Chemical Genetics of Iron Transport

描述（由申请人提供）：化学遗传学是一个新兴的领域，它利用组合化学和小分子文库来剖析复杂的生物过程。小分子可以非常快速地起作用，可以非常特异，并且可以帮助区分分子步骤的时间顺序和生物过程的分级调节。因为小分子可以改变特定基因产物的功能，所以它们可以以类似于使用诱导型显性或纯合隐性遗传突变的方式使用。大量的生物化学文献是基于过去在“反向化学遗传学”方法中采用的小分子拮抗剂的使用，以有条件地消除蛋白质功能，并在此基础上随后鉴定靶标、其作用机制及其调节。因此，哇巴因有助于确定NaK-ATP酶的催化循环，细胞松弛素B有助于确定胰岛素刺激葡萄糖摄取的分子基础，阿米洛利类似物用于纯化和确定上皮Na通道。有必要开发“正向化学遗传学”，以发现与感兴趣的途径中的关键元件合作的小分子。这一建议得到了初步数据的支持，建立了一个基于荧光的测定，以筛选哺乳动物细胞铁吸收的抑制剂。铁缺乏仍然是我国最普遍的营养问题，但最近对遗传性血色病基因的鉴定表明，每20名高加索人中就有1人携带缺陷等位基因，因此每400人中就有1人可能易患铁过载。增加对运输因素的了解以及它们如何防止铁缺乏和过载对于更广泛地解决这些重大健康问题至关重要。使用基于细胞的荧光测定，我们建议：1）使用组合文库对铁转运的不同途径的选择性抑制剂进行化学遗传筛选; 2）表征鉴定为以最高效力阻断铁摄取的化合物; 3）开发感兴趣化合物的结构-活性谱并鉴定其靶点。该项目的目标是使用化学遗传学发现铁转运的小分子抑制剂，并使用这些试剂来促进我们对铁摄取的不同途径的因素，机制和调节的理解。

DESCRIPTION (provided by applicant): Chemical genetics is an emerging field that takes advantage of combinatorial chemical and small molecule libraries to dissect complex biological processes. Small molecules can act very fast, can be very specific, and can help to distinguish the temporal order of molecular steps and the hierarchical regulation of biological processes. Because small molecules can alter the function of a specific gene product, they can be used in a manner analogous to the use of inducible dominant or homozygous recessive genetic mutations. A large body of biochemical literature is based on the past use of small molecule antagonists that were employed in "reverse chemical genetics" approaches to conditionally eliminate protein function, and on that basis to subsequently identify the target, its mechanism of action, and its regulation. Thus, ouabain helped to define the catalytic cycle of the NaK-ATPase, cytochalasin B was instrumental in defining the molecular basis for insulin's action to stimulate glucose uptake, and analogs of amiloride were used to purify and define the epithelial Na channel. There is a need to develop "forward chemical genetics" in order to discover small molecules that partner with key elements in a pathway of interest. This proposal is supported by preliminary data that establish a fluorescence-based assay to screen for inhibitors of iron uptake by mammalian cells. Iron deficiency remains the most prevalent nutritional problem in our country, yet recent identification of the gene responsible for hereditary hemochromatosis indicates that 1 in 20 Caucasians carry the defective allele and thus 1 in 400 may be susceptible to iron overload. Increased knowledge about the transport factors and how they protect against iron deficiency and overload is essential to more broadly address these significant health problems. Using the cell-based fluorescence assay, we propose to: 1) Perform chemical genetic screens for selective inhibitors of different pathways of iron transport using combinatorial libraries; 2) Characterize the compounds identified to block iron uptake with highest potency; 3) Develop structure-activity profiles on compounds of interest and identify their targets. The goals of this project are to discover small molecule inhibitors of iron transport using chemical genetics and to use these reagents to advance our understanding of the factors, mechanisms, and regulation of different pathways of iron uptake.