Design and characterization of a fluorescent ghrelin analog for imaging the growth hormone secretagogue receptor 1a

McGirr, Rebecca; McFarland, Mark S.; McTavish, Jillian; Luyt, Leonard G.; Dhanvantari, Savita

doi:10.1016/j.regpep.2011.08.011

Cited by 50 publications

(64 citation statements)

References 35 publications

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“…1) with an in vitro binding assay using HEK 293 cells over-expressing GHSR1a that we generated. Table 1 shows that the binding affinity of Cy5-ghrelin to GHS-R1a was within an order of magnitude of that of wild-type ghrelin, and comparable to our previously reported value for fluorescein-ghrelin (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19) [20]. In contrast, Cy5-des-acyl ghrelin did not show any appreciable binding to GHS-R1a.…”

Section: Receptor Binding Assays In Hek-ghs-r Cellssupporting

confidence: 87%

“…We now report the synthesis of a 19-mer ghrelin analog containing the first 19 N-terminus residues with substitutions at Ser 3 (octanoyl) to contain Dpr 3 (octanoyl), to replace the ester linkage, and Lys 19 to integrate the far-red dye, Cy5. The binding of Cy5-ghrelin shows strong affinity for GHS-R1a and the IC50 value, while not as favorable as what we reported for fluorescein ghrelin [20], is still in the nanomolar range. Therefore, it appears that any organic fluorescent dye can be incorporated at the C-terminus of truncated ghrelin analogs while preserving high-affinity binding to GHS-R1a.…”

Section: Discussioncontrasting

confidence: 70%

“…It has been shown that this modification results in improved stability while having no effect on GHS-R1a binding affinity [13]. We have previously synthesized two analogs of ghrelin, 14-mer and 19-mer, to incorporate these important structural features as well as further modifications to bear non-radioactive isotopes, 19 F and Re [25] and an optical dye [20]. Our results demonstrate the flexibility permitted by the structure of ghrelin in designing molecules for imaging GHS-R1a.…”

Section: Discussionmentioning

confidence: 71%

“…Cells were pelleted, rinsed with ice-cold 50 mM Tris-HCl (pH 7.4) to remove unbound activity and radioactivity was determined in a gamma counter. Binding curves and IC 50 values determined using GraphPad Prism 5 software (San Diego, CA) as previously described [20]. Each sample was analyzed in triplicate and assays were repeated twice for each Cy5-ghrelin analog.…”

Section: Receptor Binding Assaysmentioning

confidence: 99%

“…These results indicate that GHS-R signaling is altered in cardiovascular disease, and point toward the use of ghrelin in pharmacologic therapy of heart disease. Therefore, given the importance of GHS-R signaling and expression in the heart, we are developing ghrelin analogs for the purpose of generating agents for in situ and in vivo imaging of cardiac GHS-R. We have previously designed a fluorescent ghrelin analog consisting of a fluorescein moiety attached through a lysine to the N-terminal 18 amino acids of ghrelin (fluorescein-ghrelin (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)) [20] and have shown that it specifically binds to GHS-R1a in mouse heart tissue.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2014

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a b s t r a c tGhrelin and its receptor, the growth hormone secretagogue receptor (GHS-R), are expressed in the heart, and may function to promote cardiomyocyte survival, differentiation and contractility. Previously, we had generated a truncated analog of ghrelin conjugated to fluorescein isothiocyanate for the purposes of determining GHS-R expression in situ. We now report the generation and characterization of a far-red ghrelin analog, [Dpr 3 (octanoyl), Lys 19 (Cy5)]ghrelin (1-19), and show that it can be used to image changes in GHS-R in developing cardiomyocytes. We also generated the des-acyl analog, des-acyl [Lys 19 (Cy5)]ghrelin (1-19) and characterized its binding to mouse heart sections. Receptor binding affinity of Cy5-ghrelin as measured in HEK293 cells overexpressing GHS-R1a was within an order of magnitude of that of fluorescein-ghrelin and native human ghrelin, while the des-acyl Cy5-ghrelin did not bind GHS-R1a. Live cell imaging in HEK293/GHS-R1a cells showed cell surface labeling that was displaced by excess ghrelin. Interestingly, Cy5-ghrelin, but not the des-acyl analog, showed concentration-dependent binding in mouse heart tissue sections. We then used Cy5-ghrelin to track GHS-R expression in P19-derived cardiomyocytes. Live cell imaging at different time points after DMSO-induced differentiation showed that GHS-R expression preceded that of the differentiation marker aMHC and tracked with the contractility marker SERCA 2a. Our far-red analog of ghrelin adds to the tools we are developing to map GHS-R in developing and diseased cardiac tissues.

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Section: Receptor Binding Assays In Hek-ghs-r Cellssupporting

confidence: 87%

Section: Discussioncontrasting

confidence: 70%

Section: Discussionmentioning

confidence: 71%

Section: Receptor Binding Assaysmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

et al. 2014

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A Compact and Synthetically Accessible Fluorine‐18 Labelled Cyclooctyne Prosthetic Group for Labelling of Biomolecules by Copper‐Free Click Chemistry

2018

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A new fluorine-containing azadibenzocyclooctyne (ADIBO-F) was designed using a synthetically accessible pathway. The fluorine-18 prosthetic group was prepared from its toluenesulfonate precursor and isolated in 21-35 % radiochemical yield in 30 minutes of synthetic time. ADIBO-F has been incorporated into azide-functionalized, cancer-targeting peptides through a strain-promoted alkyne-azide cycloaddition with high radiochemical yields and purities. The final products are novel peptide-based positron emission tomography (PET) imaging agents that possess high affinities for their targets, growth hormone secretagogue receptor 1a (GHSR-1a) and gastrin-releasing peptide receptor (GRPR), with IC values of 9.7 and 0.50 nm, respectively. This is a new and rapid labelling option for the incorporation of fluorine-18 into biomolecules for PET imaging.

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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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Design and characterization of a fluorescent ghrelin analog for imaging the growth hormone secretagogue receptor 1a

Cited by 50 publications

References 35 publications

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

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

A Compact and Synthetically Accessible Fluorine‐18 Labelled Cyclooctyne Prosthetic Group for Labelling of Biomolecules by Copper‐Free Click Chemistry

Stapled ghrelin peptides as fluorescent imaging probes

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