Three-dimensional quantitative structure-activity relationship studies on UGT1A9-mediated 3-O-glucuronidation of natural flavonols using a pharmacophore-based comparative molecular field analysis model

Abstract

Glucuronidation is often recognized as one of the rate-determining factors that limit the bioavailability of flavonols. Hence, design and synthesis of more bioavailable flavonols would benefit from the establishment of predictive models of glucuronidation using kinetic parameters [e.g., K(m), V(max), intrinsic clearance (CL(int)) = V(max)/K(m)] derived for flavonols. This article aims to construct position (3-OH)-specific comparative molecular field analysis (CoMFA) models to describe UDP-glucuronosyltransferase (UGT) 1A9-mediated glucuronidation of flavonols, which can be used to design poor UGT1A9 substrates. The kinetics of recombinant UGT1A9-mediated 3-O-glucuronidation of 30 flavonols was characterized, and kinetic parameters (K(m), V(max), CL(int)) were obtained. The observed K(m), V(max), and CL(int) values of 3-O-glucuronidation ranged from 0.04 to 0.68 μM, 0.04 to 12.95 nmol/mg/min, and 0.06 to 109.60 ml/mg/min, respectively. To model UGT1A9-mediated glucuronidation, 30 flavonols were split into the training (23 compounds) and test (7 compounds) sets. These flavonols were then aligned by mapping the flavonols to specific common feature pharmacophores, which were used to construct CoMFA models of V(max) and CL(int), respectively. The derived CoMFA models possessed good internal and external consistency and showed statistical significance and substantive predictive abilities (V(max) model: q(2) = 0.738, r(2) = 0.976, r(pred)(2) = 0.735; CL(int) model: q(2) = 0.561, r(2) = 0.938, r(pred)(2) = 0.630). The contour maps derived from CoMFA modeling clearly indicate structural characteristics associated with rapid or slow 3-O-glucuronidation. In conclusion, the approach of coupling CoMFA analysis with a pharmacophore-based structural alignment is viable for constructing a predictive model for regiospecific glucuronidation rates of flavonols by UGT1A9.

Fig. 1.

Backbone of flavonol structures (see Table 1 for the definitions of the substituents). Compared with flavonols, flavones lack 3-OH on the 2-phenylbenzopyran scaffold.

Fig. 2.

Structural modifications affect the K_m values of UGT1A9-mediated 3-O-glucuronidation. A, K_m values were decreased in the presence of 1, substitutions of -OH at positions of C3′ or C4′; 2, -OMe groups at C3′, C4′, C5, or C6; or 3, -Me group at C6. B, K_m values were increased by >1-fold with the additions of 2′-OH or 6-OH. The compound numbers are labeled above the abbreviated chemical names.

Fig. 3.

Scatter plot of V_max and K_m derived from kinetics of UGT1A9-mediated 3-O-glucuronidation of flavonols.

Fig. 4.

Two hypothetically distinct flavonol orientations that are required to generate 3-O-glucuronide (1 or 3) and 7-O-glucuronide (2 or 4). Arrows indicate the site of glucuronidation. In 3 and 4, G stands for site of glucuronidation. A, B, and C indicate A-ring, B-ring, and C-ring, respectively.

Fig. 5.

A, 3-OH-specific pharmacophore model composed of one glucuronidation site (red sphere with radius of 1 Å) and two aromatic regions (yellow spheres with radius of 1.2 and 1.5 Å, respectively). B, 3-OH-specific pharmacophore model superimposed with 3HF (compound 15).

Fig. 6.

Structural alignment for constructing the 3D QSAR CoMFA model generated from the 3-OH-specific pharmacophore.

Fig. 7.

Correlation between the experimental glucuronidation parameters and the predicted ones from the CoMFA models for the training and test sets (V_max, A; CL_int, B).

Fig. 8.

A, steric and electrostatic maps from the UGT1A9 CoMFA model of V_max. Morin (compound 19; left) and isorhamnetin (compound 17; right) are shown inside the field for reference. Green indicates areas in which bulky groups are sterically favorable for glucuronidation. Blue indicates areas in which electropositive atoms are favorable for glucuronidation. Red indicates areas in which electronegative atoms are favorable for glucuronidation. B, matching of the CoMFA to experimental data. I, bulky groups at C6 increased V_max values. II, 7-OH or 7-OMe increased V_max values. III, 2′-OMe increased V_max values. IV1, 3-OH or 3-OMe increased V_max values. IV2, 4-OH or 4-OMe decreased V_max values. The compound numbers are labeled above the abbreviated chemical names.

Fig. 9.

A, steric and electrostatic maps from the UGT1A9 CoMFA model of CL_int. Morin (compound 19; left) and dioxy (compound 16; right) are shown inside the field for reference. Green indicates areas in which bulky groups are sterically favorable for glucuronidation. Blue indicates areas in which electropositive atoms are favorable for glucuronidation. Red indicates areas in which electronegative atoms are favorable for glucuronidation. B, matching of the CoMFA to experimental data. I, effect of 5-OMe on CL_int was not well defined. II, bulky groups at C6 increased CL_int values. III, 7-OMe increased CL_int values. IV, effect of 2′-OMe on CL_int was not well defined. V1, 3-OH increased CL_int values. V2, effect of 4′-OMe on CL_int was not well defined. The compound numbers are labeled above the abbreviated chemical names.