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 ) ϭ 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 M, 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 ϭ 0.738, r 2 ϭ 0.976, r pred 2 ϭ 0.735; CL int model: q 2 ϭ 0.561, r 2 ϭ 0.938, r pred 2 ϭ 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.

Flavonoid interactions during digestion, absorption, distribution and metabolism: a sequential structure–activity/property relationship-based approach in the study of bioavailability and bioactivity

“…However, a report that specifically modeled UGT1A8 or 1A10 could not be found in literature. Wu et al (2011c) also argued that it is necessary to model regiospecific glucuronidation individually to obtain a global glucuronidation rate, considering the many potential glucuronidation sites in the flavonoid structure. However, the existence of an ''accurate prediction'' model for glucuronidation, which do not model regiospecific glucuronidation , may indicate that modeling regiospecific glucuronidation has not been a successful strategy and may require further investigation and validation.…”

“…(7) Increasing the number of hydroxyl groups on both A and B rings (except for 4 0 -OH moiety) enhances the glucuronidation activity of flavones, while adding a 3-OH hydroxyl group does not seem to have an effect (Lewinsky et al, 2005;Wong et al, 2009). Using UGT1A9, one of the 9 UGT1A isoforms that has been reported to have a higher glucuronidation activity to flavonoids at the C3-OH position, Wu et al (2011c) developed a predictive 3D QSAR model with good internal and external predictabilities using the V max and CL int as dependent variables (Q 2 ¼ 0.74, R 2 training ¼ 0.98, R 2 test ¼ 0.74; Q 2 ¼ 0.56, R 2 training ¼ 0.94, R 2 test ¼ 0.63, respectively). In this model, training molecules were first aligned using a pharmacophore model that consisted of the glucuronidation site (the C3-OH moiety) and two aromatic rings (A and B rings).…”

Predicting substrate selectivity between UGT1A9 and UGT1A10 using molecular modelling and molecular dynamics approach

“…The first homology model for UGT1A1 was published in 2007 using plant protein UGT71G1 as a template in which some central loops in the NTD were missing. [71] In 2010, homology model of UGT1A1 was developed using the crystal structure of human UGT2B7 CTD and three templates (PDB code: 2VCE, [72] 1IIR [73] and 2IYA [23]) for the NTD [27]. This model showed the previously missing structural components, the envelop helices and trans-membrane helix.…”

“…Later in 2011, another group employed VvGT1 from red grape (PDB code: 2C1Z [48]) as the template to prepare a homology model of UGT1A9. [71] In the present study, the modelling of UGT enzyme was performed using multiple templates because of unavailability of a single template with good identity. BLAST and PSI-BLAST search retrieved the hits that are biased towards CTD only.…”

Absolute quantification of UGT1A1 in various tissues and cell lines using isotope label-free UPLC–MS/MS method determines its turnover number and correlates with its glucuronidation activities

“…During the drug development process, microsomes from various tissues (e.g., liver and intestine), overexpressed insect Sf9 cells, and cell line culture models are commonly used to study the in vitro glucuronidation [4, 8–11]. It is currently not possible to normalize the expression levels of UGT1A1 in different model system because the antibody available for UGT1A1 does not always have high fidelity, and certainly not highly accurate for quantitative determination.…”