Characterization of a far-red analog of ghrelin for imaging GHS-R in P19-derived cardiomyocytes

Douglas, Gregory A.F.; McGirr, Rebecca; Charlton, Carlie L.; Kagan, Dov B; Hoffman, Lisa; Luyt, Leonard G.; Dhanvantari, Savita

doi:10.1016/j.peptides.2014.01.011

Cited by 26 publications

(47 citation statements)

References 29 publications

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“…The lead peptides identified from the staple scan ( 10 and 18 ) were labelled with a sulfo‐cyanine 5 dye (Cy5) by forming an amide bond with the side chain of Lys (Table ). This allowed for the dye to be as removed as possible from the binding areas of the peptide, as well as allowing for facile attachment of the dye.…”

Section: Resultsmentioning

confidence: 99%

“…Competitive ligand binding assays were performed in triplicate using HEK293/GHS‐R1a cells and [ 125 I]Ghrelin as the competitive ligand, as previously described . Receptor binding affinities were calculated as IC 50 values as previously described …”

Section: Methodsmentioning

confidence: 99%

“…Recently, ghrelin derivatives have been developed for the purpose of molecular imaging and were determined to have similar GHS‐R1a affinity as that of the endogenous ligand, even when truncated to eight amino acids length . This makes it an ideal candidate for modification for the purpose of imaging GHSR using fluorescence microscopy . However, this shortened sequence (of only eight amino acids) is unlikely to be effective as a fluorescently labelled imaging probe, since the presence of a dye moiety would potentially interfere with binding due to the proximity to the binding pocket.…”

Section: Introductionmentioning

confidence: 99%

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Stapled ghrelin peptides as fluorescent imaging probes

et al. 2018

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Fluorescently labelled ghrelin is an effective imaging probe for ex vivo biopsy analysis, in vivo distribution studies, and cell‐based analyses. The objective of our study was to improve the receptor affinity and stability of this ghrelin probe through cyclization, thereby providing a chemical probe with advantages in specificity and sensitivity as compared to immunohistochemical approaches. Truncation of ghrelin to its first 20 essential binding amino acids simplifies chemical synthesis, but reduces the α‐helical content of the peptide, which is important for receptor recognition. To overcome this limitation, we used a “staple scan” to synthesize stable α‐helical cyclic ghrelin(1‐20) analogues using a lactam bridge in either the i, i + 4 or i, i + 7 position. Stapling improved helicity in every case when compared to the linear sequence; however, the binding affinity to the receptor was dependent on the staple position. The peptide with the greatest improvement resulted in a [θ]222/[θ]208 ratio of 0.84, and an IC50 of 7.85 nM. The lead analogue was fluorescently labeled on the C‐terminal lysine of the peptide and microscopy experiments confirmed receptor binding in cells expressing GHS‐R1a. We postulate that the lead stapled peptide can be used as a cancer cell‐specific fluorescent stain with potential research and clinical applications.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Stapled ghrelin peptides as fluorescent imaging probes

et al. 2018

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“…For image analysis, one ROI was drawn within each islet, two ROIs were drawn in the non-islet, non-ductal pancreatic areas and two ROIs were drawn in background regions of each field of view using the circle tool in ImageJ with an area of approximately 15000 pixels, as we have done previously 28– 30 . Fluorescence intensities were calculated as corrected total cell fluorescence (CTCF) as follows:…”

Section: Methodsmentioning

confidence: 99%

Characterization of 5-(2-18F-fluoroethoxy)-L-tryptophan for PET imaging of the pancreas

et al. 2016

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Purpose: In diabetes, pancreatic beta cell mass declines significantly prior to onset of fasting hyperglycemia. This decline may be due to endoplasmic reticulum (ER) stress, and the system L amino acid transporter LAT1 may be a biomarker of this process. In this study, we used 5-(2- 18F-fluoroethoxy)-L-tryptophan ( 18F-L-FEHTP) to target LAT1 as a potential biomarker of beta cell function in diabetes. Procedures: Uptake of 18F-L-FEHTP was determined in wild-type C57BL/6 mice by ex vivo biodistribution. Both dynamic and static positron emission tomography (PET) images were acquired in wild-type and Akita mice, a model of ER stress-induced diabetes, as well as in mice treated with streptozotocin (STZ). LAT1 expression in both groups of mice was evaluated by immunofluorescence microscopy. Results: Uptake of 18F-L-FEHTP was highest in the pancreas, and static PET images showed highly specific pancreatic signal. Time-activity curves showed significantly reduced 18F-L-FEHTP uptake in Akita mice, and LAT1 expression was also reduced. However, mice treated with STZ, in which beta cell mass was reduced by 62%, showed no differences in 18F-L-FEHTP uptake in the pancreas, and there was no significant correlation of 18F-L-FEHTP uptake with beta cell mass. Conclusions: 18F-L-FEHTP is highly specific for the pancreas with little background uptake in kidney or liver. We were able to detect changes in LAT1 in a mouse model of diabetes, but these changes did not correlate with beta cell function or mass. Therefore, 18F-L-FEHTP PET is not a suitable method for the noninvasive imaging of changes in beta cell function during the progression of diabetes.

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“…2 Therefore, we used Ghrelin(1-18, Lys 18 (Cy5) to quantify the expression of GHSR. Following 3 incubation with secondary antibodies, this probe was added to tissue sections for 30 min and used 4 for GHSR detection. Sections were washed with PBS, incubated 8 min with DAPI nuclear stain 5 (1:1000), and mounted with ProLong Gold antifade (Life Technologies) to prevent the tissues from 6 photobleaching.…”

Section: Introductionmentioning

confidence: 99%