Exploring efficient strategies for identifying the natural products and their derivatives that selectively regulate protein/protein interactions is one of the most actively studied topics in chemical biology. Especially, the selective activation and/or inhibition of specific phosphorylation-mediated protein/protein interactions within a signaling pathway may direct a specific biological response on the cellular level. In pursuit of such molecules, we developed a Template-Assisted Transcription Synthesis. The approach is based on our findings that (i) the bis-lysine structure such as 1 interacts with the phosphorylated compounds, and (ii) the benzyl-protected histidine significantly accelerates the peptide-based Huisgen cycloaddition, so that the structure 1 can efficiently be modified by the peptide library to strongly and selectively bind to the target phosphorylated protein in a template-assisted fashion. As a model case, we applied to preparing the Grb2/SH2 domain mimicry. The template-assisted transcription synthesis between the bis-lysine 1 and the acetylene-peptide library 2 was performed in the presence of the cyclic phosphopeptide 3 (known ligand for Grb2/SH2 domain) as the template. The clicked peptides 4a, b, and c as the template-assisted products, exhibited the micromolar-level dissociation constants to the phosphopeptide 3. Conformational restriction of the initial hit structure 4a by the solid-supported ring-closing metathesis gave the cyclic peptide 9, further enhancing the interaction with the peptide 3, i.e., Kd of 590 nM. One of the clicked product 4a, the peptidyl mimic of the Grb2-SH2 domain, was found to be internalized into A431 cancer cells, where the EGFR-induced cellular signalling is operative. It selectively bound to the phosphorylated Shc protein, one of the Grb2-SH2 interacting signaling proteins, induced A431-selective apoptosis and tumor growth inhibition. The clicked peptide 4a even exhibited the A431-selective inhibition of tumor growth without any toxic effects and inflammatory responses in the preliminary animal experiments. The results described in this symposium demonstrate the tailor-made synthesis of artificial receptors using a Template-Assisted Transcription Synthesis that is clearly distinct from previous methods. This approach may efficiently and rapidly provide small-molecular regulators that selectively control a specific cell signaling pathway or protein/protein interactions.
