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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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The binding-site barrier effect.(A) Schematic representation of the diffuse accumulation of p5 throughout the amyloid lesion. In contrast, peptide p5R accumulates as non-uniform (focal) deposits at the periphery of the amyloid mass. (B) Microautoradiographs of 125I-p5 and 125I-p5R that exemplify the consequence of a binding-site barrier on the amyloid-uptake of radiolabeled peptides.
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pone-0066181-g007: The binding-site barrier effect.(A) Schematic representation of the diffuse accumulation of p5 throughout the amyloid lesion. In contrast, peptide p5R accumulates as non-uniform (focal) deposits at the periphery of the amyloid mass. (B) Microautoradiographs of 125I-p5 and 125I-p5R that exemplify the consequence of a binding-site barrier on the amyloid-uptake of radiolabeled peptides.

Mentions: Given the enhanced helicity and resulting increased affinity for amyloid, it was anticipated that the radiolabeled peptide 125I-p5R would provide enhanced amyloid imaging in vivo. Based on biodistribution studies and SPECT imaging, the p5R peptide bound AA amyloid in mice and generated images indiscernible from images that had been produced using 125I-p5 peptide [17], [18]. However, analysis of the microdistribution of 125I-p5R peptide in mice with AA amyloid revealed that, even though the “small” discrete amyloid lesions that are common e.g., in the liver, heart, pancreas and adrenal were evenly covered by radiolabeled peptide, the large expanses of avascular, dense amyloid seen in the spleens of highly diseased animals were variably stained. This phenomenon was most evident in mice with extensive (3+ – 4+; [19]) splenic amyloid but, interestingly, not in mice with (1+ – 2+) amyloid in the spleen which suggests that this effect is not due to the lack of a binding target in the splenic amyloid. It is likely that the enhanced affinity of the p5R peptide for amyloid resulted in a binding-site barrier effect which causes a non-uniform distribution of reagent within the target site and was first observed for tumor-targeting monoclonal antibodies [39]–[42]. In these cases, the antibodies, as with the p5R peptide, are delivered to the target via the vasculature, and as the reagent passes through the vascular wall, it binds with high affinity to the first target encountered and does not penetrate into the center of the lesion [41], [42]. The affinity of p5R for AA amyloid-laden tissue homogenate in vitro (Kd estimated to be 0.15 µM) is only ∼4-fold greater than that of p5 (Kd ∼ 0.5 µM); however even this modest increase can lead to differences of more than 40 µm of tumor penetration from the blood vessel when mAbs are studied [41]. This process results in a dense accumulation of peptide at the regions of amyloid that are in the immediate vicinity of the supplying vasculature with a decreasing density of peptide in the “center” of the amyloid lesion. For probes of lower affinity, some probe can bypass the primary binding site, penetrating farther into dense target before finding a partner. This is shown schematically and with 2 examples in Fig. 7. One consequence of this effect is that larger amyloid masses or those with low surface area to volume ratios will likely accumulate less p5R peptide as compared to p5. Indeed, we demonstrated a significantly lower amount of 125I-p5R in the spleens of mice with >3+ amyloid as compared to peptide 125I-p5 (p = 0.032). The binding-site barrier effect for antibodies was overcome by using F(ab) fragments which are capable of only monovalent interactions and, therefore, have a lower affinity for the target as compared to bivalent Abs [43]. To mimic this, we first synthesized a C-terminally truncated p5R variant, p5R(1-24), that lacked 2 arginine residues. This shorter electronic signature (6 charges vs 8 charges) resulted in a relatively lower affinity for heparin. The microdistribution of this peptide in the expansive splenic AA amyloid deposits was similar to the diffuse accumulation seen with 125I-p5, supporting the hypothesis that the enhanced affinity of p5R for amyloid resulted in a barrier effect. This effect hindered penetration, altered the distribution in the amyloid and significantly decreased the accumulation of peptide in large amyloid deposits relative to that of p5. Enhanced penetration of p5R into the expansive splenic amyoid was also achieved by increasing the peptide concentration to 1 mg, which resulted in a saturation of the perivascular amyloid allowing p5R to penetrate more uniformly into the amyloid mass.


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)

The binding-site barrier effect.(A) Schematic representation of the diffuse accumulation of p5 throughout the amyloid lesion. In contrast, peptide p5R accumulates as non-uniform (focal) deposits at the periphery of the amyloid mass. (B) Microautoradiographs of 125I-p5 and 125I-p5R that exemplify the consequence of a binding-site barrier on the amyloid-uptake of radiolabeled peptides.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3672142&req=5

pone-0066181-g007: The binding-site barrier effect.(A) Schematic representation of the diffuse accumulation of p5 throughout the amyloid lesion. In contrast, peptide p5R accumulates as non-uniform (focal) deposits at the periphery of the amyloid mass. (B) Microautoradiographs of 125I-p5 and 125I-p5R that exemplify the consequence of a binding-site barrier on the amyloid-uptake of radiolabeled peptides.
Mentions: Given the enhanced helicity and resulting increased affinity for amyloid, it was anticipated that the radiolabeled peptide 125I-p5R would provide enhanced amyloid imaging in vivo. Based on biodistribution studies and SPECT imaging, the p5R peptide bound AA amyloid in mice and generated images indiscernible from images that had been produced using 125I-p5 peptide [17], [18]. However, analysis of the microdistribution of 125I-p5R peptide in mice with AA amyloid revealed that, even though the “small” discrete amyloid lesions that are common e.g., in the liver, heart, pancreas and adrenal were evenly covered by radiolabeled peptide, the large expanses of avascular, dense amyloid seen in the spleens of highly diseased animals were variably stained. This phenomenon was most evident in mice with extensive (3+ – 4+; [19]) splenic amyloid but, interestingly, not in mice with (1+ – 2+) amyloid in the spleen which suggests that this effect is not due to the lack of a binding target in the splenic amyloid. It is likely that the enhanced affinity of the p5R peptide for amyloid resulted in a binding-site barrier effect which causes a non-uniform distribution of reagent within the target site and was first observed for tumor-targeting monoclonal antibodies [39]–[42]. In these cases, the antibodies, as with the p5R peptide, are delivered to the target via the vasculature, and as the reagent passes through the vascular wall, it binds with high affinity to the first target encountered and does not penetrate into the center of the lesion [41], [42]. The affinity of p5R for AA amyloid-laden tissue homogenate in vitro (Kd estimated to be 0.15 µM) is only ∼4-fold greater than that of p5 (Kd ∼ 0.5 µM); however even this modest increase can lead to differences of more than 40 µm of tumor penetration from the blood vessel when mAbs are studied [41]. This process results in a dense accumulation of peptide at the regions of amyloid that are in the immediate vicinity of the supplying vasculature with a decreasing density of peptide in the “center” of the amyloid lesion. For probes of lower affinity, some probe can bypass the primary binding site, penetrating farther into dense target before finding a partner. This is shown schematically and with 2 examples in Fig. 7. One consequence of this effect is that larger amyloid masses or those with low surface area to volume ratios will likely accumulate less p5R peptide as compared to p5. Indeed, we demonstrated a significantly lower amount of 125I-p5R in the spleens of mice with >3+ amyloid as compared to peptide 125I-p5 (p = 0.032). The binding-site barrier effect for antibodies was overcome by using F(ab) fragments which are capable of only monovalent interactions and, therefore, have a lower affinity for the target as compared to bivalent Abs [43]. To mimic this, we first synthesized a C-terminally truncated p5R variant, p5R(1-24), that lacked 2 arginine residues. This shorter electronic signature (6 charges vs 8 charges) resulted in a relatively lower affinity for heparin. The microdistribution of this peptide in the expansive splenic AA amyloid deposits was similar to the diffuse accumulation seen with 125I-p5, supporting the hypothesis that the enhanced affinity of p5R for amyloid resulted in a barrier effect. This effect hindered penetration, altered the distribution in the amyloid and significantly decreased the accumulation of peptide in large amyloid deposits relative to that of p5. Enhanced penetration of p5R into the expansive splenic amyoid was also achieved by increasing the peptide concentration to 1 mg, which resulted in a saturation of the perivascular amyloid allowing p5R to penetrate more uniformly into the amyloid mass.

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