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-diaphorase）生物激活，并且人类实体肿瘤中含有显著升高的NQO1水平，使NQO1成为开发抗肿瘤醌类药物的一个有吸引力的靶点。我们将重点研究两组以NQO1为导向的抗肿瘤醌a)以RH1为先导化合物的aziridinylbenzoquinones和b)以17-AAG为特征的苯醌类ansamycin Hsp90抑制剂。RH1是nq01的优良底物，目前正处于1期临床试验。NQO1显著增强rh1诱导的DNA交联、细胞周期阻滞、细胞凋亡和毒性。然而，尽管NQO1激活了RH1，但在NQO1无效的细胞系和异种移植物中仍然可以观察到细胞毒性作用。RH1对nqo1缺失细胞的毒性机制尚不明确，但已被认为与单电子还原酶P450R和b5R有关。使用仅在NQO1、P450R或b5R水平上不同的等基因细胞系统，我们将在体外和体内异种移植系统中定义NQO1依赖和NQO1独立的RH1毒性机制。阐明这些机制将有助于预测RH1在不同类型肿瘤中的疗效。胰腺肿瘤含有高水平的NQO1，因此我们将确定RH1在体外胰腺肿瘤系统以及体内胰腺异种移植和原位模型中的疗效。在临床试验中的第二类醌类抗肿瘤药物是苯醌类安纳霉素Hsp90抑制剂。抑制Hsp90导致一些致癌客户蛋白的异常折叠，使Hsp90成为一个有吸引力的靶标。我们使用重组Hsp90和细胞系统的数据表明，17-AAG的醌形式似乎不是有效的Hsp90抑制剂，并且通过NQO1代谢产生对苯二酚导致更有效的Hsp90抑制和细胞毒性。分子模拟研究证实，对苯二酚类ansamyins在Hsp90蛋白的活性atp酶位点具有更有利的结合能。因此，我们将在体外和体内测试假设，即安霉素的对苯二酚形式比其母体醌更有效的Hsp90抑制剂。我们还将研究对苯二酚类ansamyins的稳定性和氧化还原特性，并验证对苯二酚类新型前药形式导致有效抑制Hsp90和抗肿瘤活性的假设。我们的研究将产生可应用于RH1以及苯醌和对苯二酚类ansamyins正在进行和未来的临床研究的数据。

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.