Development of a Real-Time Fluorescence-Based Deamination Assay and Identification of Inhibitors of Human Cytidine...

Cytidine analogues have conferred highly efficacious antimetabolites with broad utility as antiviral and anticancer agents. However, in many cases, human cytidine deaminase (CDA) converts the cytidine-based inhibitor into an inactive uridine metabolite with diminished potency. Inhibitors of CDA are useful agents to boost the efficacy of cytosine- and cytidine-containing drugs by inhibiting their rapid degradation. Toward the goal of developing CDA inhibitors, and our overarching interest in cytosine deaminase enzymes in general, we developed a real-time fluorescence-based deamination activity assay for CDA using isomorphic nucleoside analogues. Base-modified pyrimidine nucleosides that exhibit differential fluorescence properties as either the cytosine or uracil nucleobase were developed. We found that 5-benzo-2-furyl-2'-deoxycytidine is the best fluorescence reporter when implemented in a CDA enzyme activity assay, which permits detailed measurements of the kinetics of CDA activity in the presence or absence of inhibitors. Utilizing this assay, we then screened our in-house collection of 1054 fragments and found 23 hits that were further studied. Two fragment-sized CDA inhibitors with low micromolar potency (200-300 μM) and good ligand efficiency (>0.3) were identified, thereby conferring promising starting points for future inhibitor development.