A protein-targeting strategy used to develop a selective inhibitor of the E17K point mutation in the PH domain of Akt1

Deyle, Kaycie M.; Farrow, Blake; Hee, Ying Qiao; Work, Jeremy J.; Wong, Michelle; Lai, Bert; Umeda, Aiko; Millward, Steven W.; Nag, Arundhati; Das, Samir; Heath, James R.

doi:10.1038/nchem.2223

Cited by 27 publications

(22 citation statements)

References 28 publications

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“…Inh-1 binds to the active site with a ~70 nM binding affinity (k D ) and similar inhibition constant. We then employ an all synthetic in situ click epitope targeting approach [10] (Scheme 1) to identify a second peptide macrocycle ( L2 , SI Figure S3–4) that binds to a site a few angstroms away in the folded protein structure from the active site. L2 exhibits a k D ~80 nM, but no inhibitory effects.…”

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confidence: 99%

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

Angew Chem Int Ed

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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.

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confidence: 99%

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

Angew Chem Int Ed

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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%

“…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. BoNT ALC(Botulinum neurotoxin serotype Alight chain) [10] Az4-SFGHEVLNLTRN-PEG 4 -Biotin (amino acids 166-179)…”

Section: Monoclonalantibodies(mabs)raisedagainstshortpeptidementioning

confidence: 99%

“…These ligands fulfill very challenging targeting aims such as selective detection of a phosphorylated epitope[8], a single amino acid point mutation[9], and detection of sequences within malarial protein biomarkers that distinguish specific species of the Plasmodium genus or, for a different malarial biomarker, small regions of the protein that are geographically conserved. The development of the PCC agents against the malarial biomarker proteins are elaborated to illustrate the technique.…”

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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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“…31 Moreover, in situ click chemistry has been used extensively to create antibody-like protein capture agents. 32-37 …”

Section: Introductionmentioning

confidence: 99%

Ribosome-Templated Azide–Alkyne Cycloadditions: Synthesis of Potent Macrolide Antibiotics by In Situ Click Chemistry

et al. 2016

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Over half of all antibiotics target the bacterial ribosome-Nature's complex, 2.5 MDa nanomachine responsible for decoding mRNA and synthesizing proteins. Macrolide antibiotics, exemplified by erythromycin, bind the 50S subunit with nM affinity and inhibit protein synthesis by blocking the passage of nascent oligopeptides. Solithromycin (1), a third-generation semisynthetic macrolide discovered by combinatorial copper-catalyzed click chemistry, was synthesized in situ by incubating either E. coli 70S ribosomes or 50S subunits with macrolidefunctionalized azide 2 and 3-ethynylaniline (3) precursors. The ribosome-templated in situ click method was expanded from a binary reaction (i.e., one azide and one alkyne) to a sixcomponent reaction (i.e., azide 2 and five alkynes) and ultimately to a sixteen-component reaction (i.e., azide 2 and fifteen alkynes). The extent of triazole formation correlated with ribosome affinity for the anti (1,4)-regioisomers as revealed by measured K d values. Computational analysis using the Site-Identification by Ligand Competitive Saturation (SILCS) approach indicated that the relative affinity of the ligands was associated with the alteration of macrolactone+desosamine- HHS Public Access

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A protein-targeting strategy used to develop a selective inhibitor of the E17K point mutation in the PH domain of Akt1

Cited by 27 publications

References 28 publications

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

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

A General Synthetic Approach for Designing Epitope Targeted Macrocyclic Peptide Ligands

Ribosome-Templated Azide–Alkyne Cycloadditions: Synthesis of Potent Macrolide Antibiotics by In Situ Click Chemistry

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