Preclinical Validation of the Heparin-Reactive Peptide p5+14 as a Molecular Imaging Agent for Visceral Amyloidosis

Abstract

Amyloid is a complex pathologic matrix comprised principally of paracrystalline protein fibrils and heparan sulfate proteoglycans. Systemic amyloid diseases are rare, thus, routine diagnosis is often challenging. The glycosaminoglycans ubiquitously present in amyloid deposits are biochemically and electrochemically distinct from those found in the healthy tissues due to the high degree of sulfation. We have exploited this unique property and evaluated heparin-reactive peptides, such as p5+14, as novel agents for specifically targeting and imaging amyloid. Herein, we demonstrate that radiolabeled p5+14 effectively bound murine AA amyloid in vivo by using molecular imaging. Biotinylated peptide also reacted with the major forms of human amyloid in tissue sections as evidenced immunohistochemically. Furthermore, we have demonstrated that the peptide also binds synthetic amyloid fibrils that lack glycosaminoglycans implying that the dense anionic OPEN ACCESSMolecules 2015, 20 7658 motif present on heparin is mimicked by the amyloid protein fibril itself. These biochemical and functional data support the translation of radiolabeled peptide p5+14 for the clinical imaging of amyloid in patients.

Specific Amyloid Binding of Polybasic Peptides In Vivo Is Retained by β-Sheet Conformers but Lost in the Disrupted Coil and All D-Amino Acid Variants

“…There was no evidence of uptake in healthy organs or tissues, suggesting that the polybasic sheet conformation retained amyloid-specific interactions in vivo that were observed in vitro . In contrast to the helical reagent p5, and similar amyloid-reactive peptides [3], peptide p5 (sheet) has only 18 amino acids, rendering it cost-effective to produce for human use relative to peptide p5 and longer reagents [3]. Our initial studies have demonstrated that it exhibits amyloid-specific reactivity in vivo , similar to peptide p5, but previous data suggest that it may occupy a novel binding site on the amyloid fibril [13].…”

“…Amyloid-associated HS has been shown to be both biochemically and electrochemically distinct from the ubiquitous HS found in healthy (amyloid-free) tissues [2, 3]. The HS found in systemic inflammation-associated (AA) amyloid deposits in mice can be specifically targeted by using a radioiodinated single-chain fragment variable antibody (scFv), NS4F5, which binds hypersulfated HS with N-sulfation, C5-epimerization, and high degrees of 2-O- and 6-O-sulfation [4, 5].…”

“…The HS found in systemic inflammation-associated (AA) amyloid deposits in mice can be specifically targeted by using a radioiodinated single-chain fragment variable antibody (scFv), NS4F5, which binds hypersulfated HS with N-sulfation, C5-epimerization, and high degrees of 2-O- and 6-O-sulfation [4, 5]. Given that HS is invariably associated with all amyloid deposits and has a distinct pattern of hypersulfation, akin to that of heparin, we developed synthetic heparin-binding peptides as specific amyloid-targeting agents for molecular imaging [3, 6–9]. These peptides have also been shown to bind to synthetic amyloid fibrils, in the absence of HS, via electrostatic interactions with acidic amino acid side chains displayed with a well-defined periodicity on the surface of the fibrils [3, 10].…”

“…Given that HS is invariably associated with all amyloid deposits and has a distinct pattern of hypersulfation, akin to that of heparin, we developed synthetic heparin-binding peptides as specific amyloid-targeting agents for molecular imaging [3, 6–9]. These peptides have also been shown to bind to synthetic amyloid fibrils, in the absence of HS, via electrostatic interactions with acidic amino acid side chains displayed with a well-defined periodicity on the surface of the fibrils [3, 10]. …”

The pattern recognition reagents RAGE VC1 and peptide p5 share common binding sites and exhibit specific reactivity with AA amyloid in mice

“…We have developed a panel of structurally related synthetic peptides with a relatively simple heptad repeating primary structure (A-Q-X-A-Q-A-X, where X is arginine or lysine). These peptides exhibit specific binding to anionic polymers with a distinct charge distribution such as amyloid associated-hypersulfated heparan sulfate and fibrils of various types in vitro and in vivo [28,29,33]. When radiolabeled, these peptides preferentially bind AA amyloid in mice and can be visualized by using SPECT or PET imaging [28,32].…”