ABSTRACT:Testosterone and epitestosterone are endogenous steroids that differ in the configuration of the hydroxyl-bearing carbon at C-17. Testosterone is the predominant male sex hormone, whereas the role of epitestosterone is largely unclear. In humans, both androgens are excreted mainly as glucuronide conjugates and the urinary ratio of testosterone to epitestosterone (T/E), used to expose illicit testosterone abuse by male athletes, indicates the relative concentrations of the respective glucuronides. Some male athletes have T/E values greater than the accepted threshold value (4.0), even without testosterone abuse. We have analyzed athletes' urine samples and found that the main reason for such false-positive results in doping tests was a low epitestosterone glucuronide concentration not a high level of testosterone glucuronide. Sulfate conjugates of both testosterone and epitestosterone were also detected in the different urine samples. Glucuronidation assays with the 19 human UDP-glucuronosyltransferases (UGTs) of subfamilies UGT1A, UGT2A, and UGT2B revealed that UGT2B17 is the most active enzyme in testosterone glucuronidation. UGT2B17 does not glucuronidate epitestosterone, but inhibition studies revealed that it binds epitestosterone with affinity similar to that of testosterone. Epitestosterone glucuronidation is catalyzed mainly by UGT2B7, and the K m of this reaction is significantly lower than the K m of UGT2B17 for testosterone. Although UGT2B7 and UGT2B17 exhibited high, although converse, stereoselectivity in testosterone and epitestosterone glucuronidation, UGT2A1, an extrahepatic enzyme that is expressed mainly in the nasal epithelium, catalyzed the glucuronidation of both steroids at considerable rates and similar kinetics. The results shed new light on the substrate specificity and stereoselectivity of human UGTs.

The stereoselective binding and transformation of optically pure bicyclic alcohols by human UDP-glucuronosyltransferases from subfamily 2B were investigated. The enantiomers of 1-indanol, 1-tetralol, and 1-benzosuberol were synthesized by asymmetric Corey-Bakshi-Shibata reduction and subjected to glucuronidation assays. The alcohols studied were primarily glucuronidated by UGT2B7 and UGT2B17. The catalytic transformation by UGT2B17 was highly stereoselective, favoring conjugation of the (R)-enantiomers. UGT2B7, on the other hand, did not exhibit stereoselectivity toward 1-benzosuberol, the best substrate in this series. To assess binding affinities to the enzymes, the six different compounds were tested for their efficiency as inhibitors of either UGT2B7 or UGT2B17. The results of the latter analyses indicated that the affinities of both enantiomers of each pair towards UGT2B7 and UGT2B17 were of the same order of magnitude. Therefore, the findings of this study suggest that the spatial arrangement of the hydroxy group plays an important role in the glucuronic acid transfer reaction, but not necessarily in substrate binding to the UGTs.

The tricyclic sesquiterpenol (+)-longifolol served as a lead structure for the design of inhibitors of the human UDP-glucuronosyltransferase (UGT) 2B7. Twenty-four homochiral and epimeric longifolol derivatives were synthesized and screened for their ability to inhibit the enzyme. The absolute configuration at the stereogenic center C1' was determined by X-ray crystallography and 2D NMR spectroscopy (gHSQC, gNOESY). The phenyl-substituted secondary alcohol 16 b (beta-phenyllongifolol) displayed the highest affinity toward UGT2B7, and its inhibitory dissociation constant was 0.91 nM. The mode of inhibition was rapidly reversible and competitive. The inhibitor was not glucuronidated by UGT2B7 or other hepatic UGTs, presumably as a result of the high steric demand exerted by the phenyl group. Inhibition assays employing 14 other UGT isoforms suggested that inhibitor 16 b was highly selective for UGT2B7.

A set of 48 derivatives of the tricyclic sesquiterpenol alcohol isolongifolol was synthesized. The set comprised homochiral and diastereomeric alcohols, amines, chlorohydrins, as well as carboxylic acids, phosphonic acids, and their corresponding esters. The absolute configuration of the epimeric compounds was assigned by 2D NMR experiments [gradient heteronuclear single quantum correlation (gHSQC) and gradient nuclear Overhauser enhancement spectroscopy (gNOESY)] in agreement with crystallographic data. The tricyclic derivatives were assessed as inhibitors of the human UDP-glucuronosyltransferase (UGT) 2B7. The phenyl-substituted secondary alcohol 26b was the best inhibitor in this series and its competitive inhibition constant was 18 nM. Compound 26b was not glucuronidated by UGT2B7 and other hepatic UGT enzymes, presumably due to the high steric hindrance exerted by its bulky phenyl substituent. Its inhibitory activity toward 14 other UGT isoforms of subfamily 1A and 2B was determined, and the data indicated that the tricyclic secondary alcohol 26b was highly selective for UGT2B7 (true selectivity >1000).