A Pharmacophore-Based Virtual Screening Approach for the Discovery of Novel P2Y 12 Receptor Ligands

Microglia play key roles in neuroinflammation, which is a hallmark of neurodegenerative diseases such as Parkinson’s disease, Alzheimer’s disease and Multiple Sclerosis. To strengthen our understanding of the molecular mechanisms of these disorders, we need to understand the complex dynamics in microglial states. Developing new positron emission tomography (PET) tracers is exceptionally useful for this purpose, as they enable the assessment of microglia dynamics and function in the living human brain. The purinergic receptor P2Y12 (P2Y 12 R) is highly expressed in the homeostatic microglial states, and thus makes for a promising biomarker. In particular, P2Y 12 R PET tracers could complement translocator protein (TSPO) and P2X 7 receptor tracers, which image microglial density or target the pro-inflammatory state, respectively. Unfortunately, current high-affinity P2Y 12 R ligands do not enter the brain because they are charged in vivo or because they are substrates for efflux transporters. Attempts to increase brain uptake by improving their physicochemical properties have not yet resulted in a successful P2Y 12 R PET tracer. This emphasizes the need for novel chemotypes. Herein, we applied a pharmacophore-based virtual screening strategy to a library of about three million compounds. As a result, we identified the imidazo[1,2- b ]pyrazolone scaffold as a promising structural class to use as a starting point for developing P2Y 12 R ligands. After extensive hit expansion and structure-activity relationship studies, the two best ligands displayed excellent binding affinity for human P2Y 12 R ( 96 : K i = 9.61 ± 1.70 nM; 109 : K i = 3.10 ± 0.34 nM) and rat P2Y 12 R ( 96 : K i = 65.6 ± 25.9 nM; 109 : K i = 17.9 ± 12.8 nM) in a radioligand binding assay. Furthermore, imidazo[1,2- b ]pyrazolone [ 18 F] 91 was successfully radiolabeled as a proof-of-concept in sufficient radiochemical yields. Future work will assess whether the more potent ligands can be radiolabeled and used to image the P2Y 12 R in the brain. Together, these results highlight the use of pharmacophore-based strategy in PET tracer development and provide a robust basis for further hit exploration.