Rodney C. Schnur

P185ERBB2 BINDS TO GRP94 IN VIVO : DISSOCIATION OF THE P185ERBB2/GRP94 HETEROCOMPLEX BY BENZOQUINONE ANSAMYCINS PRECEDES DEPLETION OF P185ERBB2

Treatment of SKBr3 cells with benzoquinone ansamycins, such as geldanamycin (GA), depletes p185, the receptor tyrosine kinase encoded by the erbB2 gene. In the same cells, a biologically active benzoquinone photoaffinity label specifically binds a protein of about 100 kDa, and the ability of various GA derivatives to reduce the intracellular level of p185 correlates with their ability to compete with the photoaffinity label for binding to this protein. In this report, we present evidence that the 100-kDa ansamycin-binding protein is GRP94. Membrane-associated p185 exists in a stable complex with GRP94. GA binding to GRP94 disrupts this complex, leading to degradation of pre-existing p185 protein, and resulting in an altered subcellular distribution of newly synthesized p185.

Depletion of p185erbB2, Raf-1 and mutant p53 proteins by geldanamycin derivatives correlates with antiproliferative activity

Purpose: Recently, it has been shown that geldanamycin (GA), a benzoquinone ansamycin, is able to deplete mutant p53, p185erbB2 and Raf-1 proteins in cancer cells. However, the relationship between these activities of GA and its antiproliferative activity is not clear. Here we investigated the effects of 28 GA derivatives in SKBr3, a human breast cancer cell line. Methods: We performed Western blot analysis of Raf-1, p185erbB2 and mutant p53 proteins following drug treatment and correlated these findings with the cytotoxicity of the various GA derivatives. Results: We found that downregulation of Raf-1, p185erbB2 and mutant p53 proteins was correlated. Thus, a drug that was active against one oncoprotein was equally active against the two others. Inactive derivatives were identified by their inability to downregulate these oncoproteins, even at a high dose (2 μM). These inactive drugs also had no or minimal antiproliferative activity (IC50 > 3 μM). All other analogs (at a concentration of 2 μM) downregulated p53, p185erbB2, and Raf-1, and also displayed cytotoxicity (IC50 in the range 6–600␣nM). This category of drugs was further divided into more- and less-active agents by testing at lower doses (40 nM). The drugs that remained active against their molecular targets had an IC50 for antiproliferative activity of less than 40 nM. Maximal effects on mutant p53, p185erbB2 and Raf-1 were observed at doses that were 4–5 times greater than the cytotoxic IC50. Conclusions: These findings suggest that GA and its derivatives are cytostatic/cytotoxic at concentrations that also downregulate Raf-1, p185erbB2 and mutant p53, and raise the possibility that depletion of these proteins and the antiproliferative activities of GA have a common mechanism.

1,2,3,4-tetrasubstituted isoquinoline acetic acids

Abstract Differentially tetrasubstituted isoquinolines are now conveniently prepared by rearrangement of the readily obtainable indanones.