We have shown that quinones can be bioactivated by NQO1 (DT-diaphorase) and that human solid tumors contain markedly elevated NQO1 levels making NQO1 an attractive target for the development of antitumor quinones. We will focus on two groups of NQO1 directed antitumor quinones a) aziridinylbenzoquinones with RH1 as the lead compound and b) the benzoquinone ansamycin Hsp90 inhibitors typified by 17-AAG. RH1 is as an excellent substrate for NQO1 and is currently in phase 1 clinical trials. NQO1 markedly potentiates RH1-induced DNA crosslinking, cell cycle arrest, apoptosis and toxicity. However, although NQO1 activates RH1, a cytotoxic effect could still be observed in NQO1 null cell lines and xenografts. The mechanisms of RH1 toxicity in NQO1-null cells are undefined but have been suggested to depend on the one electron reductases, P450R and b5R. Using isogenic cell systems differing only in NQO1, P450R or b5R levels, we will define NQO1-dependent and NQO1-independent mechanisms of RH1 toxicity both in-vitro and in xenograft systems in-vivo. Elucidation of these mechanisms will allow predictions of the efficacy of RH1 in different types of tumors. Pancreatic tumors contain high levels of NQO1 and we will therefore define the efficacy of RH1 in pancreatic tumor systems both in-vitro and in both pancreatic xenograft and orthotopic models in-vivo. A second class of quinone antitumor agents in clinical trial are the benzoquinone ansamycin Hsp90 inhibitors. Inhibition of Hsp90 results in the aberrant folding of a number of oncogenic client proteins making Hsp90 an attractive target. Our data using both recombinant Hsp90 and cellular systems has shown that the quinone form of 17-AAG does not appear to be the active Hsp90 inhibitor and that generation of the hydroquinone via NQO1 metabolism results in more potent Hsp90 inhibition and cytotoxicity. Molecular modeling studies have confirmed more favorable binding energies of the hydroquinone ansamycins in the active ATPase site of the Hsp90 protein. We will therefore test the hypothesis both in-vitro and in-vivo that the hydroquinone forms of the ansamycins are more active Hsp90 inhibitors than their parent quinones. We will also examine the stability and redox properties of the hydroquinone ansamycins and test the hypothesis that novel prodrug forms of the hydroquinones lead to effective Hsp90 inhibition and antitumor activity. Our studies will generate data that can be applied to ongoing and future clinical studies of RH1 and both benzoquinone and hydroquinone ansamycins. ? ? ?
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