A General Synthetic Approach for Designing Epitope Targeted Macrocyclic Peptide Ligands

Abstract

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...

Inhibition of heme sequestration of histidine‐rich protein 2 using multiple epitope‐targeted peptides

“…Outside of its biological role, HRP2 holds diagnostic utility as a biomarker for acute and chronic P. falciparum infection. We previously reported on the development of protein‐catalyzed capture agents (PCCs) that bind to HRP2 . We developed several PCCs as a cocktail designed to recognize multiple sites within HRP2, including several heme‐binding epitopes (Figure B,C) .…”

“…We previously reported on the development of protein‐catalyzed capture agents (PCCs) that bind to HRP2 . We developed several PCCs as a cocktail designed to recognize multiple sites within HRP2, including several heme‐binding epitopes (Figure B,C) . We used our previously reported approach of epitope targeting combined with in situ click chemistry screening to develop three macrocyclic peptide ligands against relatively conserved motifs within the protein (Figure C,D) .…”

“…We developed several PCCs as a cocktail designed to recognize multiple sites within HRP2, including several heme‐binding epitopes (Figure B,C) . We used our previously reported approach of epitope targeting combined with in situ click chemistry screening to develop three macrocyclic peptide ligands against relatively conserved motifs within the protein (Figure C,D) . Polypeptides representing the targeted epitopes were synthesized, affixed with a click handle, and screened against combinatorial macrocyclic peptide libraries presenting the complementary click handle.…”

Designing helical peptide inhibitors of protein–protein interactions

“…Das et al have synthesized and screened libraries of cyclized peptides. [77] Their techniques provide synthetic flexibility to incorporate non-natural amino acids, allowing for post-synthesis modification of ~2 million sequences using Cu-catalyzed azide–alkyne cycloaddition or ring-closing metathesis. Screening such libraries led to the discovery of high-affinity ligands for a wide variety of protein targets.…”

“…Screening such libraries led to the discovery of high-affinity ligands for a wide variety of protein targets. [77] Further advances of this type are needed to enable high-throughput screening of large libraries constructed with diverse chemistries.…”