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

Abstract

Purpose The heparin-reactive, helical peptide p5 is an effective amyloid imaging agent in mice with systemic amyloidosis. Analogs of p5 with modified secondary structure characteristics exhibited altered binding to heparin, synthetic amyloid fibrils, and amyloid extracts in vitro. Herein, we further study the effects of peptide helicity and chirality on specific amyloid binding using a mouse model of systemic inflammation-associated (AA) amyloidosis. Procedures Peptides with disrupted helical structure [p5(coil) and p5(Pro3)], with an extended sheet conformation [p5(sheet)] or an all-D enantiomer [p5(D)], were chemically synthesized, radioiodinated, and their biodistribution studied in WT mice as well as transgenic animals with severe systemic AA amyloidosis. Peptide binding was assessed qualitatively by using small animal single-photon emission computed tomography/x-ray computed tomography imaging and microautoradiography and quantitatively using tissue counting. Results Peptides with reduced helical propensity, p5(coil) and p5(Pro3), exhibited significantly reduced binding to AA amyloid-laden organs. In contrast, peptide p5(D) was retained by non-amyloid-related ligands in the liver and kidneys of both WT and AA mice, but it also bound AA amyloid in the spleen. The p5(sheet) peptide specifically bound AA amyloid in vivo and was not retained by healthy tissues in WT animals. Conclusions Modification of amyloid-targeting peptides using D-amino acids should be performed cautiously due to the introduction of unexpected secondary pharmacologic effects. Peptides that adopt a helical structure, to align charged amino acid side chains along one face, exhibit specific reactivity with amyloid; however, polybasic peptides with a propensity for β-sheet conformation are also amyloid-reactive and may yield a novel class of amyloid-targeting agents for imaging and therapy.

Bifunctional amyloid-reactive peptide promotes binding of antibody 11-1F4 to diverse amyloid types and enhances therapeutic efficacy

“…Electrostatic interactions have been shown to provide a basis for the reactivity of peptides and proteins with multiple diverse forms of amyloid-like fibrils (37)(38)(39)(40). We have shown that polybasic peptides with a predicted α-helical or β-sheet secondary structure are reactive with ALκ, ALλ, ATTR, AA, ALECT2, Aβ (1-40), and IAPP fibrils or amyloid deposits (20,41). Furthermore, these peptides did not bind to normal tissue in any assay tested in vitro or in mice.…”

A Peptide-Fc Opsonin with Pan-Amyloid Reactivity

“…Within weeks of induction, the mice develop severe systemic AA-associated amyloidosis predominantly in the spleen and liver, but the pancreas, kidney, adrenal, heart, and vasculature are also involved. These extensive deposits can serve as a target for the in vivo quantitative evaluation of amyloid-reactive reagents such as peptides ( 13 , 14 , 17 ) and the Fc-peptide fusion reagents. Using this experimental model, we observed specific co-localization of 125 I-Fcp5 was observed in hepatosplenic AA amyloid deposits, as evidenced by the presence of black punctate staining in microautoradiographs that correlated with the anatomic distribution of amyloid (Figure 7 ).…”

“…If, however, the rapid loss of radioiodine from the amyloid deposits within 48 h postinjection reflects a short amyloid-bound half-life, improved Fc-peptide constructs may be required to enhance the peptide–amyloid interaction. To address this, we are generating reagents that increase the charge density of the amyloidophilic peptide moiety and exploring the use, either as an alternative or in conjunction with p5, of polybasic peptides with β-sheet primary structure [e.g., peptide p5 (sheet) ( 14 )].…”

“…We have developed a panel of synthetic, polybasic peptides that specifically bind to many forms of human amyloid, including AL, AA, ALect2, ATTR, Alzheimer’s disease-associated (Aβ), and islet amyloid polypeptide (AIAPP), found in patients with type 2 diabetes ( 12 – 14 ). One of these peptides, designated p5, is a non-natural 31-amino acid peptide with eight lysine residues spaced in a heptad repeat such that when an α-helical secondary structure is formed, the charged side chains align along one face of the helix ( 15 ).…”

Evaluation of the effect of d-amino acid incorporation into amyloid-reactive peptides

“…Thus, there is an unmet clinical need for an imaging agent capable of detecting many, if not all, types of systemic amyloid regardless of the anatomic site of deposition. For this reason, our recent efforts have focused on developing an imaging agent for the non-invasive, quantitative assessment of amyloid load in patients [ 8 – 10 ].…”