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。

• 批准号: 10091409
• 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
• 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/10091409
• 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

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列纳莱度胺和pomalidomide（imIDS）是高度有效的治疗方法 血液系统恶性肿瘤，例如多发性骨髓瘤（MM）或DEL（5Q）骨髓增生综合征（5Q-MDS），AS 以及FDA批准的红斑nodosom leprosum（ENL）的治疗方法。几十年来发现了米发 具有广泛但不一定是相关的影响，从诱导氧化应激，抑制 血管生成，对免疫系统的多种影响，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。理解的第二个突破 Imids的活性是IMIDS结合CRBN的发现，CRBN是CUL4-RBX1-DDB1-的底物接收器 CRBN（CRL4CRBN）E3泛素连接酶，并暴露双重活动：1）防止CRL4CRBN泛素化 它的天然底物，包括Meis2和2），以改变CRL4CRBN泛素连接酶的特异性 泛素化新靶标，尤其是淋巴样转录因子IKAROS（IKZF1）和Aiolos（IKZF3），以及 酪蛋白激酶1α（CK1α）。因此，iMIDS赋予CRL4CRBN基材的功能奖励。 接收器能够对关键治疗靶标的结合和泛素化。而IKZF1/3和CK1α提供了 对于IMID效率的合理解释，它们在解释所有细胞和临床反应方面缺乏 由imID和其他底物引起的可能存在。此外，Imids通过更改行为的发现 CRL4CRBN泛素连接酶朝向新材料的底物曲目具有变革性的潜力 毒品发现；但是，我们不了解新材料募集的分子基础。重要的 未解决的问题是1）IMIDS的完整底物曲目，2）DeGron的结构特征 由CRL4CRBN-IMID复合物识别，3）如何在分子场上进行修改，可以召集 底物选择性。在AIM 1中，我们将验证一份用A 新型的脉搏 - 硅胶质谱法。我们将发布经过验证的基板作为资源 社区将极大地支持对IMID的临床和细胞结局的深入了解 治疗。在AIM 2中，我们将结合X射线晶体学，生化重新机构和 细胞实验，以解决IMID活性的分子基础。深入了解这种新型药物 机制将指导未来医学的发展。利用我们复杂的分子和结构 理解，我们将在本提案的目标3中开发具有底物选择性改变的IMID衍生物。 通过实验室中开发的一系列定量评估，我们首次能够遵循结构和 机理指导方法合成iMID的衍生物并解释了如何微妙的化学修饰 导致底物特异性改变。综上所述，我们将定义基础的分子框架 IMID诱导的连接酶重新利用。