Design, Synthesis, and Evaluation of Novel Protease-Activated Receptor 2 (PAR2)-Targeting Imaging Agents for Cancer

Aberrant function and over-expression of protease-activated receptor 2 (PAR2), a GPCR, is associated with various cancers and inflammatory diseases. PAR2-targeting ligands have been developed with therapeutic applications but the development of imaging probes is lacking. A series of PAR2-targeted fluorescent and 18F-PET imaging agents were synthesized and assessed for PAR2-binding. A novel dye-conjugated peptide, Isox-Cha-Chg-ARK(Sulfo-Cy5)-NH2 (EC50=16nM, KD=38nM), showed \u3e10-fold increase in potency and binding affinity for PAR2 compared to the leading known fluorescent probe. A novel PET imaging 18F-labeled peptide, Isox-Cha-Chg-AR-Dpr([18F]4-FB)-NH2, is the first PAR2-targeted in vivo imaging agent. It showed significant uptake in PAR2-expressing prostate cancer cells compared to controls (P19F-standard was highly potent (EC50=13nM) and PAR2-selective. The peptide was 18F-labeled through standard prosthetic group labeling (RCY=37±3%, RCP\u3e98%, Am=20±2GBq/µmol, EOS=125±2min, n=4). These probes are useful chemical tools that could provide insight into PAR2 expression in vitro and in vivo with potential clinical applications in PAR2-related diseases

Time-Restricted Eating In Women - A Pilot Study

Background: There is a growing interest in intermittent fasting as a method of body fat loss. However, research on time-restricted eating, a type of intermittent fasting, is more limited. The purpose of this study was to assess the effects of time-restricted eating on fat mass in women. Methods: 20 healthy, young (21.3 years ± 1.2 years), body mass stable (± 2.27 kg in the past 6 months) women completed a 4-week time-restricted eating study. During the intervention, participants restricted their daily food intake to an 8-hour period, i.e., between 1200 to 2000 hours. Changes in fat mass and body mass were assessed via densitometry (Bod Pod®). Hunger, satisfaction, fullness, and adherence were assessed on a weekly basis by self-report. Results: A 0.6 kg ± 1 kg decrease in body mass occurred after the 4 weeks (p = 0.015, n = 20) but changes in fat mass were non-significant. Women who performed strength training during the study experienced a 0.7 kg ± 0.5 kg decrease in fat mass (p = 0.037, n = 5). Overall protocol adherence was high (\u3e5.5 d/wk) throughout the intervention. Hunger, satisfaction, and fullness remained constant over the study. Conclusions: Participants seemed to adapt to the time-restricted eating regime quickly and were able to sustain it over a 4-week period. Body mass and fat mass losses were small overall; however, some individuals responded substantially. Perhaps a longer intervention is needed to produce consistency. Time-restricted eating appears to have potential as a fat loss strategy; however, no definitive conclusions can be made regarding its effectiveness at present. More research is needed

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

© 2019 American Chemical Society. 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. Four of these compounds are highly potent and selective PAR2-targeting peptides (EC50 = 10 to 23 nM) that have a primary amine handle available for facile conjugation to various imaging components. We describe a peptide of the sequence Isox-Cha-Chg-ARK(Sulfo-Cy5)-NH2 as the most potent and highest affinity PAR2-selective fluorescent probe reported to date (EC50 = 16 nM, KD = 38 nM). This compound has a greater than 10-fold increase in potency and binding affinity for PAR2 compared to the leading previously reported probe and is conjugated to a red-shifted fluorophore, enabling in vitro and in vivo studies

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

© 2020 Thibeault et al. 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, β-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 β-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 β-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