Can machine learning 'transform' peptides/peptidomimetics into small molecules? A case study with ghrelin receptor ligands.

There has been considerable interest in transforming peptides into small molecules as peptide-based molecules often present poorer bioavailability and lower metabolic stability. Our studies looked into building machine learning (ML) models to investigate if ML is able to identify the 'bioactive' features of peptides and use the features to accurately discriminate between binding and non-binding small molecules. The ghrelin receptor (GR), a receptor that is implicated in various diseases, was used as an example to demonstrate whether ML models derived from a peptide library can be used to predict small molecule binders. ML models based on three different algorithms, namely random forest, support vector machine, and extreme gradient boosting, were built based on a carefully curated dataset of peptide/peptidomimetic and small molecule GR ligands. The results indicated that ML models trained with a dataset exclusively composed of peptides/peptidomimetics provide limited predictive power for small molecules, but that ML models trained with a diverse dataset composed of an array of both peptides/peptidomimetics and small molecules displayed exceptional results in terms of accuracy and false rates. The diversified models can accurately differentiate the binding small molecules from non-binding small molecules using an external validation set with new small molecules that we synthesized previously. Structural features that are the most critical contributors to binding activity were extracted and are remarkably consistent with the crystallography and mutagenesis studies.

Molecular basis for activation and biased signaling at the thrombin-activated GPCR proteinase activated receptor-4 (PAR4).

Proteinase-activated receptor (PAR)-4 is a member of the proteolytically-activated PAR family of G-protein-coupled receptors (GPCR) that represents an important target in the development of anti-platelet therapeutics. PARs are activated by proteolytic cleavage of their receptor N terminus by enzymes such as thrombin, trypsin, and cathepsin-G. This reveals the receptor-activating motif, termed the tethered ligand that binds intramolecularly to the receptor and triggers signaling. However, PARs are also activated by exogenous application of synthetic peptides derived from the tethered-ligand sequence. To better understand the molecular basis for PAR4-dependent signaling, we examined PAR4-signaling responses to a peptide library derived from the canonical PAR4-agonist peptide, AYPGKF-NH2, and we monitored activation of the Gαq/11-coupled calcium-signaling pathway, ß-arrestin recruitment, and mitogen-activated protein kinase (MAPK) pathway activation. We identified peptides that are poor activators of PAR4-dependent calcium signaling but were fully competent in recruiting ß-arrestin-1 and -2. Peptides that were unable to stimulate PAR4-dependent calcium signaling could not trigger MAPK activation. Using in silico docking and site-directed mutagenesis, we identified Asp230 in the extracellular loop-2 as being critical for PAR4 activation by both agonist peptide and the tethered ligand. Probing the consequence of biased signaling on platelet activation, we found that a peptide that cannot activate calcium signaling fails to cause platelet aggregation, whereas a peptide that is able to stimulate calcium signaling and is more potent for ß-arrestin recruitment triggered greater levels of platelet aggregation compared with the canonical PAR4 agonist peptide. These findings uncover molecular determinants critical for agonist binding and biased signaling through PAR4.

Radiofluorination of non-activated aromatic prosthetic groups for synthesis and evaluation of fluorine-18 labelled ghrelin(1-8) analogues.

The growth hormone secretagogue receptor 1a (GHSR) is differentially expressed in various disease states compared to healthy tissues and thus is a target for molecular imaging. The endogenous ligand for the GHSR is ghrelin, a 28 amino acid peptide with a unique octanoyl group on the serine-3 residue. A recently reported ghrelin analogue revealed the successful use of fluorine-containing, polycyclic aromatic groups in place of the octanoyl side chain, thereby providing potential access to new 18F-PET imaging probes. The peptide [Inp1,Dpr3(6-FN),1Nal4,Thr8]ghrelin(1-8) amide (1) showed sub-nanomolar receptor affinity (IC50 = 0.11 nM) toward the GHSR making it the strongest affinity ghrelin analogue reported to date. However, attempts to label such non-activated aromatic groups with fluoride-18 through conventional substitution methods resulted in low radiochemical yields, impractical for use in vivo. Since larger, non-activated aromatic groups appear to be of value for incorporating fluorine into ghrelin(1-8) analogues, an additional peptide bearing a 4'-fluorobiphenyl-4-carboxyl (4'-FBC) group in place of the octanoyl side chain was also of interest. Herein, we describe the radiosynthesis of [Inp1,Dpr3([18F]6-FN),1Nal4,Thr8]ghrelin(1-8) amide ([18F]1) and [Inp1,Dpr3([18F]4'-FBC),1Nal4,Thr8]ghrelin(1-8) amide ([18F]2) using a prosthetic group approach from iodonium ylide precursors as well as initial in vitro and in vivo evaluation of [18F]1 as a potential PET tracer for targeted imaging of the GHSR.

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

The growth hormone secretagogue receptor 1a (GHSR), also called the ghrelin receptor, is a G protein-coupled receptor known to play an important metabolic role in the regulation of various physiological processes, including energy expenditure, growth hormone secretion, and cell proliferation. This receptor has been implicated in numerous health issues including obesity, gastrointestinal disorders, type II diabetes, and regulation of body weight in patients with Prader-Willi syndrome, and there has been growing interest in studying its mechanism of behavior to unlock further applications of GHSR-targeted therapeutics. In addition, the GHSR is expressed in various types of cancer including prostate, breast, and testicular cancers, while aberrant expression has been reported in cardiac disease. Targeted molecular imaging of the GHSR could provide insights into its role in biological processes related to these disease states. Over the past decade, imaging probes targeting this receptor have been discovered for the imaging modalities PET, SPECT, and optical imaging. High-affinity analogues of ghrelin, the endogenous ligand for the GHSR, as well as small molecule inhibitors have been developed and evaluated both in vitro and in pre-clinical models. This review provides a comprehensive overview of the molecular imaging agents targeting the GHSR reported to the end of 2019.

A dual modality 99mTc/Re(i)-labelled T140 analogue for imaging of CXCR4 expression.

The C-X-C chemokine receptor 4 (CXCR4) has been shown to be overexpressed in at least 23 types of cancer, including prostate cancer which has been shown to have a significant distinction of expression rates between cancerous compared to healthy or benign tissue. In an attempt to exploit the difference in expression, we have synthesized a derivative of T140, a peptide antagonist for CXCR4, containing a fluorescent 4-amino-1,8-naphthalimide appended with a di-(2-picolyl)amine binding unit to chelate rhenium or technetium-99m for fluorescence or SPECT imaging. The rhenium-coordinated variant was shown to have similar binding affinity for the receptor as T140 and showed specific uptake by fluorescence microscopy in CXCR4 expressing cells. The peptide was radiolabelled with technetium-99m in decay corrected radiochemical yields ranging from 60-85%, radiochemical purities >95%, and molar activities of 36-44 GBq µmol-1. The technetium-99m labelled peptide showed two-fold higher uptake in U87 cells expressing CXCR4 compared to non-transfected cells. Ex vivo biodistribution studies were performed using the technetium-99m labelled peptide in NOD/SCID mice bearing tumors derived from U87 cells with CXCR4. Tumor uptake of 0.51 ± 0.09% ID g-1 was observed two-hours post-injection. Our novel T140 derivative is suitable for imaging of CXCR4 expression by confocal microscopy. Further structural modifications to the peptide or metal complex may result in improved biodistribution for use in SPECT imaging of CXCR4 expressing tumors.

Development and Characterization of an 18F-labeled Ghrelin Peptidomimetic for Imaging the Cardiac Growth Hormone Secretagogue Receptor.

One-third of patients with heart disease develop heart failure, which is diagnosed through imaging and detection of circulating biomarkers. Imaging strategies reveal morphologic and functional changes but fall short of detecting molecular abnormalities that can lead to heart failure, and circulating biomarkers are not cardiac specific. Thus, there is critical need for biomarkers that are endogenous to myocardial tissues. The cardiac growth hormone secretagogue receptor 1a (GHSR1a), which binds the hormone ghrelin, is a potential biomarker for heart failure. We have synthesized and characterized a novel ghrelin peptidomimetic tracer, an 18F-labeled analogue of G-7039, for positron emission tomography (PET) imaging of cardiac GHSR1a. In vitro analysis showed enhanced serum stability compared to natural ghrelin and significantly increased cellular uptake in GHSR1a-expressing OVCAR cells. Biodistribution studies in mice showed that tissue uptake of the tracer was independent of circulating ghrelin levels, and there was negligible cardiac uptake and high uptake in the liver, intestines, and kidneys. Specificity of tracer uptake was assessed using ghsr -/- mice; both static and dynamic PET imaging revealed no difference in cardiac uptake, and there was no significant correlation between cardiac standardized uptake values and GHSR1a expression. Our study lays the groundwork for further refinement of peptidomimetic PET tracers targeting cardiac GHSR1a.

Amino-Substituted 2,2'-Bipyridine Ligands as Fluorescent Indicators for ZnII and Applications for Fluorescence Imaging of Prostate Cells.

ZnII concentrations in malignant prostate tissues are much lower than in benign or healthy, suggesting that ZnII levels are a potential biomarker for prostate cancer (PCa). Five 2,2'-bipyridine ligands were synthesized containing amino substituents with varying electron-donating ability for investigation as fluorescent ZnII indicators. The excited state characteristics of the ligands were explored by UV/Vis and fluorescence spectroscopy. 3,3'-Diamino-2,2'-bipyridine (1) was previously shown to be weakly fluorescent as a result of π→π* transitions. The other four ligands have properties consistent with an n→π* intraligand charge transfer excited state. Strongly donating amino and aminophenyl (2 and 4) substituents gave low quantum yields, while weaker donating benzimidazole substituents (6 and 7) gave high quantum yields. Absorption and fluorescence wavelengths underwent bathochromic shifts upon ZnII binding in a majority of cases. Quantum yields drastically increased upon ZnII binding for 1 and 2, but decreased for 4, 6, and 7. Compounds 6 and 7 were incubated with PC-3, DU 145 and BPH-1 cells to determine their ZnII sensing abilities in a biological system. Weak fluorescence was observed in BPH-1 cells and subsequent incubation with ZnII caused fluorescence intensity to increase. No fluorescence was observed in PCa cell lines. Further investigation of these ligands may allow for quantitative determination of ZnII concentrations in ex vivo tissue samples.

A truncated RHAMM protein for discovering novel therapeutic peptides.

The receptor for hyaluronan mediated motility (RHAMM, gene name HMMR) belongs to a group of proteins that bind to hyaluronan (HA), a high-molecular weight anionic polysaccharide that has pro-angiogenic and inflammatory properties when fragmented. We propose to use a chemically synthesized, truncated version of the protein (706-767), 7 kDa RHAMM, as a target receptor in the screening of novel peptide-based therapeutic agents. Chemical synthesis by Fmoc-based solid-phase peptide synthesis, and optimization using pseudoprolines, results in RHAMM protein of higher purity and yield than synthesis by recombinant protein production. 7 kDa RHAMM was evaluated for its secondary structure, ability to bind the native ligand, HA, and its bioactivity. This 62-amino acid polypeptide replicates the HA binding properties of both native and recombinant RHAMM protein. Furthermore, tubulin-derived HA peptide analogues that bind to recombinant RHAMM and were previously reported to compete with HA for interactions with RHAMM, bind with a similar affinity and specificity to the 7 kDa RHAMM. Therefore, in terms of its key binding properties, the 7 kDa RHAMM mini-protein is a suitable replacement for the full-length recombinant protein.

Synthesis and evaluation of optical and PET GLP-1 peptide analogues for GLP-1R imaging.

A fluorescein-GLP-1 (7-37) analog was generated to determine GLP-1R distribution in various cell types of the pancreas in both strains of mice and receptor-specific uptake was confirmed by blocking with exendin-4. Biodistribution studies were carried out using 68Ga-labeled GLP-1(7-37) peptides in CD1 and C57BL/6 mice. In addition, immunocompromised mice bearing GLP-1R-expressing insulinomas were evaluated by positron emission tomography (PET) imaging and ex vivo biodistribution studies. The optical GLP-1 probe strongly colocalized with immunofluorescence for insulin and glucagon, and more weakly with amylase (exocrine pancreas) and cytokeratin 19 (ductal cells), confirming its application for in situ GLP-1R imaging in various pancreatic cell types. Insulinomas were clearly visualized by in vivo PET. Reducing the peptide positive charge decreased renal retention as well as tumor uptake. Results demonstrate the application of the developed GLP-1 peptide analogues for in situ (optical) and in vivo (PET) imaging of GLP-1R expression.

An integrated imaging probe design: the synthesis of (99m)Tc/Re-containing macrocyclic peptide scaffolds.

ß-Sheets account for over 30 % of all secondary structural conformations found in proteins. The intramolecular hydrogen bonding that exists between the two peptide strands is imperative in maintaining this secondary structure. With the proper design, cyclic peptides may act as scaffolds emulating active ß-sheet regions, enabling investigation of their importance in molecular recognition and protein aggregation. Starting from Fmoc-Lys(Fmoc)-OH, macrocyclic peptides were synthesized on a solid support, with peptide-chain elongation extending from both the alpha and epsilon amines of the lysine. The branching peptides were cyclized with a pyridyl tridentate chelation core followed by coordination using [(99m) Tc/Re(CO)3 (H2 O)3 ](+) . Variable temperature (1) H NMR spectroscopy studies were performed, demonstrating that intramolecular hydrogen bonding exists between the two sides of the uncoordinated macrocyclic peptide scaffolds. Additionally, computational modelling and circular dichroism spectroscopic analysis revealed that the peptide backbone exists in a similar conformation both before and after metal coordination. The ability to seamlessly incorporate a tridentate chelation core into the backbone of a macrocyclic peptide, without disrupting the secondary structure, can greatly assist in the design of metal-centric peptidomimetic imaging agents. This novel integrated imaging probe approach may facilitate the investigation into protein-protein interactions using macrocyclic ß-sheet scaffolds.

Evaluation of Anisole-Substituted Boron Difluoride Formazanate Complexes for Fluorescence Cell Imaging.

Evaluation of three subclasses of boron difluoride formazanate complexes bearing o-, m-, and p-anisole N-aryl substituents (Ar) as readily accessible alternatives to boron dipyrromethene (BODIPY) dyes for cell imaging applications is described. While the wavelengths of maximum absorption (λmax ) and emission (λem ) observed for each subclass of complexes, which differed by their carbon-bound substituents (R), were similar, the emission quantum yields for 7 a-c (R=cyano) were enhanced relative to 8 a-c (R=nitro) and 9 a-c (R=phenyl). Complexes 7 a-c and 8 a-c were also significantly easier to reduce electrochemically to their radical anion and dianion forms compared to 9 a-c. Within each subclass, the o-substituted derivatives were more difficult to reduce, had shorter λmax and λem , and lower emission quantum yields than the p-substituted analogues as a result of sterically driven twisting of the N-aryl substituents and a decrease in the degree of π-conjugation. The m-substituted complexes were the least difficult to reduce and possessed intermediate λmax , λem , and quantum yields. The complexes studied also exhibited large Stokes shifts (82-152 nm, 2143-5483 cm(-1) ). Finally, the utility of complex 7 c (Ar=p-anisole, R=cyano), which can be prepared for just a few dollars per gram, for fluorescence cell imaging was demonstrated. The use of 7 c and 4',6-diamino-2-phenylindole (DAPI) allowed for simultaneous imaging of the cytoplasm and nucleus of mouse fibroblast cells.

Dual-modal magnetic resonance and fluorescence imaging of atherosclerotic plaques in vivo using VCAM-1 targeted tobacco mosaic virus.

The underlying cause of major cardiovascular events, such as myocardial infarctions and strokes, is atherosclerosis. For accurate diagnosis of this inflammatory disease, molecular imaging is required. Toward this goal, we sought to develop a nanoparticle-based, high aspect ratio, molecularly targeted magnetic resonance (MR) imaging contrast agent. Specifically, we engineered the plant viral nanoparticle platform tobacco mosaic virus (TMV) to target vascular cell adhesion molecule (VCAM)-1, which is highly expressed on activated endothelial cells at atherosclerotic plaques. To achieve dual optical and MR imaging in an atherosclerotic ApoE(-/-) mouse model, TMV was modified to carry near-infrared dyes and chelated Gd ions. Our results indicate molecular targeting of atherosclerotic plaques. On the basis of the multivalency and multifunctionality, the targeted TMV-based MR probe increased the detection limit significantly; the injected dose of Gd ions could be further reduced 400x compared to the suggested clinical use, demonstrating the utility of targeted nanoparticle cargo delivery.

A survey of stapling methods to increase affinity, activity, and stability of ghrelin analogues.

The growth hormone secretagogue receptor (GHSR) is a G protein-coupled receptor which regulates various important physiological and pathophysiological processes in the body such as energy homeostasis, growth hormone secretion and regulation of appetite. As a result, it has been postulated as a potential therapeutic target for the treatment of cancer cachexia and other metabolic disorders, as well as a potential imaging agent target for cancers and cardiovascular diseases. Ghrelin is the primary high affinity endogenous ligand for GHSR and has limited secondary structure in solution, which makes it proteolytically unstable. This inherent instability in ghrelin can be overcome by incorporating helix-inducing staples that stabilize its structure and improve affinity and activity. We present an analysis of different stapling methods at positions 12 and 16 of ghrelin(1-20) analogues with the goal of increasing proteolytic stability and to retain or improve affinity and activity towards the GHSR. Ghrelin(1-20) analogues were modified with a wide range of chemical staples, including a lactam staple, triazole staple, hydrocarbon staple, Glaser staple, and xylene-thioether staple. Once synthesized, the receptor affinity and α-helicity were measured using competitive binding assays and circular dichroism spectroscopy, respectively. Generally, an increase in alpha-helicity using a flexible staple linker led to improved affinity towards GHSR. Ghrelin(1-20) analogues with a lactam, triazole, and hydrocarbon staple resulted in helical analogues with stronger affinity towards GHSR than unstapled ghrelin(1-20), a compound that lacks helical character. Compounds were also investigated for their agonist activity through ß-arrestin 1 & 2 recruitment BRET assays and for their metabolic stability through serum stability analysis.

Discovery of novel integrin ligands from combinatorial libraries using a multiplex "beads on a bead" approach.

The development of screening approaches to identify novel affinity ligands has paved the way for a new generation of molecular targeted nanomedicines. Conventional methods typically bias the display of the target protein to ligands during the screening process. We have developed an unbiased multiplex "beads on a bead" strategy to isolate, characterize, and validate high affinity ligands from OBOC libraries. Novel non-RGD peptides that target α(v)ß(3) integrin were discovered that do not affect cancer or endothelial cell biology. The peptides identified here represent novel integrin-targeted agents that can be used to develop targeted nanomedicines without the risk of increased tumor invasion and metastasis.

Discovery of Ghrelin(1-8) Analogues with Improved Stability and Functional Activity for PET Imaging.

The highest affinity ghrelin-based analogue for fluorine-18 positron emission tomography, [Inp1,Dpr3(6-FN),1Nal4,Thr8]ghrelin(1-8) amide (1), has remarkable subnanomolar receptor affinity (IC50 = 0.11 nM) toward the growth hormone secretagogue receptor 1a (GHSR). However, initial in vivo PET imaging and biodistribution of [18F]1 in mice demonstrated an unfavorable pharmacokinetic profile with rapid clearance and accumulation in liver and intestinal tissue, prompting concerns about the metabolic stability of this probe. The aims of the present study were to examine the proteolytic stability of ghrelin analogue 1 in the presence of blood and liver enzymes, structurally modify the peptide to improve stability without impeding the strong binding affinity, and measure the presently unknown functional activity of ghrelin(1-8) analogues. The in vitro stability and metabolite formation of 1 in human serum and liver S9 fraction revealed a metabolic soft spot between amino acids Leu5 and Ser6 in the peptide sequence. A focused library of ghrelin(1-8) analogues was synthesized and evaluated in a structure-activity-stability relationship study to further understand the structural importance of the residues at these positions in the context of stability and receptor affinity. The critical nature of l-stereochemistry at position 5 was identified and substitution of Ser6 with l-2,3-diaminopropionic acid led to a novel ligand with substantially improved in vitro stability while maintaining subnanomolar GHSR affinity. Despite the highly modified nature of these analogues compared to human ghrelin, ghrelin(1-8) analogues were found to recruit all G protein subtypes (Gαq/11/13/i1/oB) known to associate with GHSR as well as ß-arrestins with low micromolar to nanomolar potencies. The study of these analogues demonstrates the ability to balance desirable ligand properties, including affinity, stability, and potency to produce well-rounded candidate molecules for further in vivo evaluation.

Ghrelin proteolysis increases in plasma of men, but not women, with obesity.

AIMS: Since plasma ghrelin can undergo des-acylation and proteolysis, the aim of this study was to investigate the extent to which an enhancement of these reactions is associated to the decrease of ghrelin in plasma after food intake or in individuals with obesity. MAIN METHODS: we performed an intervention cross-sectional study, in which levels of ghrelin, desacyl-ghrelin (DAG), glucose, insulin, ghrelin des-acylation and ghrelin proteolysis were assessed in plasma before and after a test meal in 40 people (n = 21 males) with normal weight (NW, n = 20) or overweight/obesity (OW/OB, n = 20). KEY FINDINGS: Preprandial ghrelin and DAG levels were lower, whereas preprandial ghrelin proteolysis was ∼4.6-fold higher in plasma of males with OW/OB. In males, ghrelin proteolysis positively correlated with glycemia. Ghrelin and DAG levels were also lower in females with OW/OB, but preprandial ghrelin proteolysis was not different between females with NW or OW/OB. Ghrelin and DAG levels decreased postprandially in males and females, independently of BMI, and ghrelin proteolysis increased postprandially ∼2 folds only in individuals with NW. Ghrelin des-acylation remained unaffected by BMI or feeding status in both sexes. SIGNIFICANCE: Current study shows that ghrelin proteolysis increases in males with obesity as well as after meal in lean individuals. Therefore, ghrelin proteolysis may be an important checkpoint and, consequently, a putative pharmacological target to control circulating ghrelin levels in humans.

Protease-activated receptor 2 (PAR2)-targeting peptide derivatives for positron emission tomography (PET) imaging.

The proteolytically-activated G protein-coupled receptor (GPCR) protease-activated receptor 2 (PAR2), is implicated in various cancers and inflammatory diseases. Synthetic ligands and in vitro imaging probes targeting this receptor have been developed with low nanomolar affinity, however, no in vivo imaging probes exist for PAR2. Here, we report the strategic design, synthesis, and biological evaluation of a series of novel 4-fluorobenzoylated PAR2-targeting peptides derived from 2f-LIGRLO-NH2 (2f-LI-) and Isox-Cha-Chg-Xaa-NH2 (Isox-) peptide families, where the 4-fluorobenzoyl moiety acts as the 19F-standard of an 18F-labeled probe for potential use in in vivo imaging. We found that several of the 4-fluorobenzoylated peptides from the 2f-LI-family exhibited PAR2 selectivity with moderate potency (EC50 = 151-252 nM), whereas several from the Isox-family exhibited PAR2 selectivity with high potency (EC50 = 13-42 nM). Our lead candidate, Isox-Cha-Chg-Ala-Arg-Dpr(4FB)-NH2 (EC50 = 13 nM), was successfully synthesized with fluorine-18 with a radiochemical yield of 37%, radiochemical purity of >98%, molar activity of 20 GBq/µmol, and an end of synthesis time of 125 min. Biodistribution studies and preliminary PET imaging of the tracer in mice showed predominantly renal clearance. This 18F-labeled tracer is the first reported PAR2 imaging agent with potential for use in vivo. Future work will explore the use of this tracer in cancer xenografts and inflammation models involving upregulation of PAR2 expression.

Cellular Uptake of a Fluorescent Ligand Reveals Ghrelin O-Acyltransferase Interacts with Extracellular Peptides and Exhibits Unexpected Localization for a Secretory Pathway Enzyme.

Ghrelin O-acyltransferase (GOAT) plays a central role in the maturation and activation of the peptide hormone ghrelin, which performs a wide range of endocrinological signaling roles. Using a tight-binding fluorescent ghrelin-derived peptide designed for high selectivity for GOAT over the ghrelin receptor GHSR, we demonstrate that GOAT interacts with extracellular ghrelin and facilitates ligand cell internalization in both transfected cells and prostate cancer cells endogenously expressing GOAT. Coupled with enzyme mutagenesis, ligand uptake studies support the interaction of the putative histidine general base within GOAT with the ghrelin peptide acylation site. Our work provides a new understanding of GOAT's catalytic mechanism, establishes that GOAT can interact with ghrelin and other peptides located outside the cell, and raises the possibility that other peptide hormones may exhibit similar complexity in their intercellular and organismal-level signaling pathways.

Ghrelin receptor as a novel imaging target for prostatic neoplasms.

BACKGROUND: Ghrelin is a natural growth hormone secretagogue (GHS) that is co-expressed with its receptor GHSR in human prostate cancer (PCa) cells. Imaging probes that target receptors for ghrelin may delineate PCas from benign disease. The specificity of a novel ghrelin-imaging probe for PCa over normal tissue or benign disease was assessed. METHODS: A fluorescein-bearing ghrelin analogue was synthesized (fluorescein-ghrelin(1-18)), and its application for imaging was evaluated in a panel of PCa cell lines and human prostate tissue. Prostate core biopsy samples were collected from fresh surgery specimens of 13 patients undergoing radical prostatectomy. Ghrelin probe signal was detected and quantified in each sample using a hapten amplification technique and associated with pathological features. RESULTS: The ghrelin probe was taken up by GHSR-expressing LNCaP and PC-3 cells, and not in BPH cells that express low levels of GHSR. Binding was blocked by competition with excess unlabeled probe. The ghrelin probe signal was 4.7 times higher in PCa compared to benign hyperplasia tissue (P = 0.0027) and normal tissue (P = 0.0093). Furthermore, while the ghrelin probe signal was 1.9-fold higher in PIN compared to benign hyperplasia (P = 0.0022) and normal tissue (P = 0.0047), there was no significant difference in the signal of benign hyperplasia compared to normal tissue. CONCLUSION: The imaging probe fluorescein-ghrelin(1-18) is specific for PCa, and did not associate significantly with benign hyperplasia or normal prostate tissue. This data suggests that ghrelin analogues may be useful as molecular imaging probes for prostatic neoplasms in both localized and metastatic disease.