Figure 1.
The elucidation of β-glucogallin as an active component and inhibitor of AKR1B1 from E. officinalis.
(A) HPLC trace of pooled Sephadex® LH-20 fractions displaying activity against AKR1B1. The arrow indicates the fraction where activity against AKR1B1 appears to be localized. (B) The 1H NMR spectrum of the abundant active fraction from HPLC purification. The 13C spectrum and accurate mass data are shown in Figure S1 and S2 respectively. All of these data identify the active component as β-glucogallin. C) LC/MS/MS data indicate a peak at 687 m/z as a [2M aggregate + Na]. Therefore, the only compound present in this active fraction is β-glucogallin.
Figure 2.
Inhibition of AKR substrate reduction by β-glucogallin.
The IC50 was determined to be 58±3 µM (SEM) under saturating conditions of the shared substrate glyceraldehyde. β-glucogallin showed no activity against AKR1B10 and AKR1A1 at all concentrations tested under saturating conditions of glyceraldehyde as indicated. The IC50 was determined to be 17±1 µM under saturating conditions of the substrate glucose, and did not decrease significantly when the concentration of glucose was reduced 20-fold, IC50 = 13±1 µM). For all assays, inhibited enzyme activity was normalized to activity under the same conditions in the absence of inhibitor, assays were repeated in triplicate and error bars represent standard deviation from the mean. Where possible, GraphPad Prism software was used to fit normalized data to the enzyme inhibition model using nonlinear regression (method of least squares).
Figure 3.
Inhibition of AKR1B1 substrate oxidation by β-glucogallin.
A) Data were nonlinearly fit to the Michaelis-Menten model in the absence and presence of 0, 10, 20, and 40 µM β-glucogallin. B) Lineweaver-Burk plot of the same data, depicting increasing slope and y-intercept characteristic of noncompetitive inhibition. C) No significant changes were observed in the apparent Km values. D) A decreasing trend was observed in the apparent Vmax values, and the differences were found to be statistically significant as follows: *indicates P = 0.01, and ***indicates P = 0.0002. All assays were repeated in triplicate and error bars represent standard deviation from the mean. GraphPad Prism software was used to fit data to the Michaelis-Menten model using nonlinear regression (method of least squares) and to determine significance by column analysis of triplicates using the unpaired, two-tailed student’s t-test.
Figure 4.
Computational modeling studies with β-glucogallin and AKR1B1.
Modeling comparison between sorbinil (A) and β-glucogallin (B) showing ligands bound to AKR1B1. The solvent accessible surface depicts the active site anionic and specificity binding pockets, with expansion images to emphasize the detail of each pocket. The calculated binding energies for sorbinil and β-glucogallin are –32 kcal/mol and –44 kcal/mol, respectively. The active site interactions are summarized for sorbinil (C) and β-glucogallin (D) and were identified using Discovery Studio software v2.5.5 (Accelrys) and Ligand Scout v2.3. Based on the calculated binding energies and number of active site interactions β-glucogallin appears to bind AKR1B1 as effectively as the known active inhibitor sorbinil.
Figure 5.
β-glucogallin prevents sorbitol accumulation in transgenic (TG) human AKR1B1 expressing lenses ex-vivo.
Lenses extracted from TG-animals were cultured under hyperglycemic conditions in the presence or absence β-glucogallin (30 µM) or sorbinil (10 µM) over 72 hours. β-glucogallin showed potent activity preventing sorbitol accumulation by 73% as compared to untreated lenses. Comparably, the positive control sorbinil inhibited sorbitol accumulation by 97%. The amount of sorbitol in the non-transgenic controls was below the limit of detection. GraphPad Prism software was used to compare sorbitol accumulation in treated lenses to untreated controls by column analysis of duplicates using the unpaired, one-tailed student’s t-test. The * symbol denotes a statistically significant difference (P<0.05) from untreated TG lenses.