Effect of S–Se Bioisosteric Exchange on Affinity and Intrinsic Efficacy of Novel N-acylhydrazone Derivatives at the Adenosine A2A Receptor

In this work, we evaluated the conformational effect promoted by the isosteric exchange of sulfur by selenium in the heteroaromatic ring of new N-acylhydrazone (NAH) derivatives (3–8, 13, 14), analogues of the cardioactive compounds LASSBio-294 (1) and LASSBio-785 (2). NMR spectra analysis demonstrated a chemical shift variation of the iminic Csp2 of NAH S/Se-isosters, suggesting a stronger intramolecular chalcogen interaction for Se-derivatives. To investigate the pharmacological profile of these compounds at the adenosine A2A receptor (A2AR), we performed a previously validated functional binding assay. As expected for bioisosteres, the isosteric-S/Se replacement affected neither the affinity nor the intrinsic efficacy of our NAH derivatives (1–8). However, the N-methylated compounds (2, 6–8) presented a weak partial agonist profile at A2AR, contrary to the non-methylated counterparts (1, 3–5), which appeared as weak inverse agonists. Additionally, retroisosterism between aromatic rings of NAH on S/Se-isosters mimicked the effect of the N-methylation on intrinsic efficacy at A2AR, while meta-substitution in the phenyl ring of the acyl moiety did not. This study showed that the conformational effect of NAH-N-methylation and aromatic rings retroisosterism changed the intrinsic efficacy on A2AR, indicating the S/Se-chalcogen effect to drive the conformational behavior of this series of NAH.

State-Targeting Stabilization of Adenosine A2A Receptor by Fusing a Custom-Made De Novo Designed α-Helical Protein

G-protein coupled receptors (GPCRs) are known for their low stability and large conformational changes upon transitions between multiple states. A widely used method for stabilizing these receptors is to make chimeric receptors by fusing soluble proteins (i.e., fusion partner proteins) into the intracellular loop 3 (ICL3) connecting the transmembrane helices 5 and 6 (TM5 and TM6). However, this fusion approach requires experimental trial and error to identify appropriate soluble proteins, residue positions, and linker lengths for making the fusion. Moreover, this approach has not provided state-targeting stabilization of GPCRs. Here, to rationally stabilize a class A GPCR, adenosine A2A receptor (A2AR) in a target state, we carried out the custom-made de novo design of α-helical fusion partner proteins, which can fix the conformation of TM5 and TM6 to that in an inactive state of A2AR through straight helical connections without any kinks or intervening loops. The chimeric A2AR fused with one of the designs (FiX1) exhibited increased thermal stability. Moreover, compared with the wild type, the binding affinity of the chimera against the agonist NECA was significantly decreased, whereas that against the inverse agonist ZM241385 was similar, indicating that the inactive state was selectively stabilized. Our strategy contributes to the rational state-targeting stabilization of GPCRs.

Retrograde adenosine/A2A receptor signaling mediates presynaptic hippocampal LTP and facilitates epileptic seizures

A long-term change in neurotransmitter release is a widely expressed mechanism controlling neural circuits in the mammalian brain. This presynaptic plasticity is commonly mediated by retrograde signaling whereby a messenger released from the postsynaptic neuron upon activity modifies neurotransmitter release in a long-term manner by targeting a presynaptic receptor. In the dentate gyrus (DG), the main input area of the hippocampus, granule cells (GCs) and mossy cells (MCs) form a recurrent excitatory circuit that is critically involved in DG function and epilepsy. Here, we identified adenosine/A2A receptor (A2AR) as a novel retrograde signaling system that mediates presynaptic long-term potentiation (LTP) at MC-GC synapses. Using an adenosine sensor, we found that neuronal activity triggered phasic, postsynaptic TrkB-dependent release of adenosine. Additionally, epileptic seizures released adenosine in vivo, while removing A2ARs from DG decreased seizure susceptibility. Thus, adenosine/A2AR retrograde signaling mediates presynaptic LTP that may contribute to DG-dependent learning and promote epilepsy.

Efficacy of Istradefylline, an Adenosine A2A Receptor Antagonist, as Adjunctive Therapy to Levodopa in Parkinson’s Disease: A Pooled Analysis of 8 Phase 2b/3 Trials

Background: Istradefylline is a selective adenosine A2A receptor antagonist for the treatment of patients with Parkinson’s disease (PD) experiencing OFF episodes while on levodopa/decarboxylase inhibitor. Objective: This pooled analysis of eight randomized, placebo-controlled, double-blind phase 2b/3 studies evaluated the efficacy and safety of istradefylline. Methods: Istradefylline was evaluated in PD patients receiving levodopa with carbidopa/benserazide and experiencing motor fluctuations. Eight 12- or 16-week trials were conducted (n = 3,245); four of these studies were the basis for istradefylline’s FDA approval. Change in OFF time as assessed in patient-completed 24-h PD diaries at Week 12 was the primary endpoint. All studies were designed with common methodology, thereby permitting pooling of data. Pooled analysis results from once-daily oral istradefylline (20 and 40 mg/day) and placebo were evaluated using a mixed-model repeated-measures approach including study as a factor. Results: Among 2,719 patients (placebo, n = 992; 20 mg/day, n = 848; 40 mg/day, n = 879), OFF hours/day were reduced at Week 12 at istradefylline dosages of 20 mg/day (least-squares mean difference [LSMD] from placebo in reduction from baseline [95%CI], –0.38 h [–0.61, –0.15]) and 40 mg/day (–0.45 h [–0.68, –0.22], p < 0.0001); ON time without troublesome dyskinesia (ON-WoTD) significantly increased. Similar results were found in the four-study pool (OFF hours/day, 20 mg/day, –0.75 h [–1.10, –0.40]; 40 mg/day, –0.82 h [–1.17, –0.47]). Istradefylline was generally well-tolerated; the average study completion rate among istradefylline-treated patients across all studies was 89.2%. Dyskinesia was the most frequent adverse event (placebo, 9.6%; 20 mg/day, 16.1%; 40 mg/day, 17.7%). Conclusion: In this pooled analysis, istradefylline significantly improved OFF time and ON-WoTD relative to placebo and was well-tolerated.