High Throughput Screening for Identification of AKR1C3 Inhibitors

描述（由申请人提供）：该提案旨在开发一种高通量筛选（HTS）试验，该试验将鉴定醛酮还原酶AKR 1C 3的小分子抑制剂，希望它们能导致对胰岛素的更有效的细胞反应。这种抑制剂可能对II型糖尿病患者具有巨大的治疗潜力，II型糖尿病患者在外周组织如肌肉、脂肪和肝脏中患有胰岛素抵抗。所选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，其最终激活一组胰岛素应答基因。这里讨论的生物合成途径的分支从前列腺素D2（PGD 2）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，AKR 1C 3沿着另一条途径将PGD 2转化为9 α 11 β PGF 2 α，减少了可用于激活PPAR γ的15-脱氧-δ 12，14-前列腺素J2的量。因此，我们提出了一种模型，其中AKR 1C 3的上调增加了9 α 11 β PGF 2 α的产生，但牺牲了15-脱氧-δ 12，14-前列腺素J2的产生。另一方面，我们认为AKR 1C 3特异性抑制剂会减弱竞争途径，从而有利于15-脱氧-δ 12，14-前列腺素J2的产生和随后的PPARgamma刺激，导致胰岛素敏感性。为了鉴定这种抑制剂，我们将首先建立一种基于荧光的测定AKR 1C 3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PGD 2同时还原期间转化为NADP时荧光的降低。由于AKR 1C 3氧化1-苊醇，因此将在NADP转化为NADPH后进行备用试验。在检测开发完成后，我们将使其适用于HTS格式，并筛选一个试验文库以寻找抑制剂。然后将在剂量响应生化测定中测试鉴定为命中的化合物，然后进行基于细胞的测定以确认其对AKR 1C 3的抑制。已证实的抑制剂可能具有很大的价值，无论是对研究人员的实验还是对糖尿病患者的治疗。本文提出的研究旨在确定可能对胰岛素抵抗和II型糖尿病患者具有治疗潜力的化合物。这种疾病直接影响美国2000万人，只有42%的患者达到美国糖尿病协会定义的充分血糖控制。有效的治疗可以使病人得到很大的缓解。

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.