Mechanistic insights into the specificity of human cytosolic sulfotransferase 2A1 (hSULT2A1) for hydroxylated polychlorinated biphenyls through the use of fluoro-tagged probes

Abstract

Determining the relationships between the structures of substrates and inhibitors and their interactions with drug-metabolizing enzymes is of prime importance in predicting the toxic potential of new and legacy xenobiotics. Traditionally, quantitative structure activity relationship (QSAR) studies are performed with many distinct compounds. Based on the chemical properties of the tested compounds, complex relationships can be established so that models can be developed to predict toxicity of novel compounds. In this study, the use of fluorinated analogues as supplemental QSAR compounds was investigated. Substituting fluorine induces changes in electronic and steric properties of the substrate without substantially changing the chemical backbone of the substrate. In vitro assays were performed using purified human cytosolic sulfotransferase hSULT2A1 as a model enzyme. A mono-hydroxylated polychlorinated biphenyl (4-OH PCB 14) and its four possible mono-fluoro analogues were used as test compounds. Remarkable similarities were found between this approach and previously published QSAR studies for hSULT2A1. Both studies implicate the importance of dipole moment and dihedral angle as being important to PCB structure in respect to being substrates for hSULT2A1. We conclude that mono-fluorinated analogues of a target substrate can be a useful tool to study the structure activity relationships for enzyme specificity.

Keywords: 4-Hydroxy-3,5-dichlorobiphenyl; Computational chemistry; F-tagged probes; Hydroxylated polychlorinated biphenyls; Polychlorinated biphenyls; QSAR; Sulfotransferase; hSULT2A1.

Fig 1. Sulfation of 4-OHPCB 14 and its fluorinated analogues by purified recombinant hSULT2A1

Assays were conducted at pH 7.0 with 200 μM PAPS as described in methods section. Data points are the means ± standard error of duplicate reactions, and curves represent the fit of the data to a substrate inhibition equation (v = V_max/(1+ (K_m/[S])+[S]/K_i)), where S is substrate concentration, v is the initial velocity, K_m is the Michaelis constant, K_i is the substrate inhibition constant and V_max is the maximal velocity

Fig 2. A strong correlation between K_i and V_max with dipole moment is observed in 4-OH PCB 14 and its 4 fluorinated analogues

A: Visual demonstration of the correlation between the dipole moment of the four fluorinated 4-OH PCB 14 analogues and the parent 4-OH PCB 14 with their respective K_i (substrate inhibition constant) and V_max (maximal velocity) parameters. B: Visual demonstration of the correlation between the dihedral angle of the four fluorinated 4-OH PCB 14 analogues and the parent 4-OH PCB 14 with their respective Kcat/Km parameters.

Fig 3. Summary of correlation findings

A positive correlation was observed between V_max and dipole moment, while a negative correlation was observed between K_i and dipole moment. Other chemical parameters calculated for these substrates did not have significant correlations with kinetic constants for the enzyme-catalyzed reaction.