific epitopes of challenging protein biomarkers. The epitope of interest is synthesized as a polypeptide, with a detection biotin tag and a strategically placed azide (or alkyne) presenting amino acid. This synthetic epitope (SynEp) is incubated with a library of complementary alkyne or azide presenting peptides. Library elements that bind the SynEp in the correct orientation undergo the Huisgen cycloaddition, and are covalently linked to the SynEp. Hit peptides are tested against the full-length protein to identify a best binder. We describe epitope-targeted linear or macrocycle peptide ligands against 12 different diagnostic or therapeutic analytes. The general epitope targeting capability for these low molecular weight synthetic ligands enables a range of therapeutic and diagnostic applications, similar to those of monoclonal antibodies.

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Epitope Targeting of Tertiary Protein Structure Enables Target‐Guided Synthesis of a Potent In‐Cell Inhibitor of Botulinum Neurotoxin

Cited by 33 publications

References 33 publications

Epitope‐Targeted Macrocyclic Peptide Ligand with Picomolar Cooperative Binding to Interleukin‐17F

Epitope‐Targeted Macrocyclic Peptide Ligand with Picomolar Cooperative Binding to Interleukin‐17F

Monoclonal Antibodies Targeting the Alpha-Exosite of Botulinum Neurotoxin Serotype/A Inhibit Catalytic Activity

A General Synthetic Approach for Designing Epitope Targeted Macrocyclic Peptide Ligands

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