In Silico and In Vitro Assessment of Carbonyl Reductase 1 Inhibition Using ASP9521—A Potent Aldo-Keto Reductase 1C3 Inhibitor with the Potential to Support Anticancer Therapy Using Anthracycline Antibiotics

Figure 1. Structures of ASP9521 and selected known CBR1 ligands: daunorubicin (substrate), luteolin and cinnamamide-derivative Compound 15 (both inhibitors); the circles indicate structural similarities that may be the features important for CBR1 binding: H-bond acceptor (red), H-bond donor (yellow), hydrophobic aromatic ring (blue).

Figure 2. Putative binding mode of ASP9521 (orange) within CBR1 catalytic site obtained by molecular docking. Amino acid residues engaged in ligand binding throughout H-bonds (dotted yellow lines) and π-π stacking (dotted blue lines) displayed as thick sticks; NADPH and amino acid residues not involved directly in ligand binding but were pointed out as important for ligand binding in a subsequent molecular dynamics simulation (MD), displayed as thin sticks.

Figure 3. Distances between the selected ASP9521 atoms and substantial CBR1 enzymatic complex elements, measured during a 20 ns MD simulation: (a) distance between the amide oxygen atom of ASP9521 and Tyr193 side chain phenol oxygen atom of CBR1; (b) distance between an amide carbon atom of ASP9521 and C4 pyridine atom of CBR1.

Figure 4. Molecular interactions between ASP9521 and CBR1 amino acid residues observed during 20 ns of MD simulation (expressed as a percentage of the entire simulation time in which they have been observed). The colours of frames and arrows represent types of interactions: red—H-bond; green—water bridge; blue—π-π stacking; grey—π-cation; orange—other hydrophobic interactions.

Figure 5. (a) Exemplary binding mode of ASP9521 (orange) within the CBR1 catalytic site obtained via MD simulation (captured at 17.23 ns from the start of the simulation). Amino acid residues engaged in ligand binding throughout H-bonds (dotted yellow lines), π-π stacking (dotted blue lines), and pi-cation interaction (dotted grey lines) are displayed as thick sticks; NADPH and amino acid residues not involved directly in ligand binding in the selected timepoint but determined during the simulation as important for ligand binding to the active site are displayed as thin sticks; water molecules and water bridges are not displayed; (b) distances between the hydroxy group of ASP9521 and backbone oxygen atoms of Phe94 (blue line) and Met234 (red line), measured during a 20 ns MD simulation.

Figure 6. Effect of the combined administration of ASP9521 (25 μM) and DNR on the viability of A549 cells after 48 h of incubation compared to DNR without the addition of the ANT reductase inhibitor; * statistical significance −/+ ASP9521 at p < 0.05.

Figure 7. Effect of ASP9521 (25 μM) on DOX/DNR-induced damage prevention in H9c2 cells. H9c2 cells were preincubated in the presence of ASP9521 (25 μM) for 3 h. Then, DOX/DNR were added for the next 24 h. Viability of cells was determined using an MTT assay. The graph represents average cell viability expressed as percentage of control (no treated cells) ± SEM of three replications of three independent experiments; * statistical significance −/+ ASP9521 at p < 0.05.