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 MCF-7 mammary cell cultures, and in JAr choriocarcinoma cells. Recent investigations have focused on the use of mechanism-based inhibitors, such as 7alpha-substituted 1,4-androstadienediones, to biochemically probe the active site of aromatase. Inhibition kinetics were determined under initial velocity conditions using purified human placental cytochrome P450arom protein in a reconstituted system. Derivatives of 1,4-androstadiene-3,17-dione and 1,4,6-androstatriene-3,17-dione exhibited high affinity in the purified enzyme system. 7alpha-(4'-Amino)phenylthio-1,4-androstadiene-3,17-dione, abbreviated 7alpha-APTADD, demonstrated rapid time-dependent, first-order inactivation of reconstituted aromatase activity only in the presence of NADPH. The apparent K(inact) for 7alpha-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 concentration is 4.25 min. The values for the rate constant and half-time of inactivation are similar to those observed in the placental microsomal 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-IPTADD were followed by protein precipitation, solvent extraction, and column chromatography. Analysis of the isolated cytochrome P450arom by gel elctrophoresis and autoradiography demonstrated the presence of only one radioactive band, which corresponded to the protein staining band for cytochrome P450arom HPLC radiochromatographic analysis of the isolated cytochrome P450arom confirmed the presence of only one radioactive peak co-eluting with the u.v. peak for cytochrome P450arom Peptide mapping analysis by reverse-phase HPLC of digested inhibitor-cytochrome P450arom complex demonstrates that the radioactive inhibitor is covalently bound to a lipophilic fragment. In summary, these inhibitors produced enzyme-catalyzed inactivation of reconstituted aromatase activity, and radioiodinated 7alpha-IPTADD binds covalently to the cytochrome P450arom.