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A binding-site barrier affects imaging efficiency of high affinity amyloid-reactive peptide radiotracers in vivo.

Wall JS, Williams A, Richey T, Stuckey A, Huang Y, Wooliver C, Macy S, Heidel E, Gupta N, Lee A, Rader B, Martin EB, Kennel SJ - PLoS ONE (2013)

Bottom Line: Amyloid is a complex pathology associated with a growing number of diseases including Alzheimer's disease, type 2 diabetes, rheumatoid arthritis, and myeloma.The p5 peptide was posited to bind effectively to amyloid deposits, relative to similarly charged polybasic heparin-reactive peptides, because it adopted a polar α helix secondary structure.The p5R peptide had higher affinity for amyloid and visualized AA amyloid in mice by using SPECT/CT imaging; however, the microdistribution, as evidenced in micro-autoradiographs, was dramatically altered relative to the p5 peptide due to its increased affinity and a resultant "binding site barrier" effect.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of Tennessee Graduate School of Medicine, Knoxville, Tennessee, USA. jwall@utmck.edu

ABSTRACT
Amyloid is a complex pathology associated with a growing number of diseases including Alzheimer's disease, type 2 diabetes, rheumatoid arthritis, and myeloma. The distribution and extent of amyloid deposition in body organs establishes the prognosis and can define treatment options; therefore, determining the amyloid load by using non-invasive molecular imaging is clinically important. We have identified a heparin-binding peptide designated p5 that, when radioiodinated, was capable of selectively imaging systemic visceral AA amyloidosis in a murine model of the disease. The p5 peptide was posited to bind effectively to amyloid deposits, relative to similarly charged polybasic heparin-reactive peptides, because it adopted a polar α helix secondary structure. We have now synthesized a variant, p5R, in which the 8 lysine amino acids of p5 have been replaced with arginine residues predisposing the peptide toward the α helical conformation in an effort to enhance the reactivity of the peptide with the amyloid substrate. The p5R peptide had higher affinity for amyloid and visualized AA amyloid in mice by using SPECT/CT imaging; however, the microdistribution, as evidenced in micro-autoradiographs, was dramatically altered relative to the p5 peptide due to its increased affinity and a resultant "binding site barrier" effect. These data suggest that radioiodinated peptide p5R may be optimal for the in vivo detection of discreet, perivascular amyloid, as found in the brain and pancreatic vasculature, by using molecular imaging techniques; however, peptide p5, due to its increased penetration, may yield more quantitative imaging of expansive tissue amyloid deposits.

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Peptide p5R exhibits enhanced α-helical secondary structure.CD spectra of peptides p5R (A – arrow shows isodichroic point) and p5 (B) in increasing concentrations of trifluoroethanol (TFE). The ratio of 222 nm and 205 nm was calculated (C) as a measure of α-helical content for p5 (○) and p5R (•). The 222 nm/205 nm ratio for human serum albumin in PBS was 1.0 (not shown).
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pone-0066181-g002: Peptide p5R exhibits enhanced α-helical secondary structure.CD spectra of peptides p5R (A – arrow shows isodichroic point) and p5 (B) in increasing concentrations of trifluoroethanol (TFE). The ratio of 222 nm and 205 nm was calculated (C) as a measure of α-helical content for p5 (○) and p5R (•). The 222 nm/205 nm ratio for human serum albumin in PBS was 1.0 (not shown).

Mentions: The helicity of the peptides was assessed by using CD spectroscopy with varying concentrations of TFE (Figs. 2A & B). The CD spectra of both peptides contain minima at ∼205 nm and ∼220 nm but exhibit weak maxima at 195 nm indicative of a mix of α-helix and random coil. Helicity was assessed by calculating the ratio of the mean residue elipticity at 222 nm and 205 nm (Fig. 2C). Based on a CD spectrum of bovine serum albumin [30], a 222 nm/205 nm ratio of 1 was presumed to represent ∼100% helical structure. Both the p5 and p5R CD spectra ratios approached 1 as the concentration of TFE was increased to 40% by volume; however, in PBS alone, p5R exhibited 2-fold more helicity than p5 (Fig. 2C). As the TFE concentration was increased, the helicity of both peptides increased and was maximal and equivalent at 30% TFE (Fig. 2C). The spectra of peptide p5R (Fig. 2A) shows an isodichroic point (arrow) indicating a two state coil-to-helix transition. No such point was obvious in the p5 spectra.


A binding-site barrier affects imaging efficiency of high affinity amyloid-reactive peptide radiotracers in vivo.

Wall JS, Williams A, Richey T, Stuckey A, Huang Y, Wooliver C, Macy S, Heidel E, Gupta N, Lee A, Rader B, Martin EB, Kennel SJ - PLoS ONE (2013)

Peptide p5R exhibits enhanced α-helical secondary structure.CD spectra of peptides p5R (A – arrow shows isodichroic point) and p5 (B) in increasing concentrations of trifluoroethanol (TFE). The ratio of 222 nm and 205 nm was calculated (C) as a measure of α-helical content for p5 (○) and p5R (•). The 222 nm/205 nm ratio for human serum albumin in PBS was 1.0 (not shown).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3672142&req=5

pone-0066181-g002: Peptide p5R exhibits enhanced α-helical secondary structure.CD spectra of peptides p5R (A – arrow shows isodichroic point) and p5 (B) in increasing concentrations of trifluoroethanol (TFE). The ratio of 222 nm and 205 nm was calculated (C) as a measure of α-helical content for p5 (○) and p5R (•). The 222 nm/205 nm ratio for human serum albumin in PBS was 1.0 (not shown).
Mentions: The helicity of the peptides was assessed by using CD spectroscopy with varying concentrations of TFE (Figs. 2A & B). The CD spectra of both peptides contain minima at ∼205 nm and ∼220 nm but exhibit weak maxima at 195 nm indicative of a mix of α-helix and random coil. Helicity was assessed by calculating the ratio of the mean residue elipticity at 222 nm and 205 nm (Fig. 2C). Based on a CD spectrum of bovine serum albumin [30], a 222 nm/205 nm ratio of 1 was presumed to represent ∼100% helical structure. Both the p5 and p5R CD spectra ratios approached 1 as the concentration of TFE was increased to 40% by volume; however, in PBS alone, p5R exhibited 2-fold more helicity than p5 (Fig. 2C). As the TFE concentration was increased, the helicity of both peptides increased and was maximal and equivalent at 30% TFE (Fig. 2C). The spectra of peptide p5R (Fig. 2A) shows an isodichroic point (arrow) indicating a two state coil-to-helix transition. No such point was obvious in the p5 spectra.

Bottom Line: Amyloid is a complex pathology associated with a growing number of diseases including Alzheimer's disease, type 2 diabetes, rheumatoid arthritis, and myeloma.The p5 peptide was posited to bind effectively to amyloid deposits, relative to similarly charged polybasic heparin-reactive peptides, because it adopted a polar α helix secondary structure.The p5R peptide had higher affinity for amyloid and visualized AA amyloid in mice by using SPECT/CT imaging; however, the microdistribution, as evidenced in micro-autoradiographs, was dramatically altered relative to the p5 peptide due to its increased affinity and a resultant "binding site barrier" effect.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of Tennessee Graduate School of Medicine, Knoxville, Tennessee, USA. jwall@utmck.edu

ABSTRACT
Amyloid is a complex pathology associated with a growing number of diseases including Alzheimer's disease, type 2 diabetes, rheumatoid arthritis, and myeloma. The distribution and extent of amyloid deposition in body organs establishes the prognosis and can define treatment options; therefore, determining the amyloid load by using non-invasive molecular imaging is clinically important. We have identified a heparin-binding peptide designated p5 that, when radioiodinated, was capable of selectively imaging systemic visceral AA amyloidosis in a murine model of the disease. The p5 peptide was posited to bind effectively to amyloid deposits, relative to similarly charged polybasic heparin-reactive peptides, because it adopted a polar α helix secondary structure. We have now synthesized a variant, p5R, in which the 8 lysine amino acids of p5 have been replaced with arginine residues predisposing the peptide toward the α helical conformation in an effort to enhance the reactivity of the peptide with the amyloid substrate. The p5R peptide had higher affinity for amyloid and visualized AA amyloid in mice by using SPECT/CT imaging; however, the microdistribution, as evidenced in micro-autoradiographs, was dramatically altered relative to the p5 peptide due to its increased affinity and a resultant "binding site barrier" effect. These data suggest that radioiodinated peptide p5R may be optimal for the in vivo detection of discreet, perivascular amyloid, as found in the brain and pancreatic vasculature, by using molecular imaging techniques; however, peptide p5, due to its increased penetration, may yield more quantitative imaging of expansive tissue amyloid deposits.

Show MeSH
Related in: MedlinePlus