The molecular basis of IMiD induced neo-substrate recruitment to the CRL4CRBN ubiquitin E3 ligase.

IMiD 的分子基础诱导 CRL4CRBN 泛素 E3 连接酶招募新底物。

基本信息

批准号：
9899745
负责人：
Eric Sebastian Fischer
金额：
$ 39.59万
依托单位：
DANA-FARBER CANCER INST
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-03-01 至 2022-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9899745
关键词：
Address Angiogenesis Inhibition Binding Biochemical Biological Assay Biology Biophysics Cell Line Cells Cellular Assay Cellular biology Chemicals Clinical Collaborations Communities Complex Computer Models Crystallization Data Data Set Development Dysmyelopoietic Syndromes Erythema Exhibits FDA approved Failure Flow Cytometry Flowcharts Future HCT116 Cells Hematologic Neoplasms Immune system In Vitro Individual Interleukin-2 Libraries Ligase Link Literature Lymphoid Mass Spectrum Analysis Medicine Modification Molecular Molecular Structure Multiple Myeloma Mutation Analysis NF-kappa B Outcome Oxidative Stress Induction Pharmaceutical Preparations Physiologic pulse Production Property Proteins Proteomics Publishing RBX1 gene Resources Seminal Signal Transduction Specificity Spectrometry Structure Substrate Specificity System TNF gene Teratogens Testing Thalidomide Therapeutic Time Ubiquitin Ubiquitination Validation Western Blotting Work X-Ray Crystallography Zinc Fingers analog base biophysical properties casein kinase I chromosome 5q loss cytokine drug discovery drug mechanism effective therapy experimental study falls gain of function insight interest lenalidomide molecular mechanics multicatalytic endopeptidase complex novel novel therapeutics pomalidomide prevent protein degradation receptor reconstitution recruit response small molecule structural biology therapeutic target tool transcription factor ubiquitin ligase ubiquitin-protein ligase

项目摘要

Thalidomide and its analogs lenalidomide and pomalidomide (IMiDs), are highly effective treatments for hematologic malignancies such as multiple myeloma (MM) or del(5q) myelodysplastic syndrome (5q-MDS), as well as an FDA approved treatment for erythema nodosom leprosum (ENL). Over decades IMiDs were found to have broad but not necessary related effects, ranging from induction of oxidative stress, inhibition of angiogenesis, to multiple effects on the immune system such as enhanced production of cytokine interleukin-2 (IL-2) and inhibition of cytokine tumor necrosis factor (TNF). A seminal breakthrough in understanding the activity of IMiDs was the discovery that IMiDs bind CRBN, the substrate receptor of the CUL4-RBX1-DDB1- CRBN (CRL4CRBN) E3 ubiquitin ligase, and to exhibit dual activity: 1) preventing CRL4CRBN from ubiquitinating its native substrates, including MEIS2, and 2) to alter the specificity of the CRL4CRBN ubiquitin ligase to ubiquitinate new targets, notably the lymphoid transcription factors Ikaros (IKZF1) and Aiolos (IKZF3), and casein kinase 1 alpha (CK1α). Therefore IMiDs impart gain-of-function properties to the CRL4CRBN substrate receptor that enable binding and ubiquitination of key therapeutic targets. While IKZF1/3 and Ck1α provide a plausible explanation for IMiD efficacy, they fall short in explaining all of the cellular and clinical response induced by IMiDs and other substrates likely exist. Moreover, the finding that IMiDs act by altering the substrate repertoire of the CRL4CRBN ubiquitin ligase towards neo-substrates has transformative potential to drug discovery; however, we do not understand the molecular basis of neo-substrate recruitment. Important unresolved issues are 1) the complete substrate repertoire of IMiDs, 2) the structural features of the degron recognized by a CRL4CRBN-IMiD complex, and 3) how on molecular grounds, modifications to IMiDs can confer substrate selectivity. In Aim 1 we will validate a list of high-confidence substrate candidates generated with a novel pulse-SILAC mass spectrometry approach. We will publish validated substrates as a resource to the community, which will greatly facilitate the in-depth understanding of the clinical and cellular outcomes of IMiD treatment. In Aim 2, we will use a combination of X-ray crystallography, biochemical reconstitutions and cellular experiments to address the molecular basis of IMiD activity. In depth understanding of this novel drug mechanism will guide development of future medicine. Leveraging our intricate molecular and structural understanding, we will develop IMiD derivatives with altered substrate selectivity in Aim 3 of this proposal. Through a set of quantitative assays developed in the lab, we are for the first time able to follow a structure and mechanism guided approach to synthesize derivatives of IMiDs and explain how subtle chemical modifications result in altered substrate specificity. Taken together, we will define the molecular framework that underlies IMiD induced ligase repurposing.

沙利度胺及其类似物来那度胺和泊马度胺 (IMiD) 是非常有效的治疗方法血液系统恶性肿瘤，例如多发性骨髓瘤 (MM) 或 del(5q) 骨髓增生异常综合征 (5q-MDS)，以及 FDA 批准的麻风结节性红斑 (ENL) 治疗方法数十年来发现了 IMiD。具有广泛但非必要的相关作用，包括诱导氧化应激、抑制血管生成，对免疫系统产生多种影响，例如增强细胞因子白细胞介素 2 的产生 (IL-2) 和细胞因子肿瘤坏死因子 (TNF) 的抑制是理解细胞因子的重大突破。 IMiDs 的活性是发现 IMiDs 结合 CRBN（CUL4-RBX1-DDB1- 的底物受体） CRBN (CRL4CRBN) E3 泛素连接酶，并表现出双重活性：1) 防止 CRL4CRBN 泛素化其天然底物，包括 MEIS2，以及 2) 改变 CRL4CRBN 泛素连接酶的特异性泛素化新靶标，特别是淋巴转录因子 Ikaros (IKZF1) 和 Aiolos (IKZF3)，以及因此，IMiD 赋予 CRL4CRBN 底物功能获得特性。 IKZF1/3 和 Ck1α 提供了关键治疗靶点的结合和泛素化受体。对 IMiD 功效的合理解释，他们未能解释所有的细胞和临床反应此外，IMiDs 通过改变 IMiDs 发挥作用的发现可能存在。 CRL4CRBN 泛素连接酶的底物库对新底物具有变革潜力药物发现；然而，我们不了解新底物招募的分子基础。未解决的问题是 1) IMiD 的完整底物库，2) 降解决定子的结构特征被 CRL4CRBN-IMiD 复合物识别，以及 3) 在分子基础上，对 IMiD 的修饰如何赋予在目标 1 中，我们将验证使用生成的高置信度候选底物列表。新颖的脉冲 SILAC 质谱方法我们将发布经过验证的底物作为资源。社区，这将极大地促进对 IMiD 临床和细胞结果的深入了解在目标 2 中，我们将结合使用 X 射线晶体学、生化重建和细胞实验来解决 IMiD 活性的分子基础，深入了解这种新药。机制将指导未来医学的发展。据了解，我们将在本提案的目标 3 中开发具有改变的底物选择性的 IMiD 衍生物。通过实验室开发的一套定量分析，我们第一次能够遵循结构和机制引导方法合成 IMiD 衍生物并解释微妙的化学修饰是如何进行的综合起来，我们将定义潜在的分子框架。 IMiD 诱导连接酶重新利用。