Structure and Function of Heme-based Dioxygenases

摘要 色氨酸(Trp)是人体必需氨基酸中含量最低的一种。我们膳食中的大部分色氨酸是在代谢过程中 通过犬尿氨酸(KYN)途径。KYN途径的第一步也是限速步骤是由 三种基于血红素的双加氧酶，色氨酸双加氧酶(HTDO)，吲哚胺2，3-双加氧酶1(HIDO1)，以及 吲哚胺2，3-双加氧酶2(HIDO2)。最近发现，这三种双加氧酶在 癌细胞促进癌症免疫逃逸。因此，他们被认为是主要的药物靶点 癌症免疫疗法。尽管它们很重要，但这些酶的结构和功能特性仍然 难以捉摸，这阻碍了该领域的进展。这个项目的中心假设，由我们的 初步数据是：(1)三种双加氧酶的功能特性受细胞代谢产物的调节 和(Ii)每个双加氧酶都显示出不同的结构特征并具有独特的药物结合部位。我们会 通过解决两个具体目标来测试我们的假设：(I)确定与每个目标相互作用的细胞代谢物 双加氧酶和定义相关的调节机制，以及(Ii)定义三者之间的结构差异 并确定每个双加氧酶中新的小分子结合位点。我们将用一个新的高度- 基于通量质谱学的筛选技术，以识别与每种物质相互作用的代谢物 双加氧酶，用X射线结晶学和光谱技术确定其特定的分子 相互作用和功能后果。这些研究将揭示以前不为人知的细胞玩家 可能影响这些酶在癌症和癌症中的特定功能的与双加氧酶相关的人类生理学 其他疾病，从而提供新的信息，使创新的分子方法能够治疗疾病 防控。同时，我们将使用一种综合的方法，涉及广泛的生化 和生物物理技术，以及一组结构多样化的抑制剂作为探针来定义独特的结构 每种双加氧酶的特性和新的小分子结合位点。这些研究的结果将提供 能够更好地理解三种血红素的结构和功能关系的重要知识- 基于双加氧酶，并扩展我们的工具包，以合理设计酶选择性抑制剂。我们有 组建了一个专家团队，以多方面的方式开展这一创新项目。这些研究 将解决我们对潜在的生物学功能的分子机制的认识上的重大空白 这三种双加氧酶为相关的药物开发和疾病治疗提供了重要的新见解。

SUMMARY Tryptophan (Trp) is the least abundant essential amino acid. The majority of our dietary Trp is metabolized through the kynurenine (KYN) pathway. The first and rate-limiting step of the KYN pathway is catalyzed by three heme-based dioxygenases, tryptophan dioxygenase (hTDO), indoleamine 2,3-dioxygenase 1 (hIDO1), and indoleamine 2,3-dioxygenase 2 (hIDO2). Recently it was found that the three dioxygenases are expressed in cancer cells to promote cancer immune escape. Consequently they have been considered as key drug targets for cancer immunotherapy. Despite their importance, the structural and functional properties of these enzymes remain elusive, which has hindered the progress of the field. The central hypothesis of this project, as supported by our preliminary data, is (i) the functional properties of the three dioxygenases are regulated by cellular metabolites and (ii) each dioxygenase exhibits distinct structural features and possesses unique drug binding sites. We will test our hypothesis by addressing two specific aims: (i) identify cellular metabolites that interact with each dioxygenase and define the related regulatory mechanisms, and (ii) define structural differences between the three dioxygenases and determine new small molecule binding sites in each dioxygenase. We will use a new high- throughput mass spectrometry-based screening technology to identify metabolites that interact with each dioxygenase and use X-ray crystallography and spectroscopic techniques to define their specific molecular interactions and functional consequences. These studies will reveal previously unknown cellular players in dioxygenase-related human physiology that may impact the specific functions of these enzymes in cancer and other diseases, thereby offering novel information enabling innovative molecular approaches for disease prevention and control. In parallel, we will use an integrated approach, involving a wide spectrum of biochemical and biophysical techniques, and a group of structurally diverse inhibitors as probes to define unique structural features and new small molecule binding sites in each dioxygenase. The outcome of these studies will offer important knowledge enabling better understanding of structure-and-function relationships of the three heme- based dioxygenases and expanding our toolkit for rational design of enzyme-selective inhibitors. We have assembled a team of experts to carry out this innovative project with the multifaceted approach. These studies will address significant gaps in our knowledge of molecular mechanisms underlying the biological functions of the three dioxygenases and provide important new insights into related drug development and disease treatment.