The ABC-binding cassette proteins ABCB1 MDR1/P-glycoprotein (P-gp) cause resistance of cancer cells to drugs by causing their extrusion from such cells. Recently, a cytotoxic compound was found whose activity increased in direct proportion to this increased P-gp function (see reference in my other Annual Report 1 Z01 HL001003-35 LBC). This compound is a simple p-methoxyphenylisothiohydrazone of isatin, a compound normally found in the human body. I have chemically synthesized over 20 related compounds in an effort to determine the features responsible for its activity.? In addition I have examined the structure of Pgp in an effort to determine its glycosylation sites and the location of its disulfide bridges. ? ? Synthesis of NSC73306 was accomplished by simply heating commercially available p-methoxyphenylthiosemicarbazide with isatin in dilute acid and recrystallizing the product from ethanol. The 5-tritiated form was prepared in the same way by use of commercial prepared 5-tritiated isatin made from 5-bromoisatin. Functionality of both the isatin nucleus and the phenyl ring was studied by preparation of a series of compounds containing hydrogen, hydroxyl, nitro, bromo, chloro, and fluoro substituents on one side or the other of both rings. In addition several compounds were prepared by replacement of the isatin nucleus with 1- and 2-hydrindone. Additional compounds were made with allyl and hydrogen replacing the p-methoxyphenyl group and one compound was prepared with naphthisatin replacing isatin. In sum, the isatin nucleus was found to be essential for activity while more variation was allowed in the other aryl function. The addition of fluoro was found to be beneficial, perhaps by increasing solubility and other changes to increase this factor will be sought. An effort will be made to make a biotin-labelled substrate to isolate the natural proteins involved and a fluorescent derivative is under construction.? ? In related work, the P-gp protein has been examined by digestion and mass spectrometry in an attempt to identify the glycosylation sites and disulfide bridges involved. However, only about 30% of the protein was located even by use of chymotrypsin and efforts are underway to increase this by other digestion procedures.
| Ludwig, Joseph A; Szakacs, Gergely; Martin, Scott E et al. (2006) Selective toxicity of NSC73306 in MDR1-positive cells as a new strategy to circumvent multidrug resistance in cancer. Cancer Res 66:4808-15 |
