Catalytic and Inhibitory Mechanisms in Indoleamine 2,3-dioxygenase

描述（由申请方提供）：吲哚胺2，3-双加氧酶（IDO）催化L-色氨酸氧化为N-甲酰犬尿氨酸。与广谱的P450单加氧酶相比，IDO是人类中仅有的两种基于血红素的双加氧酶之一。尽管几十年的努力，其双加氧酶机制仍然难以捉摸。IDO是一种免疫抑制酶，在使癌细胞逃避免疫监视方面起着重要作用。最近，由于认识到其作为癌症治疗靶点的潜力，它吸引了大量的关注。因此，迫切需要描述IDO的两种人类同种型（称为hIDO 1和hIDO 2）的双加氧酶和抑制机制。我们计划的长期目标是（i）定义血红素双加氧酶的分子机制，填补血红素氧化学的知识空白，以及（ii）描述IDO 1/IDO 2抑制剂的抗肿瘤作用，帮助定义IDO相关的癌症生理学。本申请的目的是表征hIDO 1和hIDO 2的分子特性，以确定其催化和抑制机制。我们的中心假设是两种IDO亚型的催化和抑制机制是不同的。其理由是，该项目的成功完成将为制定针对IDO的癌症干预措施提供强有力的、概念性的和以证据为基础的指导方针。因此，拟议中的研究与NIH的使命的一部分有关，该使命涉及到基础知识的发展，这将可能有助于减轻人类残疾的负担。在强有力的初步数据的指导下，我们的假设将通过追求两个特定的目标进行测试：（i）定义hIDO 1和hIDO 2的双加氧酶机制，以及（ii）确定hIDO 1和hIDO 2的抑制机制。为了实现我们的目标，我们将采用一个多方面的方法与一套互补的光谱技术（拉曼，紫外-可见，FTIR，EPR，MS和X射线晶体学），结合计算方法（MD和QM/MM）和诱变。拟议的项目是创新的，因为（i）hIDO 1和hIDO 2的可用性使我们在进行拟议的比较研究方面处于独特的地位，以及（ii）我们实验室开发的独特的快速混合/冷冻淬火技术能够有效地表征其他实验室无法获得的关键酶中间体的结构。这项研究意义重大，因为该项目的成功完成将为未来设计针对IDO的新型治疗策略奠定坚实的基础，并促进对血红素双加氧酶化学的基本理解。 公共卫生相关性：拟议的研究集中在一个重要但知之甚少的研究领域，涉及吲哚胺2，3-双加氧酶的两种亚型，它们在使癌细胞逃避免疫监视方面发挥着重要作用。拟议的研究与公共卫生有关，因为对酶的两种异构体的催化和抑制机制的详细了解应该为开发其抑制剂用于药物干预癌症提供基础。

DESCRIPTION (provided by applicant): Indoleamine 2,3-dioxygenase (IDO) catalyzes the oxidation of L-tryptophan to N-formyl kynurenine. In contrast to the wide spectrum of P450 monooxygenases, IDO is one of the only two heme-based dioxygenases in humans. Despite decades of effort, its dioxygenase mechanism remains elusive. IDO is an immunosuppressive enzyme, which plays an important role in allowing cancer cells to escape from immune surveillance. Recently, it has attracted a great deal of attention due to the recognition of its potential as a therapeutic target for cancer. Hence, there is a critical need for the delineation of the dioxygenase and inhibitory mechanisms of the two human isoforms of IDO, named hIDO1 and hIDO2. The long term goal of our program is (i) to define the molecular mechanism of heme-based dioxygenases, filling in the knowledge gap in heme oxygen chemistry, and (ii) to delineate the antitumor effect of IDO1/IDO2 inhibitors, aiding in the definition of IDO-linked cancer physiology. The objective of this application is to characterize the molecular properties of hIDO1 and hIDO2, in order to define their catalytic and inhibitory mechanisms. Our central hypothesis is that the catalytic and inhibitory mechanisms of the two IDO isoforms are distinct. The rationale is that the successful completion of this project will offer strong, conceptual and evidence-based guidelines for the development of IDO- targeted intervention against cancer. Thus, the proposed research is relevant to that part of NIH's mission that pertains to the development of fundamental knowledge that will potentially help to reduce the burdens of human disability. Guided by strong preliminary data, our hypothesis will be tested by the pursuit of two specific aims: (i) define the dioxygenase mechanisms of hIDO1 and hIDO2, and (ii) identify the inhibition mechanisms of hIDO1 and hIDO2. To achieve our objective we will employ a multi-faceted approach with a complementary set of spectroscopic techniques (Raman, UV-Vis, FTIR, EPR, MS and X-ray crystallography), combined with computational methodologies (MD and QM/MM) and mutagenesis. The proposed project is innovative because (i) the availability of both hIDO1 and hIDO2 places us in a unique position for carrying out the proposed comparative studies, and (ii) the unique fast-mixing/freeze-quenching techniques developed in our lab enable effective structural characterization of key enzymatic intermediates that are inaccessible in other labs. The proposed research is significant because the successful completion of this project will lay a solid foundation for the future design of novel therapeutic strategies targeting IDO, as well as to advance fundamental understanding of heme-based dioxygenase chemistry. PUBLIC HEALTH RELEVANCE: The proposed studies are focused on an important, but poorly understood, area of research dealing with two isoforms of indoleamine 2,3-dioxygenase, which play an essential role in allowing cancer cells to escape from immune surveillance. The proposed research is relevant to public health, because a detailed understanding of catalytic and inhibitory mechanisms of the two isoforms of the enzyme should provide a foundation for the development of their inhibitors for pharmaceutical intervention against cancer.