Inhibition of Pancreatic α-amylase by Resveratrol Derivatives: Biological Activity and Molecular Modelling Evidence for Cooperativity between Viniferin Enantiomers

Abstract

To improve the current understanding of the role of stilbenoids in the management of diabetes, the inhibition of the pancreatic α-amylase by resveratrol derivatives was investigated. To approach in a systematic way, the mechanistic and structural aspects of the interaction, potential bioactive agents were prepared as single molecules, that were used for the biological evaluation of the determinants of inhibitory binding. Some dimeric stilbenoids-in particular, viniferin isomers- were found to be better than the reference drug acarbose in inhibiting the pancreatic α-amylase. Racemic mixtures of viniferins were more effective inhibitors than the respective isolated pure enantiomers at an equivalent total concentration, and displayed cooperative effects not observed with the individual enantiomers. The molecular docking analysis provided a thermodynamics-based rationale for the measured inhibitory ability and for the observed synergistic effects. Indeed, the binding of additional ligands on the surface of the alpha-amylase was found to decrease the dissociation constant of inhibitors bound to the active site of the enzyme, thus providing a mechanistic rationale for the observed inhibitory synergies.

Keywords: enzyme inhibition; food bioactives synergism; molecular docking; pancreatic alpha-amylase; resveratrol; stilbenoids.

Figure 1

Structures of the representative stilbenoid monomers (1–4) and dimers (5–8).

Figure 2

Synthesis of monomers 3–4 and dimers 5–8. Reagents and conditions: a) 1-IBX, DMF, r.t., 80 min; 2-NaBH₄ MeOH 0 °C, 62%; b) BBr₃, DCM, −35 °C to r.t., N₂, 22 h, 20%; c) Ac₂O, TEA, DCM, r.t., overnight, 92%; d) CAL-B (Candida antarctica lipase), toluene/n-BuOH, 40 °C, 6 h, 81%; e) IBX, DMF, r.t., 80 min, 42%; f) NH₂NH₂, MeOH, r.t., 30 min, 81%; g) 1-HRP, 30 min, 40 °C, acetone: Citrate buffer pH 5 (1:1); 2-H₂O₂, 15 min, 40 °C, overall yield 61%; h) 1-HRP, 30 min, 40 °C, acetone: Phosphate buffer pH 8 (1:1); 2-H₂O₂, 90 min, 40 °C, overall yield 21%; i) 1 - HRP, 30 min, 40 °C, acetone: Citrate buffer pH 5 (1:1); 2-H₂O₂, 90 min, 40 °C, overall yield 49%; j) FeCl₃ 6H₂O, MeOH : H₂O 1:1, 15%.

Figure 3

Chromatogram of the separation of two enantiomers of (±)-trans-δ-viniferin ((5), top) and (±)-trans-ε-viniferin ((6), bottom).

Figure 4

Inhibition of the pancreatic α-amylase by the various compounds presented in Figure 1, each at a 10 mM final concentration. The effect of a 1 mM acarbose is shown for comparison.

Figure 5

Residual enzyme activity in the presence of viniferin isomers, of their purified enantiomeric forms, and of their racemic forms. Final concentration of each inhibitor species (including acarbose and resveratrol) was 1 mM.

Figure 6

Concentration dependence of the α-amylase inhibition by purified enantiomeric forms of trans-δ-viniferin (5) and trans-ε-viniferin (6), and by equimolar mixtures of their purified enantiomers, simulating the presence of a racemic form of each species.

Figure 7

Hill plot for the inhibition of α-amylase by different enantiomeric forms of trans-δ-viniferin (5). Lines are the best fit to the experimental data, drawn by using a three-parameters Hill equation (y=a*x^n^H/([K_i]^n^H + x^n^H).

Figure 8

Top-scoring binding poses between the pig pancreatic α-amylase and the enantiomeric forms of the trans-viniferin species in the protein active site: A, (S, S)-trans-δ-viniferin; B, (R, R)-trans-δ-viniferin; C, (S, S)-trans-ε-viniferin; D, (R, R)-trans-ε-viniferin.

Figure 9

Ligand interaction diagram for the molecular docking top-scoring complexes between the pig pancreatic α-amylase and various enantiomeric forms of the trans-viniferin isomers in the protein active site. Interactions are recapitulated in the figure legend.

Figure 10

Top scoring pose of (R, R)-trans-δ-viniferin to the pig pancreatic alpha amylase with (S, S)-trans-δ-viniferin already bound to its active site.