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

The 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 (lmax) and emission (lem) observed for each subclass of complexes, which differed by their carbon-bound substituents (R), were similar, the emission quantum yields for 7a-c (R = cyano) were enhanced relative to 8a-c (R = nitro) and 9a-c (R = phenyl). Complexes 7a-c and 8a-c were also significantly easier to reduce electrochemically to their radical anion and dianion forms compared to 9a-c. Within each subclass, the o-substituted derivatives were more difficult to reduce, had shorter lmax and lem, and lower emission quantum yields than the p-substituted analogs as a result of sterically-driven twisting of the N-aryl substituents and a decrease in the degree of p conjugation. The m-substituted complexes were the least difficult to reduce and possessed intermediate lmax,lem,and quantum yields. The complexes studied also exhibited large Stokes shifts (82-152 nm, 2143-5483 cm-1). Finally, the utility of complex 7c (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 7c and 4\u27,6-diamino-2-phenylindole (DAPI) allowed for simultaneous imaging of the cytoplasm and nucleus of mouse fibroblast cells

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

Ghrelin 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

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

18F-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. Among the candidate compounds

Development of Candidates for Positron Emission Tomography (PET) Imaging of Ghrelin Receptor in Disease: Design, Synthesis, and Evaluation of Fluorine-Bearing Quinazolinone Derivatives

Molecular imaging with positron emission tomography (PET) is an attractive platform for noninvasive detection and assessment of disease. The development of a PET imaging agent targeting the ghrelin receptor (growth hormone secretagogue receptor type 1a or GHS-R1a) has the potential to lead to the detection and assessment of the higher than normal expression of GHS-R1a in diseases such as prostate, breast, and ovarian cancer. To enable the development of ¹⁸F radiopharmaceuticals, we have designed and synthesized three series of quinazolinone derivatives, resulting in the identification of two compound (<b>5i</b>, <b>17</b>) with subnanomolar binding affinity and one fluorine-bearing compound (<b>10b</b>) with picomolar binding affinity (20 pM), representing the highest binding affinity for GHS-R1a reported to date. Two lead compounds (<b>5b</b>, IC₅₀ = 20.6 nM; <b>5e</b>, IC₅₀ = 9.3 nM) were successfully ¹⁸F-radiolabeled with radiochemical purity of greater than 99%. Molecular modeling studies were performed to shed light on ligand–receptor interactions

Nitrone Modified Gold Nanoparticles: Synthesis, Characterization and Their Potential as 18F-Labeled PET Probes via I-SPANC

A bioorthogonal gold nanoparticle template displaying interfacial nitrone functional groups for bioorthogonal interfacial strain-promoted alkyne-nitrone cycloaddition (I-SPANC) reactions has been synthesized. The Nitrone-AuNPs were characterized in detail using 1H NMR spectroscopy, TEM, TGA, and XPS and a nanoparticle raw formula was calculated. The ability to control the conjugation of molecules of interest at the molecular level onto the Nitrone-AuNPs template allowed us to create a methodology for the synthesis of AuNP-based radiolabeled probes with a high degree of loading using copper free, strained-promoted cycloaddition. To this end, we also describe the synthesis of a new prosthetic group containing a strained-alkyne capable of clicking hot 18F-label onto complementary azide or nitrone labelled agents

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

© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 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-Nal4,Lys5(4-fluorobenzoyl)]G-7039 was designed as a molecular imaging tool for positron emission tomography (PET). However, this candidate was a poor binder (IC50=69 nm), required a lengthy four-step radiosynthesis, and had a cLogP above 8. To address these challenges, we now report on changes targeted at the 4th position of G-7039. A 2-fluoropropionic acid (2-FPA) group was added on to Lys5 to determine the potential binding affinity of the [18F]-2-FP radiolabeled analogue, which could be prepared by simplified radiochemistry. Lead candidate [Tyr4,Lys5(2-fluoropropionyl)]G-7039 exhibited an IC50 of 0.28 nm and low picomolar activity toward GHS-R1a. Molecular docking revealed a molecular basis for this picomolar affinity