Dissecting CRBN-substrate interactions in the mechanism of lenalidomide

 描述（由申请方提供）：来那度胺和其他免疫调节药物（IMiD）是多发性骨髓瘤、骨髓增生异常综合征伴del（5q）和其他血液学肿瘤的高效治疗药物。IMiD最近被证明通过一种新的药物机制发挥作用-调节E3泛素连接酶以特异性泛素化靶蛋白。IMiD结合cereblon（CRBN），CRBN-CRL 4 E3泛素连接酶的底物衔接子，并通过该复合物诱导两种淋巴转录因子的泛素化。这些因子对于多发性骨髓瘤细胞的存活至关重要，因此它们随后的蛋白酶体降解解释了IMiD在这种情况下的疗效。 泛素连接酶的活化， 特异性靶向蛋白质降解是一种全新的药物机制，对许多临床病症具有治疗意义。然而，在我们更广泛地应用这种机制之前，需要进一步了解CRBN和来那度胺依赖性底物之间的相互作用。具体而言，我们必须了解来那度胺是否变构增加CRBN-CRL 4对其内源性底物的总体活性，或桥接CRBN-底物界面以招募靶蛋白至CRBN。我将结合联合收割机CRBN的无偏和有针对性的诱变，以研究来那度胺通过桥接CRBN和招募底物之间的相互作用而起作用的假设。 在目标1中，我将使用扫描诱变来鉴定来那度胺诱导底物降解所必需的CRBN残基的整个图谱。在这种方法中，CRBN的每个残基突变为所有可能的氨基酸，一次一个位置。将消除来那度胺诱导的底物降解的CRBN突变映射到CRBN-IMiD复合物的晶体结构上，以鉴定与来那度胺、募集的底物和其他CRBN-CRL 4复合物成员相互作用所必需的残基。在目标2中，我将评估IMiD结合位点附近CRBN残基的靶向突变的影响。如果来那度胺通过桥接机制起作用，则应该可以鉴定该区域中影响底物募集而不影响药物结合的突变。具体而言，我将检测增加IMiD结合口袋附近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的方法，这项工作将彻底研究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型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.