De novo carborane-containing macrocyclic peptides targeting human EGFR.

L-carboranylalanine (LCba) is a unique artificial amino acid containing a cluster of ten boron atoms. Since the three-dimensional aromaticity and charge distributions of the carborane sidechain are quite different from any sidechains of proteinogenic amino acids, there is no report whether LCba can be a substrate for the translation machinery. Here we report studies on the ribosomal incorporation of LCba into peptide via initiation and elongation using the flexizyme-assisted translation system. Our results indicate that only the initiation step could tolerate LCba incorporation but elongation steps could not, very likely due to its steric bulkiness of the sidechain. Based on this knowledge, we have designed a library of macrocyclic peptides initiated by α-N-(2-choloroacetyl)-L-carboranylalanine (ClAc-LCba) and selected molecules capable of binding to human epidermal growth factor receptor (hEGFR). Two peptides that were forwarded to deeper studies exhibited affinities with KD values at 16 and 20 nM against hEGFR. Computational modeling of one of the peptides suggested that the carborane sidechain might be directly involved in the interaction with the hydrophobic β-sheet core in the EGF binding site of hEGFR, which is consistent with the mutational data where replacing LCba residue with LPhe completely eliminated the binding activity. Cell lines that stably expressing hEGFR could be stained by incubation with the C-terminal fluorescein-labeled peptides, whereas hEGFR-negative cells could not be stained. This study provides a general strategy for the de novo discovery of carborane-containing macrocyclic peptides against various tumor biomarker proteins, potentially applicable to boron neuron capture therapy.