cv buffer a akr1c3  (Bio-Rad)

Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

Article Title: The structural basis of aldo-keto reductase 1C3 inhibition by 17α-picolyl and 17( E )-picolinylidene androstane derivatives

doi: 10.1080/14756366.2025.2551979

Figure Lengend Snippet: AKR1C3 reduction of 9,10-phenanthrenequinone (PQ) in the absence or presence of either test compounds ( 1–8 ) or ibuprofen (IBU) at a final concentration of 6.25 µM. The effect of test compounds or ibuprofen on AKR1C3 activity was measured by monitoring the decrease in NADPH absorbance at 340 nm over time. Data shown represent the mean of three experiments, fit by linear regression.

Article Snippet: The column was washed in 20 CV buffer A. AKR1C3 was then eluted in 5 CV buffer A with 500 mM imidazole and applied to a Bio-Rad P10 desalting column for buffer exchange into 25 mM sodium phosphate, 150 mM NaCl, 10% glycerol, pH 8.0.

Techniques: Concentration Assay, Activity Assay

Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

Article Title: The structural basis of aldo-keto reductase 1C3 inhibition by 17α-picolyl and 17( E )-picolinylidene androstane derivatives

doi: 10.1080/14756366.2025.2551979

Figure Lengend Snippet: Effect of increasing concentrations of compounds 1 and 7 on AKR1C3 activity, represented by the decrease in NADPH absorption at A 340nm over time. Dose-response curves were obtained by measuring the effect of increasing concentrations of compound 1 (panel A) and 7 (panel B), from 0 to 500 µM (0.7, 2, 3, 7, 12, 25, 50, 100, 200, and 500 µM in 2% DMSO) on PQ reduction by AKR1C3. The concentration of PQ was constant at 0.39 µM, near the Km value obtained for AKR1C3 under the assay conditions used . IC50 values were calculated by fitting the data to a four-parameter logistic sigmoidal dose-response curve (GraphPad). All results shown are mean values from three experiments.

Article Snippet: The column was washed in 20 CV buffer A. AKR1C3 was then eluted in 5 CV buffer A with 500 mM imidazole and applied to a Bio-Rad P10 desalting column for buffer exchange into 25 mM sodium phosphate, 150 mM NaCl, 10% glycerol, pH 8.0.

Techniques: Activity Assay, Concentration Assay

Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

Article Title: The structural basis of aldo-keto reductase 1C3 inhibition by 17α-picolyl and 17( E )-picolinylidene androstane derivatives

doi: 10.1080/14756366.2025.2551979

Figure Lengend Snippet: X-ray structure of AKR1C3 in complex with NADP + and compound 7 . Chain B of the structure of AKR1C3 is shown in complex with cofactor NADP + (yellow sticks) and compound 7 (green sticks).

Article Snippet: The column was washed in 20 CV buffer A. AKR1C3 was then eluted in 5 CV buffer A with 500 mM imidazole and applied to a Bio-Rad P10 desalting column for buffer exchange into 25 mM sodium phosphate, 150 mM NaCl, 10% glycerol, pH 8.0.

Techniques:

Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

Article Title: The structural basis of aldo-keto reductase 1C3 inhibition by 17α-picolyl and 17( E )-picolinylidene androstane derivatives

doi: 10.1080/14756366.2025.2551979

Figure Lengend Snippet: Molecular interactions between compound 7 and AKR1C3 ligand binding site. Panel A: Compound 7 (green) interacts with AKR1C3 residues (white sticks) corresponding to the steroid channel (W227, L54), SP1 (F306, F311, Y319) and SP2 (W227). The 3-oxime group of compound 7 forms a direct hydrogen bond between the oxime oxygen and O2N from NADP+ and a water-mediated hydrogen bond with NADP + . This same water also mediates a hydrogen bond between the oxime nitrogen with the main chain nitrogen of Q222. The C17 picolyl group interacts with SP1 (F311, Y319) and SP2 (W227) residues as well as with M120. Panel B: 2D representation of molecular interactions between AKR1C3 and compound 7 . Residues involved in compound 7 binding were identified using an automated program, LigPlot. Residues within 4 Å of compound 7 are shown.

Article Snippet: The column was washed in 20 CV buffer A. AKR1C3 was then eluted in 5 CV buffer A with 500 mM imidazole and applied to a Bio-Rad P10 desalting column for buffer exchange into 25 mM sodium phosphate, 150 mM NaCl, 10% glycerol, pH 8.0.

Techniques: Ligand Binding Assay, Binding Assay

Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

Article Title: The structural basis of aldo-keto reductase 1C3 inhibition by 17α-picolyl and 17( E )-picolinylidene androstane derivatives

doi: 10.1080/14756366.2025.2551979

Figure Lengend Snippet: Comparison of structures of AKR1C3 in complex with compound 7 and ibuprofen. The structure of AKR1C3- 7 (blue) was aligned with the structure of AKR1C3-ibuprofen (white, PDB: 3R8G) with an RMSD of 0.254 Å. Compound 7 is shown in green and ibuprofen in orange. Select ordered waters (red for AKR1C3- 7 , white for AKR1C3-ibuprofen), the NADP + cofactor (yellow for AKR1C3- 7 , white for AKR1C3-ibuprofen) and the relative positions of select residues important in ligand binding (W227) and catalysis (Y55, H117) are shown. Hydrogen bonds are shown in blue for AKR1C3-7 and red for AKR1C3-ibuprofen.

Article Snippet: The column was washed in 20 CV buffer A. AKR1C3 was then eluted in 5 CV buffer A with 500 mM imidazole and applied to a Bio-Rad P10 desalting column for buffer exchange into 25 mM sodium phosphate, 150 mM NaCl, 10% glycerol, pH 8.0.

Techniques: Comparison, Ligand Binding Assay

Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

Article Title: The structural basis of aldo-keto reductase 1C3 inhibition by 17α-picolyl and 17( E )-picolinylidene androstane derivatives

doi: 10.1080/14756366.2025.2551979

Figure Lengend Snippet: Comparative molecular docking of non-steroidal anti-inflammatory drugs (NSAIDs) using aligned structures of AKR1C3 in complex with inhibitors ibuprofen (PDB 3R8G) or flufenamic acid (PDB 1S2C) as “receptors” in Autodock Vina. Panel A: Redocking of ibuprofen into the native structure of AKR1C3-ibuprofen. The docked pose of ibuprofen (magenta sticks) superimposes onto the experimental binding pose of ibuprofen (green sticks) with an RMSD of 0.551 Å. Panel B: Cross-docking of ibuprofen (magenta sticks) against AKR1C3-flufenamic acid (green sticks). The cross-docked pose of ibuprofen superimposes onto the experimental binding pose of ibuprofen in AKR1C3-ibuprofen with an RMSD of 0.817 Å. Panel C: Cross-docking of flufenamic acid (magenta sticks) against AKR1C3-ibuprofen (green sticks). The cross-docked pose of flufenamic acid superimposes onto the native binding pose of flufenamic acid in AKR1C3-flufenamic acid with an RMSD of 1.549 Å. Panel D: Redocking of flufenamic acid into the native structure of AKR1C3-flufenamic acid. The docked pose of flufenamic acid (magenta sticks) superimposes onto the experimental binding pose of flufenamic acid (green sticks) with an RMSD of 0.653 Å. RMSD values were calculated using Autodock Tools.

Article Snippet: The column was washed in 20 CV buffer A. AKR1C3 was then eluted in 5 CV buffer A with 500 mM imidazole and applied to a Bio-Rad P10 desalting column for buffer exchange into 25 mM sodium phosphate, 150 mM NaCl, 10% glycerol, pH 8.0.

Techniques: Binding Assay

Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

Article Title: The structural basis of aldo-keto reductase 1C3 inhibition by 17α-picolyl and 17( E )-picolinylidene androstane derivatives

doi: 10.1080/14756366.2025.2551979

Figure Lengend Snippet: Molecular docking results from Autodock Vina compared with the crystal structure of AKR1C3-7. The top ranking binding geometry predicted by Autodock Vina is shown for compound 1 (panel A) and compound 2 (panel B) in comparison with the X-ray structure of compound 7 (green sticks) in complex with AKR1C3. The NADP + cofactor is shown as yellow sticks. Selected amino acid residues involved in binding to compound 7 are shown as white sticks and labelled. Hydrogen bonds formed by the C3-oxime group of compound 7 are shown as red dashed lines.

Article Snippet: The column was washed in 20 CV buffer A. AKR1C3 was then eluted in 5 CV buffer A with 500 mM imidazole and applied to a Bio-Rad P10 desalting column for buffer exchange into 25 mM sodium phosphate, 150 mM NaCl, 10% glycerol, pH 8.0.

Techniques: Binding Assay, Comparison