Characterization of [3H]AZ12464237 as a high affinity, non-nucleotide antagonist radioligand for the P2Y12 receptor

Abstract

The purinergic receptor P2Y₁₂ (P2Y₁₂R) is a well-recognized target for anti-thrombotic agents. This receptor is also expressed in microglia, the main immune cells of the brain, where it modulates microglial activation states and inflammatory responses. To investigate P2Y₁₂R-mediated actions in the central nervous system (CNS), developing novel brain-penetrant ligands and further in vitro studies on brain tissues are essential. A radiolabeled, easily accessible tool compound would significantly advance such drug discovery efforts. Herein, we describe the ³H-labeling of a non-nucleotide P2Y₁₂R antagonist AZ12464237, and its in vitro binding properties to the receptor in membrane preparations from transfected cells, as well as on mouse brain tissues. The radioligand shows high affinity toward both the human and rat P2Y₁₂R, with K_d values of 3.12 ± 0.70 nM (human) and 16.6 ± 3.40 nM (rat), as determined by saturation binding studies. The binding kinetics of [³H]AZ12464237 are rapid with a short target residence time (∼1 min). We further confirmed the selectivity of the radioligand by performing competitive displacement studies, in which reported P2Y₁₂R ligands and other P2Y receptors ligands were tested for binding against [³H]AZ12464237. Additionally, the radioligand proved valuable for in vitro autoradiography studies on mouse brain tissues, although limited off-target binding was observed in P2Y₁₂R knock-out mouse brain. This could be traced to glycogen synthase kinase 3 α. Considering the growing interest in P2Y₁₂R as a biomarker for anti-inflammatory microglia, [³H]AZ12464237 represents a promising tool for in vitro studies, including screening assays aimed at identifying novel P2Y₁₂R ligands for CNS applications.