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Novel Mechanisms of Molecular Glue-Induced Protein Degradation
Biochemistry ( IF 2.9 ) Pub Date : 2021-07-19 , DOI: 10.1021/acs.biochem.1c00353
Shanique Alabi 1
Affiliation  

Molecular glues, as defined thus far in the targeted protein degradation (TPD) field, are monovalent small molecules (<500 Da) that reshape the surface of an E3 ligase receptor, promoting the recruitment of neosubstrates and facilitating their ubiquitination and subsequent degradation via the proteasome. The most established molecular glues in TPD are a) the immunomodulatory “IMiD” class of small molecules that bind the E3 ligase cereblon and b) the aryl sufonamides that engage DCAF15. As these molecular glues do not require a druggable pocket on the protein of interest, they are poised to allow targeting of difficult-to-drug proteins. With a catalytic mechanism of action and the ability to target nonenzymatic proteins, molecular glues have exceptional promise as a therapeutic modality.(1) Thus far, molecular glue degraders in TPD have relied on recruiting an E3 ligase receptor; however, three recent studies push the envelope of how molecular glues can be utilized to induce the degradation of neosubstrates. To discover drugs with an unrealized ability to induce degradation, Słabicki et al. studied the correlation between preclinical and clinical drugs whose cytotoxic effect necessitate the components of the E3 ligase machinery.(2) Through this exercise, they identified CR8, a pan-CDK inhibitor, whose cytotoxicity is dependent on damage-specific DNA binding protein 1 (DDB1) and induces selective degradation of cyclin K. DDB1 is an adaptor protein that coordinates the interaction between the E3 ligase substrate receptor and the CUL4A/B-RBX1 ligase core (Figure 1A, middle). Through extensive profiling using functional genomics, they identify further components of the DDB1 complex (CUL4B, NEDD8, UBEA3, and RBX1) as required for CR8 induced degradation of cyclin K. However, they could not identify an E3 ligase substrate receptor assumed to be crucial for substrate ubiquitination. Rather, they discovered CDK12, an E3 ligase unrelated component, but a known binder of CR8 as required for cyclin K degradation. Using pull-down and TR-FRET studies, they show that CDK12 has a very weak affinity for DDB1, which is strengthened by 500–1000-fold in the presence of CR8. They solved a 3.5 Å structure that confirmed the DDB1-CR8-CDK12 complex revealing a substantial 2100 Å protein–protein interface with CR8 bridging the interaction. Their structure shows that the terminal phenylpyridine ring system engages the beta-propeller C domain of DDB1, which positions cyclin K in the ubiquitination zone of the ligase normally occupied by degraded substrates (Figure 1A, bottom). Interestingly, this result was also observed by two independent groups with unique chemistry,(1,3) further highlighting those molecular glues can bypass the requirement of an E3 ligase receptor to hijack its function and induce degradation. Figure 1. Mechanisms of molecular glue induced degradation that bypass recruitment of an E3 ligase receptor. (A) CR8 hijacks the E3 ligase adaptor, DDB1, as opposed to the E3 receptor, to position cyclin K in the ubiquitination zone, promoting its proteasomal degradation. (B) BI-3802 induces polymerization of the BCL-6 BTB domain. Polymerization enhances recruitment of its cognate E3 ligase, promoting its degradation. (C) ATTECs such as 10O5 link LC-3 to mHTT, promoting its association with elongating phagophores, leading to degradation through autophagy. A second study from Słabicki et al. established induced polymerization as another mechanism by which molecular glues can induce degradation.(4) In a screen for inhibitors of factor B cell lymphoma 6 (BCL6), they confirmed a previously identified BCL6 inhibitor, BI-3802, unexpectedly induced BCL6 degradation, while a close analog did not. Hints of BI-3802 induced polymerization were established through fluorescence microscopy studies in cells expressing eGFP-BCL-6 where treatment with BI-3802 caused eGFP foci within 10 min of treatment to disappear through degradation within 100 min. In addition, size-exclusion chromatography in the presence of BI-3802 caused higher molecular weight species, while the close analog did not. Remarkably, they find that purified BCL6 in the presence of BI-3802 causes filamentous structures with a sinusoidal shape under negative stain electron microscopy. A cryo-EM structure (3.7 Å) showed that the glue binds a groove between dimers of the Bric-à-brac (BTB) domain of BCL6 and promotes polymerization primarily through hydrophobic interactions (Figure 1B). A slight modification of the solvent exposed group on the analog discouraged polymerization, explaining its inability to induce BCL6 degradation. They find that the E3 ligase, SIAH1, recognizes the BCL6 polymer and promotes its ubiquitination and degradation. Immunoprecipitation studies show that SIAH1 interacts with BCL6 in the absence of BI-3802, but their interaction is greatly enhanced in the presence of the molecular glue. Thus, through polymerization, BI-3802 increases its association with the cognate E3 ligase of BCL6, promoting its degradation. In a third and more directed approach, Li et al. used microarray screening to identify small molecules that selectively induce the interaction of mutant huntingtin protein (mHTT) and LC3, a protein important in autophagy substrate selection (Figure 1C).(5) While the proteasome is adept at degrading most proteins, it inefficiently recognizes bulky and aggregated substrates. Thus, degradation via autophagy was a more suitable approach for this aggregated target. Their screen identified 2 compounds (10O5, 8F20) that selectively induced degradation of mHTT and not WT Huntington protein, which they termed autophagy tethering compounds (ATTECs). ATTECs achieve selectivity by engaging a 72 glutamine polyQ region that is absent in the WT allele. In cells, the compounds localize to LC3B positive autophagosomes and induce degradation of mHTT in primary cultured neocortical neurons and drosophila models. Furthermore, inhibition of important autophagy mediators abrogated the function of the ATTECs, confirming its mechanism of action. Interestingly, the authors observed a hook effect, a phenomenon associated with heterobifunctional PROTACs explained by the dominance of binary (LC3:ATTEC or ATTEC:mHTT) rather than ternary (LC3:ATTEC:mHTT) protein–drug complexes at higher concentrations of the compound. Thus, it appears that ATTECs, due to their size and behavior, form a unique category that lies between traditional molecular glues (<500 Da) and PROTACS (independently engaging both target and E3 ligase). However, there is a need for more biophysical and structural studies to further solidify the proposed mechanism. These three studies reveal novel mechanisms by which molecular glues may be utilized to induce degradation of a given protein target, further diversifying the possibilities for drug development. The discovery of CR8 shows that the CRL4 E3 ligase complex can be more readily reprogrammed than previously anticipated; other E3 ligase components beyond the E3 substrate receptor may be co-opted to position target proteins in the respective ubiquitination zone. This study also creatively highlights that the drug target does not have to be the target of ubiquitination. The discoveries with BI-3802 demonstrate that induced polymerization, bearing resemblance to polymerization or aggregation observed in proteinopathies such as amyloidosis, may be a viable route to allow recruitment of a protein’s cognate E3 ligase and cause its degradation. In this example, the ability to “glue” is achieved via solvent-exposed, hydrophobic groups. Small chemical modifications of the solvent exposed region or mutations of the protein interface can completely abrogate degradation, highlighting the finicky nature of molecular glues. On one hand, this serves as a huge benefit that can lead to highly selective drugs, but on the other hand, it makes the design and identification of molecular glues extremely difficult. Indeed, thus far molecular glue identification has relied primarily on serendipity, but a a shift toward rational design is crucial to establish their general utility. Early examples are beginning to emerge, which rationalize some of these concepts to develop molecular glue degraders with new functions, but there is still much to be learned. Small molecule screening approaches such as those used in the identification of ATTECs, tied with thorough structural studies, will certainly expedite the rational discovery of new molecular glues that employ unique mechanisms for induced degradation of novel targets.(1) I would like to thank Georg Petzold, Dominico Vigil, and all scientists at Monte Rosa Therapeutics for helpful discussion and commentary. Figure 1 was created with BioRender.com. This article references 5 other publications.

中文翻译:

分子胶诱导蛋白质降解的新机制

迄今为止在靶向蛋白质降解 (TPD) 领域中定义的分子胶是单价小分子 (<500 Da),可重塑 E3 连接酶受体的表面,促进新底物的募集并促进其泛素化和随后的降解。蛋白酶体。TPD 中最成熟的分子胶是 a) 与 E3 连接酶 cereblon 结合的免疫调节“IMiD”类小分子和 b) 与 DCAF15 结合的芳基磺酰胺。由于这些分子胶不需要目标蛋白质上的可成药袋,因此它们准备好靶向难以成药的蛋白质。凭借催化作用机制和靶向非酶蛋白的能力,分子胶作为一种治疗方式具有非凡的前景。 (1) 到目前为止,TPD 中的分子胶降解剂依赖于招募 E3 连接酶受体;然而,最近的三项研究推动了如何利用分子胶诱导新底物降解的范围。为了发现具有未实现的诱导降解能力的药物,Słabicki 等人。研究了其细胞毒性作用需要 E3 连接酶机制的组件的临床前和临床药物之间的相关性。(2)通过这项练习,他们确定了 CR8,一种泛 CDK 抑制剂,其细胞毒性取决于损伤特异性 DNA 结合蛋白 1( DDB1) 并诱导细胞周期蛋白 K 的选择性降解。 DDB1 是一种衔接蛋白,可协调 E3 连接酶底物受体与 CUL4A/B-RBX1 连接酶核心之间的相互作用(图 1A,中间)。通过使用功能基因组学的广泛分析,他们确定了 DDB1 复合物的其他成分(CUL4B、NEDD8、UBEA3 和 RBX1),这是 CR8 诱导细胞周期蛋白 K 降解所需的。然而,他们无法确定被认为对底物泛素化至关重要的 E3 连接酶底物受体。相反,他们发现了 CDK12,一种与 E3 连接酶无关的成分,但它是细胞周期蛋白 K 降解所需的已知 CR8 结合剂。使用下拉和 TR-FRET 研究,他们表明 CDK12 对 DDB1 的亲和力非常弱,在 CR8 存在的情况下,这种亲和力增强了 500-1000 倍。他们解决了一个 3.5 Å 的结构,证实了 DDB1-CR8-CDK12 复合物,揭示了一个 2100 Å 的蛋白质-蛋白质界面,CR8 桥接了相互作用。它们的结构表明末端苯基吡啶环系统与 DDB1 的β-螺旋桨 C 结构域结合,它将细胞周期蛋白 K 定位在连接酶的泛素化区,通常被降解的底物占据(图 1A,底部)。有趣的是,这个结果也被两个具有独特化学性质的独立小组观察到,(1,3) 进一步强调了那些分子胶可以绕过 E3 连接酶受体的要求来劫持其功能并诱导降解。图 1. 分子胶诱导降解的机制,绕过 E3 连接酶受体的募集。(A) CR8 劫持 E3 连接酶接头 DDB1,而不是 E3 受体,将细胞周期蛋白 K 定位在泛素化区,促进其蛋白酶体降解。(B) BI-3802 诱导 BCL-6 BTB 域的聚合。聚合增强了其同源 E3 连接酶的募集,促进了其降解。(C) ATTECs 如 10O5 将 LC-3 链接到 mHTT,促进其与伸长的吞噬细胞的关联,通过自噬导致降解。Słabicki 等人的第二项研究。建立诱导聚合作为分子胶诱导降解的另一种机制。 (4) 在筛选因子 B 细胞淋巴瘤 6 (BCL6) 抑制剂时,他们证实了先前鉴定的 BCL6 抑制剂 BI-3802,意外诱导 BCL6 降解,而一个接近的模拟没有。BI-3802 诱导聚合的提示是通过荧光显微镜研究在表达 eGFP-BCL-6 的细胞中建立的,其中用 BI-3802 处理导致处理后 10 分钟内的 eGFP 病灶通过 100 分钟内的降解而消失。此外,在 BI-3802 存在下的尺寸排阻色谱法导致更高的分子量种类,而相近的类似物则没有。值得注意的是,他们发现在 BI-3802 存在下纯化的 BCL6 会在负染色电子显微镜下产生具有正弦形状的丝状结构。冷冻电镜结构 (3.7 Å) 显示胶水结合 BCL6 的 Bric-à-brac (BTB) 域的二聚体之间的凹槽,并主要通过疏水相互作用促进聚合(图 1B)。类似物上溶剂暴露基团的轻微修改会阻止聚合,解释其无法诱导 BCL6 降解。他们发现 E3 连接酶 SIAH1 识别 BCL6 聚合物并促进其泛素化和降解。免疫沉淀研究表明,在没有 BI-3802 的情况下,SIAH1 与 BCL6 相互作用,但在分子胶存在的情况下,它们的相互作用大大增强。因此,通过聚合,BI-3802 增加其与 BCL6 的同源 E3 连接酶的关联,促进其降解。在第三种更直接的方法中,Li 等人。使用微阵列筛选来鉴定选择性诱导突变亨廷顿蛋白 (mHTT) 和 LC3 相互作用的小分子,LC3 是一种在自噬底物选择中很重要的蛋白质(图 1C)。(5)虽然蛋白酶体擅长降解大多数蛋白质,但它识别效率低下庞大和聚集的基材。因此,通过自噬降解是更适合这种聚集目标的方法。他们的筛选确定了 2 种化合物(10O5、8F20),它们选择性诱导 mHTT 降解,而不是 WT Huntington 蛋白,他们将其称为自噬束缚化合物(ATTEC)。ATTECs 通过接合 WT 等位基因中不存在的 72 个谷氨酰胺 polyQ 区域来实现选择性。在细胞中,这些化合物定位于 LC3B 阳性自噬体,并在原代培养的新皮质神经元和果蝇模型中诱导 mHTT 降解。此外,抑制重要的自噬介质会取消 ATTEC 的功能,证实其作用机制。有趣的是,作者观察到了钩状效应,这是一种与异双功能 PROTAC 相关的现象,解释为二元(LC3:ATTEC 或 ATTEC:mHTT)而不是三元(LC3:ATTEC:mHTT)蛋白质-药物复合物在更高浓度的化合物中占优势. 因此,似乎 ATTECs 由于其大小和行为,形成了一个独特的类别,介于传统分子胶(<500 Da)和 PROTACS(独立参与目标和 E3 连接酶)之间。然而,需要进行更多的生物物理和结构研究,以进一步巩固拟议的机制。这三项研究揭示了分子胶可用于诱导给定蛋白质靶标降解的新机制,进一步丰富了药物开发的可能性。CR8 的发现表明 CRL4 E3 连接酶复合物比之前预期的更容易重新编程;E3 底物受体之外的其他 E3 连接酶组分可以被共同选择以将目标蛋白定位在相应的泛素化区域中。这项研究还创造性地强调了药物靶标不一定是泛素化的靶标。BI-3802 的发现表明诱导聚合,与在淀粉样变性等蛋白质病中观察到的聚合或聚集相似,可能是允许募集蛋白质的同源 E3 连接酶并导致其降解的可行途径。在这个例子中,“粘合”的能力是通过暴露在溶剂中的疏水基团实现的。溶剂暴露区域的小的化学修饰或蛋白质界面的突变可以完全消除降解,突出分子胶的挑剔性质。一方面,这是一个巨大的好处,可以产生高选择性的药物,但另一方面,它使分子胶的设计和识别变得极其困难。事实上,到目前为止,分子胶的识别主要依赖于偶然性,但向理性设计的转变对于建立它们的普遍效用至关重要。早期的例子开始出现,这使其中一些概念合理化,以开发具有新功能的分子胶降解剂,但仍有很多东西需要学习。小分子筛选方法,例如用于鉴定 ATTEC 的方法,与彻底的结构研究相结合,肯定会加速合理发现新分子胶水,这些胶水采用独特机制诱导新目标降解。 (1) 我要感谢 Georg Petzold、Dominico Vigil 和 Monte Rosa Therapeutics 的所有科学家进行有益的讨论和评论。图 1 是使用 BioRender.com 创建的。本文引用了 5 篇其他出版物。肯定会加速合理发现采用独特机制诱导新靶点降解的新分子胶。(1) 我要感谢 Georg Petzold、Dominico Vigil 和 Monte Rosa Therapeutics 的所有科学家提供有益的讨论和评论。图 1 是使用 BioRender.com 创建的。本文引用了 5 篇其他出版物。肯定会加速合理发现采用独特机制诱导新靶点降解的新分子胶。(1) 我要感谢 Georg Petzold、Dominico Vigil 和 Monte Rosa Therapeutics 的所有科学家提供有益的讨论和评论。图 1 是使用 BioRender.com 创建的。本文引用了 5 篇其他出版物。
更新日期:2021-08-10
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