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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 binds immobilized heparin and AA amyloid tissue homogenate with high affinity.(A) Peptides p5R (solid) and p5 (dashed) eluted from a heparin-agarose column with peak conductivities of 100 mS/cm (∼1.3 M NaCl) and 70 mS/cm (∼0.9 M NaCl), respectively. (B) The concentration of NaCl required to inhibit 50% binding of peptide to AA amyloid-laden tissues (IC50) was 0.9 M and 0.35 M for p5R (gray) and p5 (black), respectively. (C) Binding of p5 (open) and p5R (closed) to AA amyloid-laden tissue homogenate assayed by addition of 5 ng of 125I-p5R with increasing concentrations of unlabeled homologous peptide. Scatchard plot of p5 (D) and p5R (E) binding to AA tissue homogenate. Data were fitted using 2 linear trendlines. Kd was determined as 1/Ka (slope  = –Ka).
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pone-0066181-g003: Peptide p5R binds immobilized heparin and AA amyloid tissue homogenate with high affinity.(A) Peptides p5R (solid) and p5 (dashed) eluted from a heparin-agarose column with peak conductivities of 100 mS/cm (∼1.3 M NaCl) and 70 mS/cm (∼0.9 M NaCl), respectively. (B) The concentration of NaCl required to inhibit 50% binding of peptide to AA amyloid-laden tissues (IC50) was 0.9 M and 0.35 M for p5R (gray) and p5 (black), respectively. (C) Binding of p5 (open) and p5R (closed) to AA amyloid-laden tissue homogenate assayed by addition of 5 ng of 125I-p5R with increasing concentrations of unlabeled homologous peptide. Scatchard plot of p5 (D) and p5R (E) binding to AA tissue homogenate. Data were fitted using 2 linear trendlines. Kd was determined as 1/Ka (slope  = –Ka).

Mentions: The relative affinity of the p5 and p5R peptides for heparin was assessed in vitro using a heparin-derivatized agarose column (Fig. 3A) Reactivity for murine AA amyloid was measured by the binding of radioiodinated peptides to hepatic AA amyloid in the presence of increasing NaCl concentrations (Fig. 3B). Even though the net charge of both peptides is 8+, peptide p5R bound with greater avidity to the heparin-agarose column as indicated by the greater amount of NaCl required to elute the peptide (i.e., peak values of 70 mS/cm [∼0.9 M NaCl] vs 100 mS/cm [∼1.3 M NaCl] for p5 and p5R, respectively – Fig. 3A). Similarly, the estimated IC50 of NaCl (the molar concentration required to inhibit 50% of the peptide binding) for the interaction of peptides with AA amyloid-laden liver tissue homogenate was ∼0.35 M and ∼0.9 M for p5 and p5R, respectively (Fig. 3B). Binding curves (Fig, 3C) and Scatchard plots were generated using 5 ng of radiolabeled p5 and p5R added to AA tissue homogenate in the presence of increasing concentrations of unlabeled peptide up to 2 µg or 1 µg for p5 and p5R, respectively (Figs. D and E). Scatchard analyses revealed 2 binding sites with AA liver homogenate for both peptides (Figs, 3D and 3E), with Kd values of 0.5 µM and 0.14 µM for the high affinity binding of p5 and p5R, respectively. The low affinity binding ∼ 1 µM for each peptide was essentially equivalent.


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 binds immobilized heparin and AA amyloid tissue homogenate with high affinity.(A) Peptides p5R (solid) and p5 (dashed) eluted from a heparin-agarose column with peak conductivities of 100 mS/cm (∼1.3 M NaCl) and 70 mS/cm (∼0.9 M NaCl), respectively. (B) The concentration of NaCl required to inhibit 50% binding of peptide to AA amyloid-laden tissues (IC50) was 0.9 M and 0.35 M for p5R (gray) and p5 (black), respectively. (C) Binding of p5 (open) and p5R (closed) to AA amyloid-laden tissue homogenate assayed by addition of 5 ng of 125I-p5R with increasing concentrations of unlabeled homologous peptide. Scatchard plot of p5 (D) and p5R (E) binding to AA tissue homogenate. Data were fitted using 2 linear trendlines. Kd was determined as 1/Ka (slope  = –Ka).
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pone-0066181-g003: Peptide p5R binds immobilized heparin and AA amyloid tissue homogenate with high affinity.(A) Peptides p5R (solid) and p5 (dashed) eluted from a heparin-agarose column with peak conductivities of 100 mS/cm (∼1.3 M NaCl) and 70 mS/cm (∼0.9 M NaCl), respectively. (B) The concentration of NaCl required to inhibit 50% binding of peptide to AA amyloid-laden tissues (IC50) was 0.9 M and 0.35 M for p5R (gray) and p5 (black), respectively. (C) Binding of p5 (open) and p5R (closed) to AA amyloid-laden tissue homogenate assayed by addition of 5 ng of 125I-p5R with increasing concentrations of unlabeled homologous peptide. Scatchard plot of p5 (D) and p5R (E) binding to AA tissue homogenate. Data were fitted using 2 linear trendlines. Kd was determined as 1/Ka (slope  = –Ka).
Mentions: The relative affinity of the p5 and p5R peptides for heparin was assessed in vitro using a heparin-derivatized agarose column (Fig. 3A) Reactivity for murine AA amyloid was measured by the binding of radioiodinated peptides to hepatic AA amyloid in the presence of increasing NaCl concentrations (Fig. 3B). Even though the net charge of both peptides is 8+, peptide p5R bound with greater avidity to the heparin-agarose column as indicated by the greater amount of NaCl required to elute the peptide (i.e., peak values of 70 mS/cm [∼0.9 M NaCl] vs 100 mS/cm [∼1.3 M NaCl] for p5 and p5R, respectively – Fig. 3A). Similarly, the estimated IC50 of NaCl (the molar concentration required to inhibit 50% of the peptide binding) for the interaction of peptides with AA amyloid-laden liver tissue homogenate was ∼0.35 M and ∼0.9 M for p5 and p5R, respectively (Fig. 3B). Binding curves (Fig, 3C) and Scatchard plots were generated using 5 ng of radiolabeled p5 and p5R added to AA tissue homogenate in the presence of increasing concentrations of unlabeled peptide up to 2 µg or 1 µg for p5 and p5R, respectively (Figs. D and E). Scatchard analyses revealed 2 binding sites with AA liver homogenate for both peptides (Figs, 3D and 3E), with Kd values of 0.5 µM and 0.14 µM for the high affinity binding of p5 and p5R, respectively. The low affinity binding ∼ 1 µM for each peptide was essentially equivalent.

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