High-Affinity Probes for Androgen Receptor Imaging: From Cells and In Silico Modeling to Whole-Body Fluorescent Applications.

Castrate-resistant prostate cancer (CRPC) is driven by androgen receptor (AR) signaling. Imaging tools to monitor AR signaling dynamics are a high-priority goal. Here, we introduce ARi-FL, a series of visible- and near-infrared fluorescent AR inhibitors. Based on an aryloxy cyclohexane scaffold, a neolinker enabled amine-based conjugation to fluorophores. ARi-FL showed potent AR inhibition (IC(50) ∼ 13 nM) and allowed visualization of cytoplasmic AR, correlating with AR-expressing cells, which were blockable with excess unlabeled ligand. In vivo and ex vivo studies in human prostate cancer models confirmed ARi-FL localization in AR-positive tumors. In silico modeling across wild-type (WT) and clinically relevant AR mutants (F877L, T878A, H875Y, W742C, and F877L/T878A) revealed nanomolar binding affinities (K(d) ∼ 1-2 nM), consistent with experimental results. ARi-FL probes demonstrated high selectivity, practical yields, and stability. Taken together, ARi-FL offers a chemical imaging platform for noninvasive AR tracking with applications for studying resistance mechanisms of AR and guiding treatment decisions.