Structure-Based Screening Identifies Polyphenol Glucuronides as Cyclophilin D Ligands

(1) Background: The mitochondrial permeability transition pore (mPTP) contributes to mitochondrial dysfunction under pathological conditions. Cyclophilin D (CypD), a key regulator of sustained mPTP opening, represents an important molecular target. However, systematic strategies for selecting an appropriate CypD crystal structure for virtual screening and efficiently identifying CypD-binding natural polyphenols remain limited. (2) Methods: Six CypD crystal structures were evaluated by receiver operating characteristic analysis using reported CypD inhibitors and approximately 4000 decoys. The optimal receptor structure was subsequently used to screen 484 dietary polyphenols by molecular docking. Selected top-ranked candidates were further characterized by 15N–1H HSQC NMR titration and surface plasmon resonance (SPR). (3) Results: PDB 6R8L exhibited the best virtual-screening performance, particularly in early enrichment. Several polyphenol glucuronides ranked among the top candidates. NMR analysis showed that apigenin 7-glucuronide and luteolin 7-glucuronide induced pronounced chemical shift perturbations at CypD residues F60, M61, I117, and W121, with F60, M61, and W121 located within or near the docking-predicted S1 binding region. SPR independently confirmed direct binding, yielding KD values of 66 μM and 84 μM for apigenin 7-glucuronide and luteolin 7-glucuronide, respectively. (4) Conclusions: Integrating systematic receptor evaluation, molecular docking, and complementary NMR and SPR characterization provides a practical workflow for identifying and characterizing natural CypD-binding compounds. Apigenin 7-glucuronide and luteolin 7-glucuronide were identified as micromolar CypD-binding candidates, highlighting polyphenol glucuronides as a chemical class warranting further investigation in CypD ligand discovery.