Comparison of a Novel Homogeneous Cyclic Amp Assay and a Luciferase Assay for Measuring Stimulating Thyrotropin-Receptor Autoantibodies

Objective: Stimulating thyrotropin-receptor antibodies (TSAb) cause Graves’ disease (GD). We tested a novel homogeneous fluorescent 3′,5′ cyclic adenine monophosphate (cAMP) assay for the detection of TSAb in a bioassay. Methods: Chinese hamster ovary (CHO) cell lines expressing either a chimeric (MC4) or wild-type (WT) TSH-R were incubated with the adenyl cyclase activator forskolin, a human TSAb monoclonal antibody (M22), and with sera from GD patients. Intracellular cAMP levels were measured using a Bridge-It® cAMP assay, and the results were compared with a luciferase-based bioassay. Results: Both cell lines were stimulated with forskolin concentrations (0.006–200 µM) in a dose-dependent manner. The linear range in the MC4 and WT cells was 0.8–25 and 3.1–50 µM, respectively. Levels of cAMP and luciferase in forskolin-treated MC4 and WT cells were positively correlated (r = 0.91 and 0.84, both p < 0.001). The 50% maximum stimulatory concentration of forskolin was more than 16-fold higher for the CHO-WT cells than the CHO-MC4 cells in the cAMP assay and 4-fold higher in the luciferase assay. Incubation of both cell lines with M22 (0.006–50 ng/mL) resulted in a dose-dependent increase in cAMP levels with linear ranges for the MC4 and WT cells of 0.8–12.5 and 0.2–3.125 ng/mL, respectively. Comparison of cAMP and luciferase levels in M22-treated MC4 and WT cells also showed a positive correlation (r = 0.88, p < 0.001 and 0.75, p = 0.002). A positive correlation was also noted when using patient samples (r = 0.96, p < 0.001) that were all TSH-R-Ab binding assay positive. Conclusion: The novel, rapid, simple-to-perform cAMP assay provides TSAb-mediated stimulatory results comparable to a luciferase-based bioassay.

β-Fluorofentanyls are pH-Sensitive Mu Opioid Receptor Agonists

The concept recently postulated by Stein and coworkers (Science 2017, 355, 966) that mu opioid receptor (MOR) agonists possessing amines with attenuated basicity show pH-dependent activity and can selectively act at damaged, low pH tissues has been additionally supported by in vitro studies reported here. We synthesized and tested analogs of fentanyl possessing one or two fluorine atoms at the beta position of the phenethylamine side chain, with additional fluorines optionally added to the benzene ring of the side chain. These compounds were synthesized in 1 to 3 steps from commercial building blocks. The novel bis-fluorinated analog RR-49 showed superior pH sensitivity, with full efficacy relative to DAMGO, but with 19-fold higher potency (EC₅₀) in a MOR cAMP assay at pH 6.5 versus 7.4. Such compounds hold significant promise as analgesics for inflammatory pain with reduced abuse potential.

β-Fluorofentanyls are pH-Sensitive Mu Opioid Receptor Agonists

The concept recently postulated by Stein and coworkers (Science 2017, 355, 966) that mu opioid receptor (MOR) agonists possessing amines with attenuated basicity show pH-dependent activity and can selectively act at damaged, low pH tissues has been additionally supported by in vitro studies reported here. We synthesized and tested analogs of fentanyl possessing one or two fluorine atoms at the beta position of the phenethylamine side chain, with additional fluorines optionally added to the benzene ring of the side chain. These compounds were synthesized in 1 to 3 steps from commercial building blocks. The novel bis-fluorinated analog RR-49 showed superior pH sensitivity, with full efficacy relative to DAMGO, but with 19-fold higher potency (EC₅₀) in a MOR cAMP assay at pH 6.5 versus 7.4. Such compounds hold significant promise as analgesics for inflammatory pain with reduced abuse potential.

Uridine Triphosphate Thio-analogues Inhibit Platelet P2Y12 Receptors and Aggregation

Platelet P2Y12 is an important ADP receptor that is involved in agonists-induced platelet aggregation and is an important target for the development of anti-platelet aggregation drugs. Here the effects of thio-analogues of uridine triphosphate (UTP) on ADP-induced platelet aggregation are characterised. Using human platelet rich plasma we demonstrate that UTP inhibits P2Y12 but not P2Y1 receptors and antagonises ADP-induced platelet aggregation in a conc.-dependent manner with an IC50 value of ~250 mM against ADP (10 mM). An 8-fold increase in the platelet inhibitory activity was observed with 2-thio analogue of UTP (2S-UTP) with an IC50 value of 30 mM. A 33-fold increase in anti-platelet aggregation activity was observed with 4-thio analogue (4S-UTP) with an IC50 value of 7.5 mM. However, a 3-fold decrease in activity was observed by introducing an isobutyl group at the 4S- position. A complete loss in anti-platelet aggregation activity was observed with thio-modification of gamma phosphate of the sugar moiety which yields an enzymatically stable analogue. The interaction of UTP analogues with P2Y12 receptors was further verified by P2Y12 receptor binding assay and cAMP assay. The novel data demonstrate for the first time that 2- and 4-thio analogues of UTP are potent P2Y12 receptor antagonists that can be useful candidates for therapeutic intervention.