Development of New GI-specific Histone Deacetylase Inhi*

描述（由申请人提供）：拟议研究的主要目的是开发新的组蛋白脱乙酰酶（HDAC）抑制剂，对人胃肠道和近端器官（如前列腺、胰腺、肝脏和肾脏）具有离散选择性。这一目标得到了两个关键发现的推动。首先，在美国，结直肠癌是男性癌症死亡的第二大原因，女性癌症死亡的第三大原因。其次，已经表明，萝卜硫苷（GPN）（西兰花中的主要化学预防剂）经历黑芥子酶依赖性代谢以提供弱HDAC抑制剂。有证据强烈表明，GPN衍生的HDAC抑制剂是在结构上类似于下文所示的C的半胱氨酸缀合物。很可能HDAC抑制代谢物不仅与西兰花消费有关，而且与许多其他蔬菜的摄入有关，这些蔬菜提供能够进行GI定位代谢的芥子油苷，如下所示。我们的目标是产生新的物质，分享萝卜硫苷的新的作用机制，但更有效的HDAC抑制剂，从而更有用的化学预防剂。我们将首先制备衍生自GPN的半胱氨酸-缀合物的许多衍生物，以验证半胱氨酸衍生的羧酸盐负责HDAC活性位点内的Zn 2+配位（并且因此，酶失活）的假设。从伯胺文库中，我们将制备ITC类似物（如下文所示的B）和半胱氨酸-1连接的缀合物（C型）。将在无细胞测定（Fluor de Lys）和人结肠癌细胞（HCT 116）中测试文库的每个成员的ITC和半胱氨酸缀合物的HDAC抑制。显示抗HDAC活性的药物将是进一步合成和生物化学研究的候选者。具体地，将构建这些活性剂的芥子油苷前体（下文所示的形式A）。将通过自动反相HPLC评估所得芥子油苷经历黑芥子酶触发转化为相应ITC的能力，并且如果适用，我们将在无细胞测定和HCT 116细胞中检查所述黑芥子酶活化剂的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.