INTERACTION OF AN ESTRAMUSTINE PHOTOAFFINITY ANALOG WITH CYTOSKELETALPROTEINS IN PROSTATE CARCINOMA-CELLS

To identify specific drug targets of the antimitotic drug estramustine , a photoaffinity analogue, arbamyl]estradiol-3-N-bis(2-chloroethyl)ca rbamate, was synthesized and reacted in competition assays with cytosk eletal protein preparations. By attaching the photoaffinity ligand to the 17 beta-position of the steroid D-ring, the cytotoxic properties o f the drug were maintained. In cytoskeletal protein preparations from human prostate carcinoma cells (DU 145) or a clonally selected, estram ustine-resistant cell line (E4), the major microtubule-associated prot ein (MAP) present was MAP4. In both cytoskeletal fractions and reconst ituted microtubules, carbamyl]estradiol-3-N-bis(2-chloroethyl)carbamat e bound to both MAP4 and tubulin. From competition assays, the apparen t binding constant for MAP4 from DU 145 cells was 15 mu M. Similar cal culations for tubulin gave values of 13 mu M (bovine brain), 19 mu M ( DU 145 wild-type cells), and 25 mu M (E4 cells). The identification of these cytoskeletal proteins as specific drug targets provides a direc t explanation for the antimicrotubule and antimitotic effects of estra mustine.