Synthesis and Evaluation of a PET Radiotracer [¹⁸F]LY3000328 Selectively Targeting the Cysteine Protease Cathepsin S

Cathepsin S (Cat S) is a cysteine protease broadly expressed in the brain and a key mediator of neuroinflammatory processes, making it an attractive target for molecular imaging agents and drug development. Here, we report the development and evaluation of [¹⁸F]LY3000328, a positron emission tomography (PET) radiotracer that selectively targets Cat S and is derived directly from the clinical Cat S inhibitor LY3000328. [¹⁸F]LY3000328 was synthesized via a ruthenium-assisted late-stage ¹⁸F-deoxyfluorination of the corresponding phenol precursor. In vitro autoradiography, the marked reduction in tracer uptake under blocking conditions suggests specific binding of [¹⁸F]LY3000328 to Cat S, and the significantly higher uptake in transgenic 5xFAD mouse brain sections indicates upregulation of Cat S in Alzheimer's disease (AD), consistent with previously reported findings. However, in vivo PET imaging in wild-type mice revealed low blood-brain barrier (BBB) penetration and no significant reduction under blocking conditions, suggesting that limited BBB permeability may constrain its efficiency as a brain PET tracer. Despite its limited brain penetration, these results suggest that [¹⁸F]LY3000328 may serve as a useful starting scaffold for further optimization of physicochemical and pharmacokinetic properties toward Cat S-targeted PET imaging of neuroinflammation in AD and potentially other neurodegenerative disorders.