Identification, Characterization, and Optimization of Integrin αvβ6-Targeting Peptides from a One-Bead One-Compound (OBOC) Library: Towards the Development of Positron Emission Tomography (PET) Imaging Agents

Abstract

The current translation of peptides identified through the one-bead one-compound (OBOC) technology into positron emission tomography (PET) imaging agents is a slow process, with a major delay between ligand identification and subsequent lead optimization. This work aims to streamline the development process of 18F-peptide based PET imaging agents to target the integrin αvβ6. By directly identify αvβ6–targeting peptides from a 9-mer 4-fluorobenzoyl peptide library using the on-bead two-color (OBTC) cell-screening assay, a total of 185 peptide beads were identified and 5 beads sequenced for further evaluation. The lead peptide 1 (VGDLTYLKK(FB), IC50 = 0.45 ± 0.06 μM, 25% stable in serum at 1 h) was further modified at the N-, C-, and bi-termini. C-terminal PEGylation increased the metabolic stability (>95% stable), but decreased binding affinity (IC50 = 3.7 ± 1 μM) was noted. C-terminal extension (1i, VGDLTYLKK(FB)KVART) significantly increased binding affinity for integrin αvβ6 (IC50 = 0.021 ± 0.002 μM), binding selectivity for αvβ6-expressing cells (3.1 ± 0.8:1), and the serum stability (>99% stable). Our results demonstrate the challenges in optimizing OBOC-derived peptides, indicate both termini of 1 are sensitive to modifications, and show that further modification of 1 is necessary to demonstrate utility as an 18F-peptide imaging agent.

Biological Relevance of RGD‐Integrin Subtype‐Specific Ligands in Cancer

“…It is important to note that other RGD-integrins become under spotlight as for example the αvβ6 integrin. [162] Selective Inhibitors and PET tracers have been designed, [163][164][165][166][167] which will be useful to detect and treat epithelial carcinoma for example. Integrins remain therapeutic targets in oncology (as assessed by 2 recent reviews [168,169] ), but we have to reconsider their roles in the area of personalized medicine.…”

MuTaTo<sup>&copy</sup>—A Novel Concept for Curing Cancer

“…We have improved the half-life in mice blood from 4 minutes in the case of one peptide to 3 hours when 8 copies of the same peptide were connected to 8-arm PEG (data not shown). The peptides that are connected to PEG are also partly protected from digestion with an increase in their metabolic stability in serum [14]. Although they are big, they are very flexible and hydrophilic due to the long PEG arms (Figure 1).…”

Incorporation of Fluorine into an OBOC Peptide Library by Copper-Free Click Chemistry toward the Discovery of PET Imaging Agents

“…The synthesis of one-bead one-compound (OBOC) libraries is a combinatorial method introduced by Lam et al in 1991 that involves split-and-mix synthesis to create millions of candidates from which peptide–receptor interactions can be discovered. Although the screening of classic OBOC libraries produces target-specific peptides, − post-identification modification of these peptides to convert them into imaging agents often decreases binding affinity and creates the necessity for further modifications. − However, few ventures have been made into making OBOC libraries of imaging agents. − …”

“…This enables high-throughput screening of millions of potential imaging agents. Tang et al recently created a focused OBOC peptide library containing both the α v β 6 -targeting motif and a C-terminal 4-fluorobenzoyl group for imaging and had success in discovering lead peptides toward developing new 18 F PET agents …”

“…Exciting new advancements in technologies for tracking synthetic histories of solid-phase syntheses with DNA tags or RFID microtransponders are highly complementary to this sort of application and will remove the sequencing bottleneck encountered, which would result in higher numbers of imaging agent hits to study. , Improvements in the methodology to address some of the challenges encountered in development of this library will be explored in future generations of OBOC imaging agent libraries. Focused library synthesis would aid in the throughput of sequence deconvolution when part of the sequence is known as well as likely produce higher affinity hits. , Other library conformations where the imaging moiety is dispersed through the peptide sequences could also be explored to promote discovery of imaging agents where the imaging moiety is integrated into the receptor-binding pocket.…”