Dual-Purpose Linker for Alpha Helix Stabilization and Imaging Agent Conjugation to Glucagon-Like Peptide-1 Receptor Ligands

Abstract

Peptides display many characteristics of efficient imaging agents such as rapid targeting, fast background clearance, and low non-specific cellular uptake. However, poor stability, low affinity, and loss of binding after labeling often preclude their use in vivo. Using the glucagon-like peptide-1 receptor (GLP-1R) ligands exendin and GLP-1 as a model system, we designed a novel alpha helix stabilizing linker to simultaneously address these limitations. The stabilized and labeled peptides showed an increase in helicity, improved protease resistance, negligible loss or an improvement in binding affinity, and excellent in vivo targeting. The ease of incorporating azidohomoalanine in peptides and efficient reaction with the dialkyne linker enables this technique to potentially be used as a general method for labeling alpha helices. This strategy should be useful for imaging beta cells in diabetes research and in developing and testing other peptide targeting agents.

Quantitative Impact of Plasma Clearance and Down-regulation on GLP-1 Receptor Molecular Imaging

“…High levels of receptor expression (> 50,000 receptors per cell), rapid internalization, and an easily accessible cell surface location make GLP-1 receptor an attractive target for BCM quantitation, and simulations help identify the most important areas for improvement. Lowering non-specific interactions through peptide stabilization[43–44] and/or accounting for this signal through ratio imaging[80] will help increase the precision and sensitivity of beta cell mass measurements for the early detection of type 1 diabetes.…”

“…Expression of GLP-1R in beta cells, high vascularization of tissue within the islets of Langerhans, rapid binding kinetics, and in vivo target specificity have led to the development of multiple radiolabeled exendin molecules to track the beta cell mass as well as detection of insulinomas[24, 33–34, 37–42]. Owing to its fast binding kinetics, rapid uptake in target tissue, and ability to be stabilized to resist protease degradation, exendin and related peptides have been investigated as molecular imaging agents for measuring beta cell mass in vivo [24, 36, 43–44]. However, current approaches lack the sensitivity required for clinical application[25, 41, 45], so more development is needed.…”

A Helix-Stabilizing Linker Improves Subcutaneous Bioavailability of a Helical Peptide Independent of Linker Lipophilicity

“…Careful consideration was taken when choosing dyes, as dye structure and molecular charge can greatly impact non-specific cellular and plasma protein interactions 49, 50 . Cy7 and Alexa Fluor 680 were chosen based on plasma protein binding of the free dye as determined by rapid equilibrium dialysis as well as previously published data on Cy7 exendin and s -AF680 exendin 32, 51 . All fluorescent peptides were purified with moderate yield (40–60%) and the exact mass confirmed by ESI-MS (SI).…”

“…Changes in proteolytic stability due to helix stabilization were investigated through a trypsin digest adapted from a previously published protocol 32 as well as serum and plasma stability measurements (SI). Digests of stabilized and non-stabilized peptides (Fig.…”

“…Similar to previously published work, either one or two AHA substitutions were made to generate single-mutant and double-mutant exendin 32, 51 . A methionine residue at the 14 th position was substituted with AHA during solid phase peptide synthesis based on its position pointing away from the binding pocket of GLP-1R 48 .…”