Synthesis of Novel Fluorinated Xanthine Derivatives with High Adenosine A2B Receptor Binding Affinity.

The G protein-coupled adenosine A2B receptor is suggested to be involved in various pathological processes accompanied by increased levels of adenosine as found in inflammation, hypoxia, and cancer. Therefore, the adenosine A2B receptor is currently in focus as a novel target for cancer therapy as well as for noninvasive molecular imaging via positron emission tomography (PET). Aiming at the development of a radiotracer labeled with the PET radionuclide fluorine-18 for imaging the adenosine A2B receptor in brain tumors, one of the most potent and selective antagonists, the xanthine derivative PSB-603, was selected as a lead compound. As initial biodistribution studies in mice revealed a negligible brain uptake of [3H]PSB-603 (SUV3min: 0.2), structural modifications were performed to optimize the physicochemical properties regarding blood-brain barrier penetration. Two novel fluorinated derivatives bearing a 2-fluoropyridine (5) moiety and a 4-fluoro-piperidine (6) moiety were synthesized, and their affinity towards the four adenosine receptor subtypes was determined in competition binding assays. Both compounds showed high affinity towards the adenosine A2B receptor (Ki (5) = 9.97 ± 0.86 nM; Ki (6) = 12.3 ± 3.6 nM) with moderate selectivity versus the other adenosine receptor subtypes.

Development of a Radiofluorinated Adenosine A2B Receptor Antagonist as Potential Ligand for PET Imaging.

The adenosine A2B receptor has been proposed as a novel therapeutic target in cancer, as its expression is drastically elevated in several tumors and cancer cells. Noninvasive molecular imaging via positron emission tomography (PET) would allow the in vivo quantification of this receptor in pathological processes and most likely enable the identification and clinical monitoring of respective cancer therapies. On the basis of a bicyclic pyridopyrimidine-2,4-dione core structure, the new adenosine A2B receptor ligand 9 was synthesized, containing a 2-fluoropyridine moiety suitable for labeling with the short-lived PET radionuclide fluorine-18. Compound 9 showed a high binding affinity for the human A2B receptor (Ki(A2B) = 2.51 nM), along with high selectivities versus the A1, A2A, and A3 receptor subtypes. Therefore, it was radiofluorinated via nucleophilic aromatic substitution of the corresponding nitro precursor using [18F]F-/K2.2.2./K2CO3 in DMSO at 120 °C. Metabolic studies of [18F]9 in mice revealed about 60% of radiotracer intact in plasma at 30 minutes p.i. A preliminary PET study in healthy mice showed an overall biodistribution of [18F]9, corresponding to the known ubiquitous but low expression of the A2B receptor. Consequently, [18F]9 represents a novel PET radiotracer with high affinity and selectivity toward the adenosine A2B receptor and a suitable in vivo profile. Subsequent studies are envisaged to investigate the applicability of [18F]9 to detect alterations in the receptor density in certain cancer-related disease models.

Radiosynthesis and in vivo evaluation of a fluorine-18 labeled pyrazine based radioligand for PET imaging of the adenosine A2B receptor.

On the basis of a pyrazine core structure, three new adenosine A2B receptor ligands (7a-c) were synthesized containing a 2-fluoropyridine moiety suitable for 18F-labeling. Compound 7a was docked into a homology model of the A2B receptor based on X-ray structures of the related A2A receptor, and its interactions with the adenosine binding site were rationalized. Binding affinity data were determined at the four human adenosine receptor subtypes. Despite a rather low selectivity regarding the A1 receptor, 7a was radiolabeled as the most suitable candidate (Ki(A2B) = 4.24 nM) in order to perform in vivo studies in mice with the aim to estimate fundamental pharmacokinetic characteristics of the compound class. Organ distribution studies and a single PET study demonstrated brain uptake of [18F]7a with a standardized uptake value (SUV) of ≈1 at 5 min post injection followed by a fast wash out. Metabolism studies of [18F]7a in mice revealed the formation of a blood-brain barrier penetrable radiometabolite, which could be structurally identified. The results of this study provide an important basis for the design of new derivatives with improved binding properties and metabolic stability in vivo.

PSA-stratified detection rates for [68Ga]THP-PSMA, a novel probe for rapid kit-based 68Ga-labeling and PET imaging, in patients with biochemical recurrence after primary therapy for prostate cancer.

PURPOSE: [68Ga]Tris(hydroxypyridinone)(THP)-PSMA is a novel radiopharmaceutical for one-step kit-based radiolabelling, based on direct chelation of 68Ga3+ at low concentration, room temperature and over a wide pH range, using direct elution from a 68Ge/68Ga-generator. We evaluated the clinical detection rates of [68Ga]THP-PSMA PET/CT in patients with biochemically recurrent prostate cancer after prostatectomy. METHODS: Consecutive patients (n=99) referred for evaluation of biochemical relapse of prostate cancer by [68Ga]THP-PSMA PET/CT were analyzed retrospectively. Patients underwent a standard whole-body PET/CT (1 h p.i.), followed by delayed (3 h p.i.) imaging of the abdomen. PSA-stratified cohorts of positive PET/CT results, standardized uptake values (SUVs) and target-to-background ratios (TBRs) were analyzed, and compared between standard and delayed imaging. RESULTS: At least one lesion suggestive of recurrent or metastatic prostate cancer was identified on PET images in 52 patients (52.5%). Detection rates of [68Ga]THP-PSMA PET/CT increased with increasing PSA level: 94.1% for a PSA value of ≥10 ng/mL, 77.3% for a PSA value of 2 to <10 ng/mL, 54.5% for a PSA value of 1 to <2 ng/mL, 14.3% for a PSA value of 0.5 to <1 ng/mL, 20.0% for a PSA value of >0.2 to <0.5, and 22.2% for a PSA value of 0.01 to 0.2 ng/mL. [68Ga]THP-PSMA uptake (SUVs) in metastases decreased over time, whereas TBRs improved. Delayed imaging at 3 h p.i. exclusively identified pathologic findings in 2% of [68Ga]THP-PSMA PET/CT scans. Detection rate was higher in patients with a Gleason score ≥8 (P=0.02) and in patients receiving androgen deprivation therapy (P=0.003). CONCLUSIONS: In this study, [68Ga]THP-PSMA PET/CT showed suitable detection rates in patients with biochemical recurrence of prostate cancer and PSA levels ≥ 2 ng /mL. Detections rates were lower than in previous studies evaluating other PSMA ligands, though prospective direct radiotracer comparison studies are mandatory particularly in patients with low PSA levels to evaluate the relative performance of different PSMA ligands.

Imaging Characteristics and First Experience of [68Ga]THP-PSMA, a Novel Probe for Rapid Kit-Based Ga-68 Labeling and PET Imaging: Comparative Analysis with [68Ga]PSMA I&T.

PURPOSE: [68Ga]Trishydroxypyridinone (THP)-prostate-specific membrane antigen (PSMA) is a novel tracer that can be labeled in one step by cold reconstitution of a kit with unprocessed generator eluate, targeting PSMA via the lysine-urea-glutamate (KuE) motif. The aim of this study was to evaluate the human imaging characteristics of [68Ga]THP-PSMA. PROCEDURES: [68Ga]THP-PSMA positron emission tomography (PET)/x-ray computed tomography (CT) was performed in 25 patients with biochemical recurrence after radical prostatectomy for prostate cancer. Urinary and biliary excretion and tumor lesion uptake were quantified using standardized uptake values (SUVs). Imaging characteristics were assessed in terms of non-target organ uptake, background activity, target-to-background ratios (TBRs) of tumor lesions, and frequency of bladder halo artifacts. Findings were compared to a matched cohort of 25 patients undergoing PET/CT with the established agent [68Ga]PSMA I&T. RESULTS: Physiologic uptake of [68Ga]THP-PSMA was significantly lower in salivary glands (P < 0.0001), liver (P < 0.0001), spleen (P < 0.0001), and kidneys (P < 0.0001) than with [68Ga]PSMA I&T. While biliary tracer excretion of [68Ga]THP-PSMA was negligible, urinary tracer excretion of [68Ga]THP-PSMA was fast, and significantly higher than for [68Ga]PSMA I&T, contributing to a higher frequency of bladder artifacts. Malignant lesion uptake of [68Ga]THP-PSMA assessed as either SUV or TBR was significantly lower than with [68Ga]PSMA I&T. CONCLUSION: [68Ga]THP-PSMA yields suitable in vivo uptake characteristics. The simplified synthesis method for [68Ga]THP-PSMA may facilitate wider application and higher patient throughput with PSMA imaging. However, direct intraindividual comparison studies are needed to assess the relative performance of [68Ga]THP-PSMA vs other PSMA ligands in terms of clinical detection rate and image quality.

Development of Potent and Metabolically Stable APJ Ligands with High Therapeutic Potential.

The apelin ligand receptor system is an important target to develop treatment strategies for cardiovascular diseases. Although apelin exhibits strong inotropic effects, its pharmaceutical application is limited because no agonist with suitable properties is available. On the one hand, peptide ligands are too instable, and on the other hand, small-molecule agonists show only low potency. This study describes the development of apelin (APJ) receptor agonists with not only high activity but also metabolic stability. Several strategies including capping of termini, insertion of unnatural amino acids, cyclization, and lipidation were analyzed. Peptide activity was tested using a Ca2+ -mobilization assay and the degradation of selected analogues was analyzed in rat plasma. The best results were obtained by N-terminal lipidation of a 13-mer apelin derivative. This analogue displayed a half-life of 29 h in rat plasma, compared with 0.025 h for the wild-type peptide. Furthermore, in vivo pharmacokinetics revealed a clearance of 0.049 L h-1 kg-1 and a half-life of 0.36 h. In summary, amino acid substitution and fatty acid modification resulted in a potent and 1000-fold more stable peptide that exhibits high pharmaceutical potential.

Molecular tools to characterize adiponectin activity.

Within the past years, numerous hormones were found to be secreted by adipose tissue. As these adipokines exert different physiological effects with great importance in obesity, they provide new strategies for the treatment of obesity associated disorders. Adiponectin is one of the most promising targets due to its protective properties in glucose and lipid metabolism, which are mediated by the adiponectin receptor 1 and 2. Within the past decades, substantial progress in understanding the molecular function of this unique ligand-receptor system could be achieved. This review summarizes the most important approaches for the investigation of adiponectin activity. Even though many insights into adipokine function could be achieved, clarification of the detailed mode of action is still challenging. For this reason, this review gives an overview of frequently used methods, which led to the molecular characterization of adiponectin and might help to get more detailed insights into the broad aspects of obesity.

Two motifs with different function regulate the anterograde transport of the adiponectin receptor 1.

The anterograde trafficking of GPCR has been described as a tightly controlled process involving specific amino acid sequences that mediate the receptor transport. In this study, we investigated whether the cell surface delivery of the adiponectin receptor 1, a newly identified class of heptahelix receptors different from G protein-coupled receptors, is regulated. Sequential N-terminal deletion revealed that the export of the AdipoR1 from the endoplasmic reticulum (ER) is controlled by distinct parts of the receptor N-terminus. Strong evidence is provided that the ER exit is mediated by two specific sequences, a F(X)(3)F(X)(3)F and a D(X)(3)LL motif. Disruption of these motifs led to a substantial accumulation of the AdipoR1 in the ER. Mutation of similar motifs in the AdipoR1 C-terminus did not result in aberrant receptor localization, suggesting that these motifs are sequence and position specific to the AdipoR1 N-terminus. Further analysis of the regulation mechanism identified an interaction with the chaperone BiP and additionally, strong evidence is provided that both motifs exert different biological function in the AdipoR1 ER export. In conclusion, our data demonstrate that the receptor transport shares similar ER exit motifs although AdipoR are structurally different from GPCR. However, since even two specific sequences are identified, the anterograde trafficking of the AdipoR1 seems to be regulated in a more complex manner.

Adiponectin receptor 1 interacts with both subunits of protein kinase CK2.

Adiponectin is an adipose tissue-derived hormone that is involved in the inhibition of metabolic syndrome, protection of hypertension, and suppression of atherosclerosis. Since these effects are not understood in detail, adiponectin signaling has to be clarified for therapeutic applications. Adiponectin activities are mediated by its two receptors adiponectin receptor 1 and adiponectin receptor 2, which consist of seven transmembrane helices. Previous studies revealed the beta subunit of protein kinase CK2 as an interaction partner of the adiponectin receptor 1 N-terminus using a yeast-two-hybrid screen, co-immunoprecipitation, ELISA experiments, and co-localization studies. Inhibition of CK2 activity by 2-dimethylamino-4,5,6,7-tetrabromo-1H-benz-imidazole led to a decrease of ACC phosphorylation and indicates an important role of CK2 in adiponectin signaling. CK2 is characterized as a heterotetramer that consists of two regulatory beta and two catalytic alpha subunits, but a holoenzyme-independent role for both subunits is described as well. Therefore, we analyzed the role of the catalytic subunit in this interaction by co-immunoprecipitation and bimolecular fluorescence complementation studies and found CK2 alpha as an interaction partner of the receptor. Treatment with full-length adiponectin resulted in no dissociation of the catalytic alpha subunit. Consequently, our data suggest an interaction of the adiponectin receptor 1 with the tetrameric complex and identified protein kinase CK2 as a key player in adiponectin signaling.

Dimerization of adiponectin receptor 1 is inhibited by adiponectin.

AdipoR1 and AdipoR2 are newly discovered members of the huge family of seven-transmembrane receptors, but both receptors are structurally and functionally different from G-protein-coupled receptors. Little is known about the oligomerization of the AdipoRs. Here, we show the presence of endogenous AdipoR1 dimers in various cell lines and human muscle tissue. To directly follow and localize the dimerization, we applied bimolecular fluorescence complementation (BiFC) in combination with flow cytometry. We visualized and quantified AdipoR1 homodimers in HEK293 cells. Moreover, we identified a GxxxG dimerization motif in the fifth transmembrane domain of the AdipoR1. By mutating both glycine residues to phenylalanine or glutamic acid, we were able to modulate the dimerization of AdipoR1, implicating a role for the GxxxG motif in AdipoR1 dimerization. Furthermore, we tested whether the AdipoR1 ligand adiponectin had any influence on receptor dimerization. Interestingly, we found that adiponectin decreases the receptor dimerization in a concentration-dependent manner. This effect is mainly mediated by segments of the collagen-like domain of full-length adiponectin. Accordingly, this is the first direct read-out signal of adiponectin at the AdipoR1 receptor, which revealed the involvement of specific amino acids of both the receptor and the ligand to modulate the quaternary structure of the AdipoR1.

Protein kinase CK2 interacts with adiponectin receptor 1 and participates in adiponectin signaling.

Adiponectin is an adipokine with anti-atherogenic, anti-diabetic and insulin sensitizing properties. Its effects on energy homeostasis, glucose and lipid metabolism are mediated by two ubiquitously expressed seven-transmembrane receptors, AdipoR1 and -R2. With the exception of APPL1 and RACK1, no intracellular binding partners of adiponectin receptors are reported and thus signaling pathways downstream of these receptors remain largely unknown. To incorporate adiponectins protective potential in drug development it is essential to understand adiponectin signaling cascades in detail. A yeast two-hybrid approach employing AdipoR1s cytoplasmatic N-terminus led to the identification of the regulatory subunit of protein kinase CK2. We confirmed the interaction in co-immunoprecipitation, ELISA experiments and co-localization analysis in mammalian cells. Furthermore we could localize the interaction site in an N-terminal basic region close to the transmembrane domain. In adiponectin stimulation experiments of C2C12 mouse myotubes and MCF7 cells incorporating CK2 inhibitor 2-dimethylamino-4,5,6,7-tetrabromo-1H-benz-imidazole (DMAT) we found a modulator role of CK2 in adiponectin signaling. Accordingly we identified the regulatory subunit of protein kinase CK2 as a novel intracellular partner of AdipoR1 and have strong evidence of CK2 as an effector molecule in adiponectin signaling. Since CK2 is involved in signaling cascades of other adipokines and hormones, e.g. leptin and insulin, our findings suggest a possible key function in crosstalk between adiponectin and insulin signaling pathways and could provide further insight into the anti-diabetic effects of adiponectin.