Development of Bicyclo[3.1.0]hexane-Based A3 Receptor Ligands: Closing the Gaps in the Structure–Affinity Relationships

Abstract

The adenosine A3 receptor is a promising target for treating and diagnosing inflammation and cancer. In this paper, a series of bicyclo[3.1.0]hexane-based nucleosides was synthesized and evaluated for their P1 receptor affinities in radioligand binding studies. The study focused on modifications at 1-, 2-, and 6-positions of the purine ring and variations of the 5′-position at the bicyclo[3.1.0]hexane moiety, closing existing gaps in the structure–affinity relationships. The most potent derivative 30 displayed moderate A3AR affinity (Ki of 0.38 μM) and high A3R selectivity. A subset of compounds varied at 5′-position was further evaluated in functional P2Y1R assays, displaying no off-target activity.

Still in Search for an EAAT Activator: GT949 Does Not Activate EAAT2, nor EAAT3 in Impedance and Radioligand Uptake Assays

“…To further validate whether GT949 truly activates EAAT2, radioligand uptake assays were performed. The protocols for the dose response curve (DRC) and kinetic assay (Figure ) were adapted from previous work done in our lab . TFB-TBOA (10 μM) was taken as a negative control in all radioligand uptake assays.…”

“…The protocols for the dose response curve (DRC) and kinetic assay (Figure 3) were adapted from previous work done in our lab. 23 TFB-TBOA (10 μM) was taken as a negative control in all radioligand uptake assays. Indeed, TFB-TBOA, which has been described as a nonselective competitive inhibitor, acting on EAAT1, 2, and 3, 18,19 clearly reduced radioligand (L-[3,4-3 H]glutamic acid) uptake in both EAAT2 and EAAT3 (Figure 3), in accordance with the normal signal-to-noise ratio found in the literature.…”

“…The plates were dried at 55 °C, after which MicroscintTM-20-cocktail was added (PerkinElmer, Groningen, The Netherlands). Intracellular radioactivity was determined by scintillation spectrometry using a MicroBeta 2 2450 Microplate Counter (PerkinElmer, Groningen, The Netherlands) …”

“…Intracellular radioactivity was determined by scintillation spectrometry using a MicroBeta 2 2450 Microplate Counter (PerkinElmer, Groningen, The Netherlands). 23 Radioligand Uptake Assay: 24-Well Assay. The dose− response assay was performed as described recently.…”

Microwave-Assisted Synthesis, Structure, and Preliminary Biological Evaluation of Novel 6-Methoxy-5,6-dihydro-5-azapurines

“…Therefore, compounds 21 were tested at 1 μM in radioligand displacement experiments against four subtypes of adenosine receptors, namely, A 1 , A 2A , A 2B , and A 3 , as previously reported ( Figure S7 and Table S3 , Supporting Information ). 55 In addition, compounds were screened at 10 μM against P2X7R purinergic receptors in an YO-PRO-1 uptake assay 56 ( Figure S8 , Supporting Information ). Performed experiments, however, revealed that synthesized 5,6-dihydro-5-azapurines 21 have little to no affinity/inhibitory activity toward these five purinergic receptors.…”