Discovery of non-steroidal aldo-keto reductase 1D1 inhibitors through automated screening and in vitro evaluation

Steroid hormones regulate a wide range of physiological processes in the human body. However, exposure to xenobiotics can disrupt the hormonal balance by inhibition of enzymes involved in hormone synthesis or metabolism. Aldo-keto reductase 1D1 (AKR1D1) plays a key role in bile acid and steroid hormone metabolism by catalyzing the reduction of the double bond between C4 and C5 atoms of Δ(4)-steroids. In our previous work, we developed a model to screen for steroid-like xenobiotics that inhibit AKR1D1. In the current study, we used this model to screen for novel non-steroidal inhibitors. By applying an automatized screening approach, based on molecular docking and scoring in combination with post-docking refinement, 45 compounds were detected as potential hits and selected for in vitro evaluation. Among them, zardaverine was identified as the most potent inhibitor, with an IC₅₀ value of 2.32 ± 1.27 μM. Other moderate inhibitors included carbamazepine, larotrectinib, endosulfan II, megastigmatrienone A, and mizolastine. The structural diversity of the identified inhibitors demonstrates that the binding site of AKR1D1 is rather promiscuous and can accommodate a broad range of ligands. These findings underscore the importance of toxicity screening and potential to identify structurally different AKR1D1 inhibitors.