Allosteric modulation of the adenosine A2A receptor by cholesterol

Cholesterol is a major component of the cell membrane and commonly regulates membrane protein function. Here, we investigate how cholesterol modulates the conformational equilibria and signaling of the adenosine A2A receptor (A2AR) in reconstituted phospholipid nanodiscs. This model system conveniently excludes possible effects arising from cholesterol-induced phase separation or receptor oligomerization and focuses on the question of allostery. GTP hydrolysis assays show that cholesterol weakly enhances the basal signaling of A2AR while decreasing the agonist EC50. Fluorine nuclear magnetic resonance (19F NMR) spectroscopy shows that this enhancement arises from an increase in the receptor’s active state population and a G-protein-bound precoupled state. 19F NMR of fluorinated cholesterol analogs reveals transient interactions with A2AR, indicating a lack of high-affinity binding or direct allosteric modulation. The combined results suggest that the observed allosteric effects are largely indirect and originate from cholesterol-mediated changes in membrane properties, as shown by membrane fluidity measurements and high-pressure NMR.

KANPHOS: A Database of Kinase-Associated Neural Protein Phosphorylation in the Brain

Protein phosphorylation plays critical roles in a variety of intracellular signaling pathways and physiological functions that are controlled by neurotransmitters and neuromodulators in the brain. Dysregulation of these signaling pathways has been implicated in neurodevelopmental disorders, including autism spectrum disorder, attention deficit hyperactivity disorder and schizophrenia. While recent advances in mass spectrometry-based proteomics have allowed us to identify approximately 280,000 phosphorylation sites, it remains largely unknown which sites are phosphorylated by which kinases. To overcome this issue, previously, we developed methods for comprehensive screening of the target substrates of given kinases, such as PKA and Rho-kinase, upon stimulation by extracellular signals and identified many candidate substrates for specific kinases and their phosphorylation sites. Here, we developed a novel online database to provide information about the phosphorylation signals identified by our methods, as well as those previously reported in the literature. The “KANPHOS” (Kinase-Associated Neural Phospho-Signaling) database and its web portal were built based on a next-generation XooNIps neuroinformatics tool. To explore the functionality of the KANPHOS database, we obtained phosphoproteomics data for adenosine-A2A-receptor signaling and its downstream MAPK-mediated signaling in the striatum/nucleus accumbens, registered them in KANPHOS, and analyzed the related pathways.

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.

Pentoxifylline as a Potential Adjuvant Therapy for COVID-19: Impeding the Burden of the Cytokine Storm

The outburst of inflammatory response and hypercoagulability are among the factors contributing to increased mortality in severe COVID-19 cases. Pentoxifylline (PTX), a xanthine-derived drug registered for the treatment of vascular claudication, has been reported to display broad-spectrum anti-inflammatory and immunomodulatory properties via adenosine A2A receptor (A2AR)-related mechanisms, in parallel to its rheological actions. Prior studies have indicated the efficacy of PTX in the treatment of various pulmonary diseases, including the management of acute respiratory distress syndrome of infectious causes. Therefore, PTX has been proposed to have potential benefits in the treatment of SARS-CoV-2 symptoms, as well as its complications. The aim of this review is to discuss available knowledge regarding the role of PTX as a complementary therapeutic in SARS-CoV-2.