A Chemical Epitope‐Targeting Strategy for Protein Capture Agents: The Serine 474 Epitope of the Kinase Akt2

Nag, Arundhati; Das, Samir; Yu, Mary Beth; Deyle, Kaycie M.; Millward, Steven W.; Heath, James R.

doi:10.1002/anie.201305882

Cited by 20 publications

(19 citation statements)

References 26 publications

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“…We increased the interaction footprint of the PCC with Akt2 by expanding it into two distinct triligands through in situ click chemistry screens. One of the triligands, tri_a, was shown to allosterically activate Akt enzymatic activity in in vitro kinase assays .…”

Section: Introductionmentioning

confidence: 90%

“…These synthetic peptides have some similarities to monoclonal antibodies, but are a fraction of the size and can exhibit a high level of stability . A recent advance of this technology permits the targeted development of a PCC against a specific epitope of a given protein . Unlike the case for small molecule ligands, the generalized PCC epitope targeting strategy does not rely on the presence of a hydrophobic binding pocket.…”

Section: Introductionmentioning

confidence: 99%

“…Aberrant Akt signaling is implicated in diabetes and in many cancers, making it an attractive drug and diagnostic target . We previously reported the development of a PCC targeting the C‐terminal hydrophobic motif of Akt2 that includes the Ser474 residue . Phosphorylation of Akt at Ser474 leads to allosteric activation of Akt and increases the kinase activity tenfold .…”

Section: Introductionmentioning

confidence: 99%

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Degradation of Akt using protein-catalyzed capture agents

et al. 2016

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Abnormal signaling of the protein kinase Akt has been shown to contribute to human diseases such as diabetes and cancer, but Akt has proven to be a challenging target for drugging. Using iterative in situ click chemistry we recently developed multiple protein catalyzed capture (PCC) agents that allosterically modulate Akt enzymatic activity in a protein based assay. Here we utilize similar PCCs to exploit endogenous protein degradation pathways. We use the modularity of the anti-Akt PCCs to prepare Proteolysis Targeting Chimeric molecules (PROTACs) that are shown to promote the rapid degradation of Akt in live cancer cells. These novel PROTACs demonstrate that the epitope targeting selectivity of PCCs can be coupled with non-traditional drugging moieties to inhibit challenging targets.

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Section: Introductionmentioning

confidence: 90%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Degradation of Akt using protein-catalyzed capture agents

et al. 2016

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“…We provide adetailed description of the screening process and demonstrate its generality through the identification of 12 epitope targeted PCC agents.T hese ligands fulfill very challenging targeting aims such as selective detection of ap hosphorylated epitope, [8] as ingle amino acid point mutation, [9] ag enus specific sequence in am alarial protein biomarker, and au niversally conserved small region of ag eographically variable malarial biomarker. Thep rocess of development of the PCC agents against malarial biomarker proteins is elaborated to illustrate the technique.Macrocyclic peptide libraries have yielded superior performing PCC agents,a nd so are described in detail.…”

Section: Monoclonalantibodies(mabs)raisedagainstshortpeptidementioning

confidence: 99%

“…Thee pitope targeted in situ click screen is as ingle generation screen, with results that are filtered through one or more anti-screens.T he OBOC peptide libraries, [7] which are comprehensive in 18 natural or non-natural amino acids ( % 2million sequences), are screened against abiotin tagged scrambled sequence of the same length as the SynEp,o ra n off-target peptide representing adifferent epitope of the same protein (Table S1 in the Supporting Information). Nonspecific binders from the anti-screen are identified colorimetrically by treatment of the screened library with an anti- X. p-Akt2 (Protein kinase B2;s trategy targets region adjacent to p-Ser474) [8] ITPPDRYDSLGLLELQRTHFPQF [pS-(Zn 2 L)]YSASIRE (amino acids 450-481 of pAkt2) wkvkl (Li)3 .6 mm XI. Akt1 E17K (Protein kinase Bwith oncogenic point mutation) [9] Biotin-PEG 5 -PEVAIVKEGWLKKRGK Y-Pra-KTWRPRYFLLKNDG yleaf (Li)6 1nm 54 nm XII.…”

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

Farrow

Wong

Malette³

et al. 2015

Angewandte Chemie

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Botulinum neurotoxin (BoNT) serotype A is the most lethal known toxin and has an occluded structure which prevents direct inhibition of its active site before it enters the cytosol. We combine in situ click target-guided synthesis with synthetic epitope-targeting to exploit the tertiary structure of the BoNT protein as a landscape for assembling a competitive inhibitor. A substrate mimicking peptide macrocycle is used as a direct inhibitor of BoNT. An epitope targeted in situ click screen is utilized to identify a second peptide macrocycle ligand that binds to an epitope that, in the folded BoNT structure, is active site adjacent. A second in situ click screen identifies a molecular bridge between the two macrocycles. The resulting divalent inhibitor exhibits an inhibition constant of 165 pM in vitro against the BoNT/A catalytic chain. The inhibitor is carried into cells by the intact holotoxin, and demonstrates protection and rescue of BoNT intoxication in a human neuron model. Keywordstarget-guided synthesis; botulinum neurotoxin; combinatorial screening; epitope targeting; peptides Botulinum neurotoxin (BoNT) serotype A is the most lethal known toxin and is produced by some species of the bacterial genus Clostridium. BoNT/A is a chemodenervating zincdependent protease that prevent the Ca 2+ -triggered release of acetylcholine in neuromuscular junctions, by cleaving one of the three SNARE proteins required for synaptic vesicle

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A Chemical Epitope‐Targeting Strategy for Protein Capture Agents: The Serine 474 Epitope of the Kinase Akt2

Cited by 20 publications

References 26 publications

Degradation of Akt using protein-catalyzed capture agents

Degradation of Akt using protein-catalyzed capture agents

A General Synthetic Approach for Designing Epitope Targeted Macrocyclic Peptide Ligands

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

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