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

沙利度胺及其类似物来那度胺和泊马度胺(IMiDS)是治疗食道癌的高效药物。 恶性血液病，如多发性骨髓瘤(MM)或del(5q)骨髓增生异常综合征(5q-MDS)，AS 以及FDA批准的治疗结节性麻风红斑(ENL)。几十年来，人们发现了IMIDS 有广泛但不必要的相关影响，包括诱导氧化应激，抑制 血管生成，对免疫系统的多种影响，如增加细胞因子IL-2的产生 (IL-2)和抑制细胞因子肿瘤坏死因子(TNF)。在理解这一问题上的重大突破 IMids的活性是发现IMids与CUL4-RBX1-DDB1的底物受体CRBN结合。 CRBN(CRL4CRBN)E3泛素连接酶，并具有双重活性：1)阻止CRL4CRBN泛素化 它的天然底物，包括MEIS2，和2)改变CRL4CRBN泛素连接酶的特异性 泛素化新靶点，特别是淋巴转录因子Ikaros(IKZF1)和Aiolos(IKZF3)，以及 酪蛋白激酶1α(CK1α)。因此，IMID赋予CRL4CRBN衬底以函数增益特性 能够结合和泛素化关键治疗靶点的受体。而IKZF1/3和CK1α则提供了 对imid疗效的合理解释，它们不能解释所有的细胞和临床反应。 由IMIDS和其他底物诱导的可能存在。此外，IMIDS的发现是通过改变 CRL4CRBN泛素连接酶对新底物的底物库具有转化潜力 药物发现；然而，我们不了解新底物招募的分子基础。重要 尚未解决的问题是1)完整的IMIDS衬底体系，2)降解物的结构特征 被CRL4CRBN-IMiD复合体识别，以及3)基于分子基础，对IMiD的修饰如何能够提供 底物选择性。在目标1中，我们将验证使用 新型PULSE-SILAC质谱学方法。我们将把经过验证的底物作为资源发布到 社区，这将极大地促进对IMiD临床和细胞结局的深入了解 治疗。在目标2中，我们将使用X射线结晶学、生化重组和 研究IMiD活性的分子基础的细胞实验。对这种新药的深入了解 机制将引导未来医学的发展。利用我们复杂的分子和结构 了解到，我们将在本提案的目标3中开发具有改变底物选择性的IMID衍生物。 通过实验室开发的一套定量分析，我们第一次能够跟踪结构和 机制导引方法合成IMIDS的衍生物并解释如何进行微妙的化学修饰 导致底物专一性改变。综上所述，我们将定义作为基础的分子框架 IMIDD诱导的连接酶重新定位。

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.