Dissecting CRBN-substrate interactions in the mechanism of lenalidomide

 描述(申请人提供)：来那度胺和其他免疫调节药物(IMids)是治疗多发性骨髓瘤、伴有del(5q)的骨髓增生异常综合征和其他血液肿瘤的高效药物。IMID最近被证明通过一种新的药物机制发挥作用--调节E3泛素连接酶来特异性泛素化目标蛋白。IMID结合CRBN-CRL4 E3泛素连接酶的底物接头Cereblon(CRBN)，并通过该复合体诱导两种淋巴转录因子的泛素化。这些因素对多发性骨髓瘤细胞的生存至关重要，因此它们随后的蛋白酶体降解解释了IMiDS在这种情况下的疗效。激活泛素连接酶以 具体地说，靶蛋白的降解是一种全新的药物机制，对许多临床疾病具有治疗意义。然而，在我们能够更广泛地应用这一机制之前，需要进一步了解CRBN和来那度胺依赖底物之间的相互作用。具体地说，我们必须了解来那度胺是变构增加CRBN-CRL4在其内源底物上的整体活性，还是桥接CRBN-底物界面以招募靶蛋白到CRBN。我将结合CRBN的无偏突变和靶向突变来研究来那度胺通过连接CRBN和招募的底物之间的相互作用而起作用的假设。在目标1中，我将使用扫描突变来鉴定来那度胺诱导底物降解所必需的CRBN残基的整个图景。在这种方法中，CRBN的每个残基都被突变为所有可能的氨基酸，一次一个位置。消除来那度胺诱导的底物降解的CRBN突变将被映射到CRBN-IMiD复合体的晶体结构上，以确定与来那度胺、招募底物和其他CRBN-CRL4复合体成员相互作用所必需的残基。在目标2中，我将评估与IMiD结合位点相邻的CRBN残基的靶向突变的效果。如果来那度胺通过一种桥接机制起作用，应该可以在不影响药物结合的情况下识别该区域的突变，这些突变影响底物募集。具体地说，我将测试增加IMiD结合口袋附近的四个非极性氨基酸的空间体积对来那度胺诱导的底物降解、底物募集和来那度胺结合的影响。我还将测试将IMiD结合位点周围的四个带电残基突变为丙氨酸的效果。通过假设驱动和无偏倚的方法，这项工作将深入研究CRBN和来那度胺诱导的底物之间的相互作用。了解这些相互作用是将这种新的药物机制应用于其他分子靶点和临床条件的关键。

 DESCRIPTION (provided by applicant): Lenalidomide and other immunomodulatory drugs (IMiDs) are highly effective treatments for multiple myeloma, myelodysplastic syndrome with del(5q), and other hematologic neoplasms. IMiDs were recently shown to act by a novel drug mechanism-modulation of an E3 ubiquitin ligase to specifically ubiquitinate target proteins. IMiDs bind cereblon (CRBN), the substrate adaptor for the CRBN-CRL4 E3 ubiquitin ligase, and induce the ubiquitination of two lymphoid transcription factors by this complex. These factors are essential for the survival of multiple myeloma cells, so their subsequent proteasomal degradation explains the efficacy of IMiDs in this condition. Activation of an ubiquitin ligase to specifically target proteins for degradation is a completely novel drug mechanism with therapeutic implications for many clinical conditions. However, further understanding of the interactions between CRBN and lenalidomide-dependent substrates is needed before we can apply this mechanism more broadly. Specifically, we must understand whether lenalidomide allosterically increases the overall activity of CRBN-CRL4 on its endogenous substrates or bridges the CRBN-substrate interface to recruit target proteins to CRBN. I will combine unbiased and targeted mutagenesis of CRBN to investigate the hypothesis that lenalidomide works by bridging interactions between CRBN and recruited substrates. In Aim 1, I will use scanning mutagenesis to identify the entire landscape of CRBN residues essential for lenalidomide-induced substrate degradation. In this approach, each residue of CRBN is mutated to all possible amino acids, one position at a time. CRBN mutations which abrogate lenalidomide-induced substrate degradation will be mapped onto the crystal structure of the CRBN-IMiD complex to identify residues essential for interactions with lenalidomide, recruited substrates, and other CRBN-CRL4 complex members. In Aim 2, I will evaluate the effects of targeted mutations of CRBN residues adjacent to the IMiD binding site. If lenalidomide works by a bridging mechanism, it should be possible to identify mutations in this region which affect substrate recruitment without affecting drug binding. Specifically, I will test the effects of increasing the steric bulk of four non-polar amino acids adjacent to the IMiD-binding pocket on lenalidomide-induced substrate degradation, substrate recruitment, and lenalidomide binding. I will also test the effects of mutating four charged residues surrounding the IMiD binding site to alanine. Through hypothesis driven and unbiased approaches, this work will thoroughly investigate the interactions between CRBN and lenalidomide-induced substrates. Understanding these interactions is key to applying this novel drug mechanism to other molecular targets and clinical conditions.