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解剖复杂的生物过程。小分子可以非常快地起作用，可以非常具体，并且可以帮助区分分子步骤的时间顺序和生物过程的分层调节。因为小分子可以改变特定基因产物的功能，它们可以以类似于使用诱导显性或纯合隐性基因突变的方式使用。大量的生化文献是基于过去使用的小分子拮抗剂，这些拮抗剂被用于“反向化学遗传学”方法中，以有条件地消除蛋白质功能，并在此基础上随后确定靶标，其作用机制及其调控。因此，瓦巴因有助于确定Na - 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作用的分子基础，阿米洛利类似物用于纯化和确定上皮Na通道。有必要发展“正向化学遗传学”，以发现在感兴趣的途径中与关键元素合作的小分子。这一建议得到了初步数据的支持，该数据建立了一种基于荧光的检测方法，以筛选哺乳动物细胞铁摄取抑制剂。缺铁仍然是我国最普遍的营养问题，但最近对遗传性血色素沉着病基因的鉴定表明，每20个白种人中就有1个携带有缺陷的等位基因，因此每400人中就有1人可能易患铁超载。提高对运输因素及其如何防止缺铁和铁超载的认识，对于更广泛地解决这些重大健康问题至关重要。利用基于细胞的荧光分析，我们建议：1)使用组合文库对铁转运不同途径的选择性抑制剂进行化学遗传筛选；2)鉴定出阻断铁吸收的最有效化合物；3)开发感兴趣的化合物的结构-活性谱并确定其目标。该项目的目标是利用化学遗传学发现铁转运的小分子抑制剂，并利用这些试剂来推进我们对铁摄取不同途径的因素、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.