New GI-specific Histone Deacetylase Inhibitors

描述(申请人提供)：拟议研究的主要目标是开发新的组蛋白脱乙酰酶(HDAC)抑制剂，对人体胃肠道和近端器官，如前列腺、胰腺、肝脏和肾脏具有离散的选择性。这一目标得到了两个关键发现的推动。首先，结直肠癌是美国男性和女性的第二大癌症死亡原因和第三大癌症死亡原因。第二，已有研究表明，西兰花中的主要化学预防药物葡萄糖萝卜素(GPN)经历了依赖于芥子酸酶的代谢，从而提供了一种弱的HDAC抑制剂。强有力的证据表明，GPN衍生的HDAC抑制剂是半胱氨酸偶联物，结构类似于下面所示的C。HDAC抑制代谢物很可能是有益的化学预防效果的原因，不仅与食用西兰花有关，而且与摄入许多其他蔬菜有关，这些蔬菜提供如下所示的能够在胃肠道局部代谢的硫代葡萄糖苷。我们的目标是产生新的物质，这些物质与葡萄糖胺的新作用机制相同，但却是更有效的HDAC抑制剂，因此也是更有用的化学预防药物。我们将首先制备一些来自GPN的半胱氨酸偶联物的衍生物，以验证半胱氨酸衍生的羧酸盐负责HDAC活性部位内的锌离子配位(从而导致酶失活)的假设。然后，我们将从伯胺库中制备ITC类似物(如下图所示的B)和半胱氨酸-1标记的偶联物(C型)。该文库每个成员的ITC和半胱氨酸结合物将在无细胞分析(Fluor De Lys)和人结肠癌细胞(HCT116)中测试HDAC抑制。显示抗HDAC活性的试剂将是进一步合成和生化研究的候选对象。具体地说，将构建这些活性物质的硫代葡萄糖苷前体(如下所示的形式A)。生成的硫代葡萄糖苷通过芥子酶触发转化为相应ITC的能力将通过自动反相高效液相色谱法进行评估，如果适用，我们将在无细胞检测和HCT 116细胞中检测所述芥子酶激活剂的HDAC抑制谱。

DESCRIPTION (provided by applicant): The primary objective of the proposed research is to develop new inhibitors of histone deacetylase (HDAC) with discrete selectivity for the human GI tract and proximal organs such as prostate, pancreas, liver and kidney. This goal is promoted by two key findings. First, colorectal cancer is the second leading cause of death from cancer in males and the third in females in the U.S. Secondly, it has been shown that glucoraphanin (GPN), the primary chemopreventive agent in broccoli undergoes myrosinase-dependent metabolism to afford a weak HDAC inhibitor. Evidence strongly suggests the GPN-derived HDAC inhibitor to be a cysteine conjugate similar in structure to C shown below. It is highly likely that the HDAC inhibiting metabolite is responsible for the beneficial chemopreventive effects associated not only with broccoli consumption but also the ingestion of many other vegetables that provide glucosinolates capable of GI-localized metabolism as shown below. Our goal is to generate new substances that share glucoraphanin's novel mechanism of action but are more potent HDAC inhibitors and thus more useful chemopreventive agents. We will first prepare a number of derivatives of the cysteine-conjugate derived from GPN to verify the hypothesis that the cysteine-derived carboxylate is responsible for Zn2+ coordination (and hence, enzyme inactivation) within the HDAC active site. From a library of primary amines, we will then prepare ITC analogs (like B shown below) and cysteine-1 inked conjugates (of type C). ITC and cysteine conjugates for each member of the library will be tested for HDAC inhibition in cell free assays (Fluor de Lys) and human colon cancer cells (HCT116). Agents that display anti-HDAC activity will be candidates for further synthetic and biochemical study. Specifically, glucosinolate precursors (of form A shown below) to these active agents will be constructed. The ability of resulting glucosinolates to undergo myrosinase-triggered conversion to the corresponding ITC will be assessed by automated reverse phase HPLC and, if applicable, we will examine the HDAC inhibition profile of said myrosinase-activated agents in both cell free assays and in HCT 116 cells.