A Holistic Approach to Identifying a Positron Emission Tomography (PET) Tracer Candidate for In Vivo Imaging of Purinergic P2X7 Receptor in Neuroinflammation

High Resolution Image Download MS PowerPoint Slide The central role of neuroinflammation in the pathogenesis of neurodegenerative diseases and brain disorders has spurred the development of Positron Emission Tomography (PET) radiotracers to investigate neuroimmune mechanisms noninvasively in vivo. Because it is expressed in glia, the purinergic P2X7 receptor (P2X7R) is a validated target for in vivo imaging of neuroinflammation, an alternative to the 18 kDa translocator protein, which is currently the standard target for neuroinflammation in clinical practice. However, clinically validated P2X7R PET radiotracers remain needed. The present study aimed to identify a novel molecular scaffold for developing an effective P2X7R PET radiotracer, starting from three chemotypes with antagonist activity in the nanomolar range at human P2X7R, to exploit structural diversity and meet the multidimensional key attributes that a CNS PET radiotracer must have. Thus, we evaluated the selected chemotypes across a range of properties, including radioligand binding affinity at the human P2X7 receptor, off-target selectivity, in vitro metabolic stability, and nonspecific binding to brain tissue. Our study pointed to compound 2 (2-chloro-3-methoxy- N -[2-morpholino-2-[6-(trifluoromethyl)pyridin-3-yl]ethylbenzamide) as a promising molecular scaffold to deliver an effective PET tracer because of its nanomolar affinity for human cloned P2X7R, broad off-target selectivity, high in vitro metabolic stability, passive permeability across two model membrane monolayers, limited interaction with blood-brain barrier efflux transporters, and brain free fraction predictive of low in vivo nonspecific binding. To ensure robust translatability, we also evaluated the binding affinity of compound 2 in human meningiomas by autoradiography and found that the compound binds to native P2X7R with high affinity (IC 50 = 72 nM).