Active-site-directed inactivation of aromatase from human placental microsomes by brominated androgen derivatives

Abstract

Several brominated androgen derivatives were tested for their ability to inactivate microsomal aromatase from term human placenta. In the experimental protocol, the microsomal homogenate was incubated either with androstenedione or a brominated derivative of androstenedione (16alpha-bromo-6-ketoandrostenedione, 16alpha-bromoandrostenedione, 7alpha-(3'-bromoacetoxypropyl)androstenedione, 6alpha-bromoandrostenedione, or 6beta-bromoandrostenedione) and reduced nicotinamide adenine dinucleotide phosphate in a nitrogen saturated buffer composed of glycerol, ethylenediaminetetraacetic acid, and dithiothreitol in tris(hydroxymethyl)aminomethane hydrochloride (pH 7.4) under nitrogen at 4 degrees C with shaking. After the incubation period, the microsomes were recovered by centrifugation and washed once before determining aromatase specific activity. The brominated androgen derivatives which inactivated aromatase were 7alpha-(3'-bromoacetoxypropyl)androstenedione and 6alpha-bromoandrostenedione. The structures of 6alpha- and 6beta-bromoandrostenedione were unequivocally established by single crystal x-ray diffraction techniques. The extent of the enzyme inactivation by 6alpha-bromoandrostenedione was linearly proportional to the logarithm of its concentration. The evidence that this inactivation occurs at the aromatase active site is that androstenedione, when coincubated with 6alpha-bromoandrostenedione, protected aromatase from this inactivation. Progesterone provided much less protection than androstenedione. Furthermore, both 6alpha- and 6beta-bromoandrostenedione are competitive inhibitors of androstenedione aromatization, as determined by a Lineweaver-Burk plot, and 6alpha-bromoandrostenedione gives the same type I cytochrome P-450 binding spectrum with placental microsomes as androstenedione. These data suggest that 6alpha-bromandrostenedione is effective as an active-site-directed inhibitor of placental microsomal aromatase.

Synthesis and Biochemical Properties of 6-Bromoandrostenedione Derivatives with a 2,2-Dimethyl or 2-Methyl Group as Aromatase Inhibitors

“…The 2,2-dimethyl-6b-and 6a-bromo steroids 4 and 5 were extremely powerful, and their affinity to the active site was about 3 times higher than that of the natural substrate AD (apparent K i for 4 and 5 vs. apparent K m for AD, 14 and 10 nM vs. 33 nM). Introduction of a 2,2-dimethyl function to 6b-bromoAD (1) markedly increased the binding affinity to the active site (K i : 68 nM 15) vs. 14 nM for 1 vs. 4), as observed in a series of AD, 19) 4-hydroxyAD, 19) and 6-oxoAD, 20) 1880 Vol. 27, No.…”

“…Treatment of 2,2-dimethylAD (3) with NBS in the presence of benzoylperoxide according to the previous method, 15) used for the preparation of 6b-bromoAD (1), gave the 6b-bromo derivative 4 in a good yield (Fig. 2).…”

“…The stereochemistry at C-6 of the products was confirmed by 1 H-NMR spectrometry according to the previous reports. 15,27) We have previously reported that 2a-methylAD (6) is a good substrate for aromatase, producing 2-methyl estrogen 21) ; thus, we next focused on the synthesis of the 6b- Fig. 2.…”

“…and 6b-bromo-ADs 15) are the earliest discovered suicide substrates. The three former steroids act by oxygenation of the 19-angular methyl moiety in the inactivation of human placental aromatase in a suicide manner, 12,[16][17][18] whereas the inactivation mechanism for the 6b-bromo inhibitor 1 (Fig.…”

Gas Chromatography-Mass Spectrometric Determination of Activity of Human Placental Aromatase Using 16.ALPHA.-Hydroxyandrostenedione as a Substrate.

“…16a-OH AD, 12,13) 4-hydroxyandrostenedione (4-OH AD), 14) and 6-oxoandrostenedione (6-oxo AD) 15) were synthesized by previously reported methods. [2,4,6,6, Enzyme Preparation Human term placental microsomes (sedimented after 60 min at 105000 g) were obtained as described by Ryan.…”