CDA Inhibitors(University of Minnesota)

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.

Fungal chitin deacetylase (CDA) plays a crucial role in pathogen-plant interactions, which is regarded as an innovative and promising target for fungicides. In this study, a pharmacophore-based virtual screening strategy was employed to identify compounds VS-24 and VS-25 as potent inhibitors against Puccinia striiformis f. sp. tritici CDA (PstCDA). Further bioassays demonstrated that VS-24 exhibited a protective effect of 61.2% against rice blast at 100 μg/mL, while VS-25 showed a superior protective effect of 45.5% against corn rust at 5 μg/mL, both superior to the reported CDA inhibitor benzohydroxamic acid (BHA). Molecular dynamics simulations revealed that multiple key interactions involving the Zn²⁺ ion and residues His207 and Tyr152 of PstCDA are critical for the binding of VS-24 or VS-25, with electrostatic interactions contributing most significantly to the binding free energy. Finally, toxicity predictions confirmed the potential biosafety of VS-24 and VS-25. Overall, this study identified two promising lead compounds targeting fungal CDA to control plant diseases.