Design, Synthesis, and Preclinical Evaluation of a High-Affinity F-Labeled Radioligand for Myocardial Growth Hormone Secretagogue Receptor Before and After Myocardial Infarction.

The peptide hormone ghrelin is produced in cardiomyocytes and acts through the myocardial growth hormone secretagogue receptor (GHSR) to promote cardiomyocyte survival. Administration of ghrelin may have therapeutic effects on post-myocardial infarction (MI) outcomes. Therefore, there is a need to develop molecular imaging probes that can track the dynamics of GHSR in health and disease to better predict the effectiveness of ghrelin-based therapeutics.

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.

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

Ghrelin 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.

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, [Inp,Dpr(6-FN),1Nal,Thr]ghrelin(1-8) amide (), has remarkable subnanomolar receptor affinity (IC = 0.11 nM) toward the growth hormone secretagogue receptor 1a (GHSR). However, initial PET imaging and biodistribution of 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.

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.

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-NH (2f-LI-) and Isox-Cha-Chg-Xaa-NH (Isox-) peptide families, where the 4-fluorobenzoyl moiety acts as the F-standard of an F-labeled probe for potential use in in vivo imaging.

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.

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 F-PET imaging probes.

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.

Incorporation of Fluorine into an OBOC Peptide Library by Copper-Free Click Chemistry toward the Discovery of PET Imaging Agents.

A one-bead one-compound (OBOC) library of peptide-based imaging agents was developed where a F-containing moiety was added onto the N-terminus of octamer peptides through copper-free click chemistry prior to screening of the library. This created a library of complete imaging agents that was screened against CXCR4, a receptor of interest for cancer imaging. The screen directly resulted in the discovery of a peptide-based imaging agent with an IC of 138 μM.

A Hyaluronan-binding Peptide (P15-1) Reduces Inflammatory and Catabolic Events in IL-1β-treated Human Articular Chondrocytes.

Inflammation plays a critical role in osteoarthritis (OA). It stimulates catabolic events in articular chondrocytes and prevents chondrogenic precursor cells from repairing cartilage lesions, leading to accelerated cartilage degradation. Therefore, the identification of novel factors that reduce catabolic events in chondrocytes and enhances chondrogenic differentiation of precursor cells in an inflammatory environment may provide novel therapeutic strategies for the treatment of OA.

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.

Nitrone-Modified Gold Nanoparticles: Synthesis, Characterization, and Their Potential as F-Labeled Positron Emission Tomography Probes via I-SPANC.

A novel bioorthogonal gold nanoparticle (AuNP) template displaying interfacial nitrone functional groups for bioorthogonal interfacial strain-promoted alkyne-nitrone cycloaddition reactions has been synthesized. These nitrone-AuNPs were characterized in detail using H nuclear magnetic resonance spectroscopy, transmission electron microscopy, thermogravimetric analysis, and X-ray photoelectron spectroscopy, and a nanoparticle raw formula was calculated. The ability to control the conjugation of molecules of interest at the molecular level onto the nitrone-AuNP template allowed us to create a novel methodology for the synthesis of AuNP-based radiolabeled probes.

A study of Tc/Re-tricarbonyl complexes of 4-amino-1,8-naphthalimides.

Three 4-amino-1,8-naphthalimide analogues were synthesized, consisting of the tridentate chelators di-2-picolylamine, (pyridin-2-ylmethyl)glycinate, and iminodiacetate conjugated to the naphthalimide scaffold. Coordination with fac-Tc/Re(CO) resulted in metal complexes with overall charges of -1, 0, or +1. Upon coordination of Re(i), the initial naphthalimide-based fluorescence is largely maintained for both negative and neutral complexes compared to their free ligand forms, while an increase in fluorescence quantum yield was observed for the positively charged complex.

Single Amino Acid Replacement in G-7039 Leads to a 70-fold Increase in Binding toward GHS-R1a.

The growth hormone secretagogue receptor type 1a (GHS-R1a) is a class A rhodopsin-like G protein coupled receptor (GPCR) that is expressed in a variety of human tissues and is differentially expressed in benign and malignant prostate cancer. Previously, the peptidomimetic [1-Nal ,Lys (4-fluorobenzoyl)]G-7039 was designed as a molecular imaging tool for positron emission tomography (PET). However, this candidate was a poor binder (IC =69 nm), required a lengthy four-step radiosynthesis, and had a cLogP above 8.

High Affinity Fluorescent Probe for Proteinase-Activated Receptor 2 (PAR2).

PAR2 is a proteolytically activated G protein-coupled receptor (GPCR) that is implicated in various cancers and inflammatory diseases. Ligands with low nanomolar affinity for PAR2 have been developed, but there is a paucity of research on the development of PAR2-targeting imaging probes. Here, we report the development of seven novel PAR2-targeting compounds.

F-Labeled PET Probe Targeting Enhancer of Zeste Homologue 2 (EZH2) for Cancer Imaging.

The enzyme enhancer of zeste homologue 2 (EZH2) plays a catalytic role in histone methylation (H3K27me3), one of the epigenetic modifications that is dysregulated in cancer. The development of a positron emission tomography (PET) imaging agent targeting EZH2 has the potential to provide a method of stratifying patients for epigenetic therapies. In this study, we designed and synthesized a series of fluoroethyl analogs based upon the structure of EZH2 inhibitors UNC1999 and EPZ6438.

A dual modality Tc/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 Chimera That Curbs Appetite.

A chimeric drug design approach, merging the structure of an antagonist and an inverse agonist, results in a new molecular scaffold for targeting the ghrelin receptor (GHSR).

Development and Characterization of an F-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.

Peptidomimetic growth hormone secretagogue derivatives for positron emission tomography imaging of the ghrelin receptor.

The ghrelin receptor is a seven-transmembrane (7-TM) receptor known to have an increased level of expression in human carcinoma and heart failure. Recent work has focused on the synthesis of positron emission tomography (PET) probes designed to target and image this receptor for disease diagnosis and staging. However, these probes have been restricted to small-molecule quinalizonones and peptide derivatives of the endogenous ligand ghrelin.