Biochemical and Molecular Studies on DT Diaphorase

描述（由申请人提供）：我们已经证明醌类可以被NQO1（DT-心肌黄酶）生物活化，并且人实体瘤含有显著升高的NQO1水平，使得NQO1成为开发抗肿瘤醌类的有吸引力的靶标。我们将集中在两组NQO 1导向的抗肿瘤醌类化合物a）以RH 1为先导化合物的氮丙啶基苯醌和B）以17-AAG为代表的苯醌安莎霉素Hsp90抑制剂。RH1是NQO1的优良底物，目前正处于1期临床试验中。NQO1显著增强RH1诱导的DNA交联、细胞周期阻滞、凋亡和毒性。然而，尽管NQO1激活RH1，但在NQO1无效细胞系和异种移植物中仍可观察到细胞毒性作用。NQO1-null细胞中RH1毒性的机制尚不明确，但已被认为取决于单电子还原酶P450 R和b5 R。使用仅在NQO1、P450 R或b5 R水平上不同的同基因细胞系统，我们将定义NQO1依赖性和NQO1独立性的RH1毒性机制，包括体外和体内异种移植系统。阐明这些机制将允许预测RH1在不同类型肿瘤中的疗效。胰腺肿瘤含有高水平的NQO1，因此我们将在体外和胰腺异种移植物和原位模型中定义RH1在胰腺肿瘤系统中的功效。临床试验中的第二类醌类抗肿瘤剂是苯醌安莎霉素Hsp90抑制剂。Hsp90的抑制导致许多致癌客户蛋白的异常折叠，使得Hsp90成为有吸引力的靶标。我们使用重组Hsp90和细胞系统的数据表明，醌形式的17-AAG似乎不是活性Hsp90抑制剂，通过NQO1代谢产生的氢醌导致更有效的Hsp90抑制和细胞毒性。分子模拟研究已经证实了对苯二酚安莎霉素在Hsp90蛋白的活性ATP酶位点的更有利的结合能。因此，我们将在体外和体内检验安莎霉素的氢醌形式比它们的母体醌类更有效的Hsp90抑制剂的假设。我们还将研究氢醌安莎霉素的稳定性和氧化还原性质，并测试新的前药形式的氢醌导致有效的Hsp90抑制和抗肿瘤活性的假设。我们的研究将产生的数据，可以应用于正在进行的和未来的临床研究RH1和苯醌和氢醌安莎霉素。

DESCRIPTION (provided by applicant): 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.