Identification of small molecule inhibitors of the DDI2 protease

DDI2 蛋白酶小分子抑制剂的鉴定

• 批准号: 10638837
• 负责人: Gary L. Kleiger
• 金额: $ 73.1万
• 依托单位: VIRGINIA COMMONWEALTH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-10 至 2027-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10638837
• 关键词: Achievement; Acquired Immunodeficiency Syndrome; Address; Affect; Apoptosis; Applications Grants; Area; Binding; Biological Assay; Biophysics; Cancer Patient; Catalytic Domain; Cell Survival; Cells; Cellular Assay; Clinic; Clinical Trials; Data; Detection; Development; Disease remission; Dose; Drug Targeting; Dryness; Enzymes; Event; Exhibits; FDA approved; Fruit; Funding; Future; Genes; Goals; Grant; HIV Protease; Human; Immune system; Length; Libraries; Marketing; Measurement; Modality; Multiple Myeloma; Mus; Normal Cell; Pathway interactions; Patient-Focused Outcomes; Patients; Peptide Hydrolases; Play; Powder dose form; Predisposition; Production; Protease Inhibitor; Proteasome Inhibition; Proteasome Inhibitor; Protein Kinase; Proteins; Protocols documentation; Recovery; Reproducibility; Risk; Role; Signal Transduction Pathway; System; Testing; Therapeutic; Transcript; Ubiquitin; Up-Regulation; Validation; Work; Xenograft Model; cancer cell; cancer therapy; cheminformatics; counterscreen; expectation; high throughput screening; immune modulating agents; inhibitor; liquid chromatography mass spectrometry; malignant breast neoplasm; multicatalytic endopeptidase complex; programs; protein degradation; proteostasis; response; scaffold; scale up; screening; side effect; small molecule inhibitor; synergism; targeted cancer therapy; transcription factor; tumor; tumor growth

PROJECT SUMMARY Proteasome inhibitors are currently being used in the clinic against Multiple myeloma. This approach is thought to work at least in part because cancer cells appear to rely more heavily on proteasomes than do normal cells. However, our previous studies showed that proteasome inhibition invokes an adaptive program driven by the transcription factor NRF1 which upregulates proteasome genes resulting in the recovery of proteasome activity, thus limiting the efficacy of this approach. Consistent with this notion, our preliminary data suggest that depletion of NRF1 potentiates the action of proteasome inhibition therapy in a breast cancer xenograft model. Taking advantage of the fact that a protease DDI2 is essential for NRF1 activation, here we propose to identify small molecule inhibitors of this protease that could be used to enhance the efficacy of proteasome inhibitors. Moreover, based on our preliminary data that showed depleting DDI2 in itself retards tumor growth, DDI2 inhibitors could also find use as a single-agent. Based on our pilot screen that demonstrates feasibility and consistent with the goals of PAR-20-271, this 4-year project will pursue the following specific aims. In AIM 1, we will express and purify DDI2 protein and perform high-throughput screens using a protein thermal shift (PTS) assay to identify compounds that bind DDI2. In AIM 2, we will perform hit selection, confirmation and profiling using a panel of secondary assays that includes orthogonal and counter-screen assays as well as biophysical assays. In AIM 3, we will perform hit validation, hit expansion, probe selection and profile the mechanism of action of hits, followed by cellular assays to assess DDI2 target engagement, impact on the DDI2-NRF1 axis and cancer cell apoptosis.

项目总结 蛋白酶体抑制剂目前正用于临床治疗多发性骨髓瘤。这 这种方法被认为至少在一定程度上是因为癌细胞似乎更依赖于 蛋白酶体比正常细胞多。然而，我们之前的研究表明，蛋白酶体 抑制调用由转录因子NRF1驱动的适应性程序，该程序 上调蛋白酶体基因导致蛋白酶体活性的恢复，从而限制 这种方法的有效性。与这一概念一致，我们的初步数据表明 缺失NRF1增强蛋白酶体抑制治疗在乳腺癌中的作用 异种移植模型。利用蛋白酶DDI2对NRF1是必不可少的事实 激活，在这里我们建议识别这种蛋白水解酶的小分子抑制剂，它可能是 用于增强蛋白酶体抑制剂的疗效。此外，根据我们的初步数据， 这表明消耗DDI2本身会抑制肿瘤的生长，DDI2抑制剂也可以用来 一个单一的代理人。基于我们的试点屏幕，演示了可行性，并与 根据PAR-20-271的目标，这个为期4年的项目将实现以下具体目标。在目标1中，我们 将表达和纯化DDI2蛋白，并使用一种蛋白进行高通量筛选 热位移(PTS)分析以确定与DDI2结合的化合物。在AIM 2中，我们将表演HIT 使用包括正交法的一组二次化验进行选择、确认和分析 以及反筛选分析和生物物理分析。在AIM 3中，我们将表演HIT 验证、HIT扩展、探头选择和描述HITS的作用机制，随后 通过细胞分析评估DDI2靶参与、对DDI2-NRF1轴的影响和 癌细胞的凋亡。