Stapled ghrelin peptides as fluorescent imaging probes

Abstract

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.

A Decade’s Progress in the Development of Molecular Imaging Agents Targeting the Growth Hormone Secretagogue Receptor

“…Recently, ghrelin (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20) was cyclized through a lactam bridge between Glu 12 and Lys 16 for improved stability of secondary structure, and labeled with the SulfoCy5 dye off the C-terminal lysine residue (Figure 5). 80 This stapled peptide, cyclo-12, 16[Dpr(octanoyl) 3 , Glu 12 , Lys(SulfoCy5) 20 ]ghrelin(1-20) (8), was reported to have an IC 50 value of 1.0 nM, which is an improved affinity compared to any previously reported fluorescent ghrelin analogue, and was found to image receptor expression in 3 ovarian cancer cell lines: OvCar8 GHSR positive cells as well as parental OvCar3 and HEYA8 cells.…”

Designing stapled peptides to inhibit protein‐protein interactions: An analysis of successes in a rapidly changing field

“…scanned all possible lactam staple positions ( i , i + 4 and i , i + 7) on a series of ghrelin peptides; testing their affinity for the growth hormone secretagogue receptor type 1a (GHS‐R1a) to determine their potential use as imaging probes. [ 141 ] Examination of deposited stapled peptide sequences in the PDB (Table S2) suggests there is no inherently preferred position for the staple in the overall amino acid sequence.…”

“…[140] Correspondingly, Lalonde et al scanned all possible lactam staple positions (i,i + 4 and i,i + 7) on a series of ghrelin peptides; testing their affinity for the growth hormone secretagogue receptor type 1a (GHS-R1a) to determine their potential use as imaging probes. [141] Examination of deposited stapled peptide sequences in the PDB (Table S2) (Table 1, PDB code 5WGQ). [12] Similarly, it was shown for the nonhelical stapled bicyclic peptide Grb7-B4 binding to the Grb7-SH2 subdomain that interactions between the RCM staple and protein were required for high-affinity binding ( Table 1, PDB code 5EEQ).…”

The development of peptide–boron difluoride formazanate conjugates as fluorescence imaging agents

“…Fluorescence quantum yields were determined using the comparative method described by Fery-Forgues and coworkers 33 using the standard [Ru(bpy) 3 ][PF 6 ] 2 . 34 Cell imaging studies For 5: OVCAR-8 cells stably transfected with GHSR-1a (as previously described 36 ) were released from the tissue culture ask by trypsin free dissociation buffer (Fischer Scientic) and seeded onto coverslips in a 12-well tissue culture plate at a cell density of 50 000 cells per well. The cells were incubated overnight in Roswell Park Memorial Institute medium (RPMI) containing 10% fetal bovine serum (FBS) at 37 C with 5% CO 2 .…”