High Throughput Screening for Identification of AKR1C3 Inhibitors

用于鉴定 AKR1C3 抑制剂的高通量筛选

• 批准号: 7290911
• 负责人: Brian Lavan
• 金额: $ 15.63万
• 依托单位: METABOLEX, INC.
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-06-01 至 2009-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7290911
• 关键词: 9-deoxy-delta-9-prostaglandin D2; Address; Adipose tissue; Affect; Agonist; American; Attenuated; Biochemical; Biological Assay; Cell Line; Cells; Clinical Treatment; Diabetes Mellitus; Dinoprost; Dose; Enzyme-Linked Immunosorbent Assay; Enzymes; Fluorescence; Gene Expression; Genes; Hand; Heart; Human; Individual; Insulin; Insulin Resistance; Lead; Libraries; Ligands; Liver; Measures; Metabolic Pathway; Methods; Modeling; Molecular Profiling; Muscle; Muscle Cells; NADP; Non-Insulin-Dependent Diabetes Mellitus; Nuclear; Oxidation-Reduction; PPAR gamma; Pathway interactions; Patients; Peripheral; Permeability; Peroxisome Proliferator-Activated Receptors; Production; Prostaglandin D2; Reaction; Recombinants; Research; Research Personnel; Route; Screening procedure; Signal Transduction Pathway; Structure; System; Testing; Therapeutic; Thinking; Tissues; Transcriptional Activation; United States; Up-Regulation; Week; base; carbonyl reductase (NADPH); diabetes mellitus therapy; diabetic; glycemic control; high throughput screening; inhibitor/antagonist; insulin sensitivity; insulin signaling; oxidation; response; small molecule; tool; transcription factor

DESCRIPTION (provided by applicant): This proposal aims to develop a high throughput screening (HTS) assay that will identify small molecule inhibitors for the aldo-keto reductase AKR1C3, in hopes that they would lead to a more potent cellular response to insulin. Such inhibitors could be of great therapeutic potential to Type II diabetics, who suffer from insulin resistance in peripheral tissues such as muscle, adipose and liver. The selected compounds could also serve as useful tools for studying the insulin signal transduction pathway, especially concerning the nuclear transcription factor PPARgamma, which ultimately activates a set of insulin-responsive genes. The branch of the biosynthetic pathway addressed here starts with prostaglandin D2 (PGD2). This compound is normally converted to 15-deoxy-delta12,14-prostaglandin J2 - a potent PPARgamma ligand. However, especially in diabetic muscle tissue, AKR1C3 converts PGD2 along an alternative pathway to 9alpha11betaPGF2alpha, diminishing the amount of 15-deoxy-delta12,14-prostaglandin J2 available for PPARgamma activation. We have therefore proposed a model whereby the up-regulation of AKR1C3 increases the production of 9alpha11betaPGF2alpha at the expense of the production of 15-deoxy-delta12,14-prostaglandin J2. On the other hand, we believe that AKR1C3-specific inhibitors would attenuate the competing pathway, thus favoring production of 15-deoxy-delta12,14-prostaglandin J2 and the consequent stimulation of PPARgamma, leading to insulin sensitivity. To identify such inhibitors we will first establish a fluorescence-based assay for measuring AKR1C3 activity that will take advantage of the redox function of the enzyme. It will measure the decreasing fluorescence of NADPH as it is converted to NADP during the concomitant reduction of PGD2. A backup assay will follow the conversion of NADP to NADPH, as AKR1C3 oxidizes 1-acenapthenol. After the assay is developed, we will then adapt it for HTS format and screen a pilot library in search of inhibitors. Compounds identified as hits will then be tested in a dose response biochemical assay followed by a cell-based assay to confirm their inhibition of AKR1C3. Confirmed inhibitors may be of great value, both experimentally to researchers and therapeutically to diabetes patients. The research proposed here aims to identify compounds that are likely to have therapeutic potential for individuals suffering from insulin resistance and Type II diabetes. This ailment directly affects 20 million people in the United States, with only 42% of patients attaining sufficient glycemic control, as defined by the American Diabetes Association. An effective therapy could provide much relief to the patients.

描述（由申请人提供）：本提案旨在开发一种高通量筛选（HTS）试验，以鉴定醛酮还原酶AKR1C3的小分子抑制剂，希望它们能导致对胰岛素更有效的细胞反应。这种抑制剂对II型糖尿病患者有很大的治疗潜力，II型糖尿病患者在肌肉、脂肪和肝脏等外周组织中遭受胰岛素抵抗。所选择的化合物也可以作为研究胰岛素信号转导途径的有用工具，特别是关于核转录因子PPARgamma，它最终激活了一组胰岛素应答基因。生物合成途径的分支从前列腺素D2 （PGD2）开始。该化合物通常转化为15-脱氧- δ 12,14-前列腺素J2 -一种有效的PPARgamma配体。然而，特别是在糖尿病肌肉组织中，AKR1C3将PGD2转化为9alpha11betaPGF2alpha，减少了可用于PPARgamma激活的15-deoxy-delta12,14-前列腺素J2的量。因此，我们提出了一个模型，即AKR1C3的上调增加了9alpha11betaPGF2alpha的产生，而牺牲了15-deoxy-delta12,14-前列腺素J2的产生。另一方面，我们认为akr1c3特异性抑制剂会减弱竞争通路，从而有利于15-deoxy-delta12,14-prostaglandin J2的产生和随后的PPARgamma刺激，导致胰岛素敏感性。为了确定这样的抑制剂，我们将首先建立一个基于荧光的测定AKR1C3活性的方法，该方法将利用酶的氧化还原功能。它将测量NADPH在伴随PGD2的还原过程中转化为NADP时荧光的减弱。当AKR1C3氧化1-苊烯醇时，备份分析将遵循NADP向NADPH的转化。在试验开发完成后，我们将使其适应HTS格式，并筛选一个试点文库以寻找抑制剂。确定为命中的化合物将在剂量反应生化试验中进行测试，然后进行基于细胞的试验以确认其对AKR1C3的抑制作用。已证实的抑制剂对研究人员和糖尿病患者的治疗都有很大的价值。这里提出的研究旨在确定可能对患有胰岛素抵抗和II型糖尿病的个体具有治疗潜力的化合物。这种疾病直接影响到美国2000万人，根据美国糖尿病协会的定义，只有42%的患者达到了足够的血糖控制。一种有效的治疗方法可以给病人带来很大的缓解。