Interaction of an estramustine photoaffinity analogue with cytoskeletal proteins in prostate carcinoma cells

To identify specific drug targets of the antimitotic drug estramustine, a photoaffinity analogue, 17-O-[[2-[3-(4-azido-3-[125I] iodophenyl)propionamido]ethyl]carbamyl]estradiol-3-N-bis(2- chloroethyl)carbamate, was synthesized and reacted in competition assays with cytoskeletal protein preparations. By attaching the photoaffinity ligand to the 17 beta-position of the steroid D-ring, the cytotoxic properties of the drug were maintained. In cytoskeletal protein preparations from human prostate carcinoma cells (DU 145) or a clonally selected, estramustine-resistant cell line (E4), the major microtubule-associated protein (MAP) present was MAP4. In both cytoskeletal fractions and reconstituted microtubules, 17-O-[[2-[3-(4-azido-3-[125I]iodophenyl)propionamido] ethyl]carbamyl]estradiol-3-N-bis(2-chloroethyl)carbamate bound to both MAP4 and tubulin. From competition assays, the apparent binding constant for MAP4 from DU 145 cells was 15 microM. Similar calculations for tubulin gave values of 13 microM (bovine brain), 19 microM (DU 145 wild-type cells), and 25 microM (E4 cells). The identification of these cytoskeletal proteins as specific drug targets provides a direct explanation for the antimicrotubule and antimitotic effects of estramustine.