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.
Target and click: Peptide ligands targeted to the C‐terminal motif of the kinase Akt2 were obtained by combining phosphate recognition of a dinuclear zinc(II) complex with in situ click chemistry to target this epitope. The peptide ligands (shown as XXXXX) selectively bind the C‐terminal polypeptide of Akt2, and are selective for Akt2 relative to the Akt1 and Akt3 isoforms. The ligands differentially modulate Akt2 activity.
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. Graphical abstractCommunication: Match made in situ. We describe development of 12 peptide ligands against distinct epitopes of challenging proteins. This development is enabled by a general synthetic strategy. A synthetic epitope with an azide is screened against an alkyne containing macrocyclic library to identify the best binder. These synthetic ligands can enable antibody like therapeutic and diagnostic applications, with a small molecular footprint.Correspondence to: James R. Heath, heath@caltech.edu.[d] These authors have equal contribution. of 5-mer linear or macrocyclic peptides against a synthetic epitope (SynEp), which is a 10 to 30 amino acids long peptide representing a modified variant of the epitope of interest. The library elements are designed to present an azide (or alkyne) click handle, and a complementary alkyne (or azide) presenting amino acid is strategically substituted into the SynEp. During a screen, a library element that interacts with the SynEp in the right orientation, undergoes 1,3-dipolar cycloaddition to covalently bond to the epitope. The precise orbital alignment of terminal alkynes and azides required for the cycloaddition increases the entropic penalty of orientation, making the un-catalyzed reaction non-spontaneous under ambient conditions. This limitation is exploited here: we rely on specific interactions between the SynEp and the library element to overcome this entropic penalty so that the reaction proceeds un-catalyzed. HHS Public AccessWe provide a detailed description of the screening process and demonstrate its generality through the identification of 12 epitope targeted PCC agents. 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 protein...
Zielen und Klick: Peptidliganden, die auf das C‐terminale Epitop der Kinase Akt2 abzielen, wurden erhalten, indem die Phosphaterkennung durch einen zweikernigen Zink(II)‐Komplex mit In‐situ‐Klick‐Chemie kombiniert wurde. Die Peptidliganden (gezeigt als XXXXX) binden selektiv das C‐terminale Polypeptid von Akt2 und sind außerdem auch selektiv für Akt2 gegenüber den Akt1‐ und Akt3‐Isoformen. Die Liganden zeigen eine differenzielle Modulation der Akt2‐Aktivität.
Macrocycle peptides are promising constructs for imaging and inhibiting extracellular, and cell membrane proteins, but their use for targeting intracellular proteins is typically limited by poor cell penetration. We report the development of a cell-penetrant high-affinity peptide ligand targeted to the phosphorylated Ser474 epitope of the (active) Akt2 kinase. This peptide can function as an allosteric inhibitor, an immunoprecipitation reagent, and a live cell immunohistochemical staining reagent. Two cell penetrant stereoisomers were prepared and shown to exhibit similar target binding affinities and hydrophobic character but 2-3-fold different rates of cell penetration. Experimental and computational studies resolved that the ligands’ difference in cell penetration could be assigned to their differential interactions with cholesterol in the membrane. These results expand the tool kit for designing new chiral-based cell-penetrant ligands.
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