ZM-241385

Ischemic damage to the retina during development can lead to irreversible neuronal loss, driven in part by excitotoxic mechanisms and energy deprivation. While two‐day caffeine exposure has previously been shown to confer neuroprotection in this context, it remained unclear whether acute administration during an ischemic event could yield similar benefits. In this study, we used an ex vivo model of oxygen and glucose deprivation (OGD) in chick embryo retinas to investigate whether a single, acute dose of caffeine applied during the insult reduces cell death and engages intracellular survival pathways. Results showed a significant reduction in OGD‐induced cytotoxicity by caffeine exposure. However, unlike in the context of chronic exposure, acute caffeine exposure did not increase BDNF expression. Furthermore, antioxidant agents failed to mimic caffeine's protective effects, and acute caffeine exposure did not induce the expression of antioxidant response genes, nor oxidative stress or VEGF expression, suggesting that oxidative stress mitigation is not the primary mechanism for this effect. However, pharmacological antagonism of adenosine A 2A receptors with ZM241385 reproduced the neuroprotective effects of caffeine and reduced extracellular glutamate levels during OGD. These findings indicate that acute caffeine administration protects the developing retina primarily through A 2A receptor antagonism and inhibition of glutamate excitotoxicity, rather than by activating canonical survival signaling pathways. This supports the potential use of caffeine as a rapid neuroprotective agent during acute ischemic events in the immature central nervous system.

Structure determination of G protein-coupled receptors (GPCRs) plays an important role in accelerating drug development against this medically important protein family. This study outlines the development of a new fusion tool to enable structure determination of GPCRs in inactive conformations by cryo-EM. Initially, a PDB mining approach was applied to select eight naturally occurring proteins with the intention of fusing them into the intracellular loop 3 (ICL3) of GPCRs to create a suitable fiducial marker for cryo-EM workflows. During the selection process, candidates with known high-affinity protein binders were prioritized to enable a further increase in the protein mass of the fiducial marker. Fusion constructs were generated with adenosine receptor A 2A (A 2A R) and were assessed for expression and aggregation levels. For the two best-performing new fusion constructs, ligand binding was characterized to ensure that the fusion tag did not significantly affect protein behaviour. A 2A R with a β-lactamase fusion in ICL3 and binding partner β-lactamase inhibitory protein II (BLIPII) was then selected to solve an antagonist-bound structure. The overall map was resolved to an average of 3.2 Å resolution with continuous helices connecting the β-lactamase to helices 5 and 6 of A 2A R. Focused refinement of the A 2A R region improved the local resolution and map detail in the orthosteric site, thereby allowing confident modelling of the antagonist ligand, ZM241385, which matches previously described X-ray crystallographic structures. This new fusion provides an alternative option for GPCR structure determination, with several potential benefits compared with existing tools, such as a more favourable position relative to the GPCR to reduce potential clashes.