Inhibition of rat liver steroid 5 alpha-reductase by 3-androstene-3-carboxylic acids: mechanism of enzyme-inhibitor interaction.
The interactions of a series of newly discovered inhibitors of delta 4-3-oxo-steroid 5 alpha-reductase (SR; EC 1.3.1.30), the 3-androstene-3-carboxylic acids (steroidal acrylates), have been studied by using a solubilized rat liver enzyme preparation. As exemplified by one member of this series, 17 beta-[N,N-diisopropyl-carbamoyl)androst-3,5-diene-3-carboxylic acid (1a), the dead-end inhibition patterns of selected compounds in this class are best evaluated by a linear uncompetitive kinetic model versus either substrate, testosterone (T) or NADPH. These results were interpreted within the context of the preferentially ordered kinetic mechanism for rat liver SR to arise from the association of inhibitor to the binary complex of enzyme and NADP+. This proposed inhibition mechanism was supported by data from double-inhibition experiments implicating the synergistic binding of steroidal acrylate and NADP+ to SR. Further evidence for the preferential formation of this ternary complex was obtained from filtration binding assays with [3H]-1a, where radioligand association to protein was greatly enhanced in the presence of NADP+. The amount of [3H]-1a binding to protein was proportional to the specific activity of SR in the enzyme preparations, and the estimated dissociation constant from binding data by Scatchard analysis (Kd = 25 nM) was comparable to the inhibition constants estimated for SR activity (Ki = 12-26 nM). From the pH profile for inhibition of the solubilized liver SR with 1a, it is proposed that the anion of the steroidal acrylate (pK1 = 4.7 +/- 0.2) is the active inhibitory species, coordinating to a protonated active site functionality (pK2 = 7.5 +/- 0.1). On the basis of data from similar experiments with structural analogues of 1a, the determinants for binding recognition and inhibitory potency are compared to structural features of the putative enzyme-bound intermediate states. These compounds represent a potential therapeutic alternative in the treatment of 5 alpha-dihydrotestosterone specific androgen dependent disease states.
Levy
M A
MA
Department of Medicinal Chemistry, Smith Kline & French Laboratories, King of Prussia, Pennsylvania 19406.
Brandt
M
M
Heys
J R
JR
Holt
D A
DA
Metcalf
B W
BW
D058891:5-alpha Reductase Inhibitors; D000179:Acrylates / Q000378:metabolism; D000730:Androstadienes / Q000494:pharmacology*; D000736:Androstenes / Q000494:pharmacology*; D000818:Animals; D002384:Catalysis; D002850:Chromatography, Gel; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver / Q000187:drug effects / Q000201:enzymology*; D008297:Male; D009249:NADP / Q000378:metabolism; D011467:Prostate / Q000187:drug effects / Q000201:enzymology; D051381:Rats; D012995:Solubility
D058891:5-alpha Reductase Inhibitors; D000179:Acrylates; D000730:Androstadienes; D000736:Androstenes; C064406:17-(N,N,-diisopropylcarbamoyl)androst-3,5-diene-3-carboxylic acid; D009249:NADP