STEROIDAL INHIBITORS AS CHEMICAL PROBES OF THE ACTIVE-SITE OF AROMATASE

Androstenedione analogs containing 7alpha-substituents have proven to be potent inhibitors of aromatase in human placental microsomes, in MC F-7 mammary cell cultures, and in JAr choriocarcinoma cells. Recent in vestigations have focused on the use of mechanism-based inhibitors, su ch as 7alpha-substituted 1,4-androstadienediones, to biochemically pro be the active site of aromatase. Inhibition kinetics were determined u nder initial velocity conditions using purified human placental cytoch rome P450arom protein in a reconstituted system. Derivatives of 1,4-an drostadiene-3,17-dione and 1,4,6-androstatriene-3,17-dione exhibited h igh affinity in the purified enzyme system. (4'-Amino)phenylthio-1,4-a ndrostadiene-3,17-dione, abbreviated 7alpha-APTADD, demonstrated rapid time-dependent, first-order inactivation of reconstituted aromatase a ctivity only in the presence of NADPH. The apparent K(inact) for 7alph a-APTADD is 11.8 nM, the first-order rate of inactivation is 2.72 x 10 (-3) sec-1, and the half-time of inactivation at infinite inhibitor co ncentration is 4.25 min. The values for the rate constant and half-tim e of inactivation are similar to those observed in the placental micro somal assay system. Further studies were performed with radioiodinated 7alpha-(4'-iodo)phenylthio-1,4-androstadienedione, 7alpha-IPTADD, and the reconstituted aromatase system. Incubations with [I-125]7alpha-IP TADD were followed by protein precipitation, solvent extraction, and c olumn chromatography. Analysis of the isolated cytochrome P450arom by gel elctrophoresis and autoradiography demonstrated the presence of on ly one radioactive band, which corresponded to the protein staining ba nd for cytochrome P450arom HPLC radiochromatographic analysis of the i solated cytochrome P450arom confirmed the presence of only one radioac tive peak co-eluting with the u.v. peak for cytochrome P450arom Peptid e mapping analysis by reverse-phase HPLC of digested inhibitor-cytochr ome P450arom complex demonstrates that the radioactive inhibitor is co valently bound to a lipophilic fragment. In summary, these inhibitors produced enzyme-catalyzed inactivation of reconstituted aromatase acti vity, and radioiodinated 7alpha-IPTADD binds covalently to the cytochr ome P450arom.