Department of Chemistry, University of California, Berkeley, Berkeley, CA 94720, USANovartis-Berkeley Translational Chemical Biology Institute, Berkeley, CA 94720, USAInnovative Genomics Institute, Berkeley, CA 94704, USA
Department of Chemistry, University of California, Berkeley, Berkeley, CA 94720, USANovartis-Berkeley Translational Chemical Biology Institute, Berkeley, CA 94720, USAInnovative Genomics Institute, Berkeley, CA 94704, USA
Department of Chemistry, University of California, Berkeley, Berkeley, CA 94720, USANovartis-Berkeley Translational Chemical Biology Institute, Berkeley, CA 94720, USAInnovative Genomics Institute, Berkeley, CA 94704, USA
Novartis-Berkeley Translational Chemical Biology Institute, Berkeley, CA 94720, USANovartis Institutes for BioMedical Research, Emeryville, CA 94608, USA
Novartis-Berkeley Translational Chemical Biology Institute, Berkeley, CA 94720, USANovartis Institutes for BioMedical Research, Cambridge, MA 02139, USA
Novartis-Berkeley Translational Chemical Biology Institute, Berkeley, CA 94720, USANovartis Institutes for BioMedical Research, Cambridge, MA 02139, USA
Novartis-Berkeley Translational Chemical Biology Institute, Berkeley, CA 94720, USANovartis Institutes for BioMedical Research, Cambridge, MA 02139, USA
Department of Chemistry, University of California, Berkeley, Berkeley, CA 94720, USANovartis-Berkeley Translational Chemical Biology Institute, Berkeley, CA 94720, USAInnovative Genomics Institute, Berkeley, CA 94704, USADepartment of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA
We use chemoproteomic platforms to discover a covalent molecular glue degrader 我们使用化学蛋白质组学平台发现一种共价分子胶粘剂降解剂
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We identify a cysteine-reactive covalent glue degrader EN450 我们确定了一种半胱氨酸反应性的共价粘合剂降解剂 EN450
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EN450 glues together E2 ubiquitin-conjugating enzyme UBE2D with NFKB1 EN450 使用 E2 泛素连接酶 UBE2D 与 NFKB1 粘合在一起
Summary 摘要
Targeted protein degradation has arisen as a powerful therapeutic modality for degrading disease targets. While proteolysis-targeting chimera (PROTAC) design is more modular, the discovery of molecular glue degraders has been more challenging. Here, we have coupled the phenotypic screening of a covalent ligand library with chemoproteomic approaches to rapidly discover a covalent molecular glue degrader and associated mechanisms. We have identified a cysteine-reactive covalent ligand EN450 that impairs leukemia cell viability in a NEDDylation and proteasome-dependent manner. Chemoproteomic profiling revealed covalent interaction of EN450 with an allosteric C111 in the E2 ubiquitin-conjugating enzyme UBE2D. Quantitative proteomic profiling revealed the degradation of the oncogenic transcription factor NFKB1 as a putative degradation target. Our study thus puts forth the discovery of a covalent molecular glue degrader that uniquely induced the proximity of an E2 with a transcription factor to induce its degradation in cancer cells. 针对蛋白质降解已经成为一种强大的治疗模式，用于降解疾病靶标。虽然蛋白质降解靶向嵌合物（PROTAC）设计更具模块化性，但发现分子粘合剂降解物却更具挑战性。在这里，我们将共价配体库的表型筛选与化学蛋白质组学方法相结合，快速发现了一种共价分子粘合剂降解物及其相关机制。我们确定了一种半胱氨酸反应性共价配体 EN450，以 NEDDylation 和蛋白酶体依赖的方式损害了白血病细胞的生存能力。化学蛋白质组学分析显示 EN450 与 E2 泛素连接酶 UBE2D 中的一个变构 C111 发生共价相互作用。定量蛋白质组学分析显示了致癌转录因子 NFKB1 的降解作为一个潜在的降解靶标。因此，我们的研究提出了一种共价分子粘合剂降解物的发现，其独特地诱导了 E2 与转录因子的接近，从而在癌细胞中诱导其降解。
Most small-molecule drugs in the clinic operate through classical occupancy-driven pharmacology that consists of small molecules binding to deep active site binding pockets and resulting in functional modulation of the target. However, many therapeutic target proteins have been deemed “undruggable” since they do not possess well-defined, functionally relevant binding pockets, thus rendering these proteins inaccessible to classical drug discovery approaches.
Targeted protein degradation using heterobifunctional proteolysis-targeting chimeras (PROTACs) or molecular glues has arisen as a powerful alternative therapeutic modality aiming at degradation instead of inhibition of the disease target.
While heterobifunctional PROTACs still require protein-targeting ligands that are capable of binding to the target protein with decent potency, monovalent molecular glue degraders can exploit shallower protein interfaces to induce ternary complex formation and subsequent ubiquitination and degradation of specific proteins.
While molecular glue degraders are potentially more attractive and drug-like, most molecular glue degraders have been discovered fortuitously and rational discovery of molecular glue degraders has remained challenging. 临床上大多数小分子药物通过经典的占位驱动药理学发挥作用，即小分子结合到深层活性位点结合口袋，导致靶点的功能调节。然而，许多治疗靶蛋白被认为是“难药化”的，因为它们没有明确定义的、功能相关的结合口袋，因此这些蛋白对经典药物发现方法是不可及的。利用异双功能蛋白降解靶向融合物（PROTACs）或分子胶已成为一种强大的替代治疗模式，旨在降解疾病靶点而不是抑制它。虽然异双功能 PROTACs 仍需要能够具有相当强效的结合到靶蛋白的蛋白靶向配体，但单价分子胶降解剂可以利用较浅的蛋白界面诱导三元复合物形成，随后泛素化和降解特定蛋白。 虽然分子胶黏剂降解剂可能更具吸引力和药物样性，但大多数分子胶黏剂降解剂都是偶然发现的，而对分子胶黏剂降解剂的理性发现仍然具有挑战性。
Recent studies by Mayor-Ruiz et al. have showcased innovative phenotypic screening paradigms for rapidly discovering small molecules that exert anti-cancer activity through molecular glue degrader mechanisms.
These screens for anti-cancer small molecules consisted of counter screens for an attenuated phenotype in hyponeddylation cell lines to identify molecules that exerted their phenotypes through a Cullin E3 ligase-dependent mechanism. However, the backend mechanistic elucidation of the ternary complex components—identifying the degraded target and ubiquitin-proteasome component that were brought together by the small molecule—required whole genome-wide CRISPR screens, which can sometimes be laborious and may yield indirect targets in addition to direct targets of the small molecule.
Covalent chemoproteomic approaches have arisen as powerful platforms for coupling phenotypic screening of covalent electrophile libraries with rapid mechanistic deconvolution.
As such, we conjectured that coupling the screening of a covalent ligand library for molecular glue degraders with backend chemoproteomic and quantitative proteomic approaches would provide rapid discovery of molecular glue degraders and their ternary complex components and downstream mechanisms. In this study, we phenotypically screened a library of covalent ligands for anti-proliferative compounds and used chemoproteomic platforms to discover a covalent molecular glue degrader that uniquely relies on an E2 ubiquitin-conjugating enzyme. 共价化学蛋白质组学方法已经成为将共价亲电子库的表型筛选与快速机械解构相结合的强大平台。因此，我们推测，将共价配体库的筛选与后端化学蛋白质组学和定量蛋白质组学方法相结合，将快速发现分子胶粘剂降解剂及其三元复合物组分和下游机制。在这项研究中，我们对一系列共价配体进行了表型筛选，寻找抗增殖化合物，并利用化学蛋白质组学平台发现了一种共价分子胶粘剂降解剂，其独特依赖于E2泛素连接酶。
Results 结果
To discover covalent molecular glue degraders, we screened a library of 750 cysteine-reactive covalent ligands for anti-proliferative effects in HAP1 leukemia cancer cells (Figure 1A and Table S1). We identified 11 hits from our primary screen, of which three of these hits—EN222, EN450, and EN266—showed reproducible impairments in HAP1 cell proliferation by greater than 90% (Figure 1B). Among these three hits, we next sought to identify if these compounds may be exerting their anti-proliferative effects through a Cullin E3 ubiquitin ligase-dependent mechanism. To this end, we counter-screened our hit compounds in UBE2M knockdown hyponeddylation lines alongside dCeMM1, a positive control molecular glue degrader previously identified by Mayor-Ruiz et al. that induces degradation of RBM39 in a CRL4DCAF15-dependent manner (Figures 1C and 1D).
As such, compounds that show attenuated phenotypes in these hyponeddylation cell lines would indicate a Cullin E3 ligase-dependent mechanism. EN266, EN450, and the positive control glue degrader dCeMM1, but not EN222, showed significant attenuation of anti-proliferative effects in HAP1 cells with EN450 showing the most robust effects (Figures 1D and 1E). EN450 is a fragment ligand containing a cysteine-reactive acrylamide warhead (Figure 1E). EN450 as well as dCeMM1 also showed significant attenuation of anti-proliferative effects upon pre-treatment of HAP1 cells with the NEDDylation inhibitor MLN4924 and the proteasome inhibitor bortezomib, further cementing that EN450 was exerting its anti-proliferative effects through a Cullin E3 ligase and proteasome-dependent mechanism (Figures 1F and 1G). 为了发现共价分子粘合剂降解剂，我们在HAP1白血病癌细胞中筛选了一个包含750个半胱氨酸反应性共价配体的文库，以寻找抗增殖效应（图1A和表S1）。我们从初筛中鉴定出11个命中物，其中三个命中物——EN222、EN450和EN266——显示出HAP1细胞增殖受到超过90%的可重复损害（图1B）。在这三个命中物中，我们接下来试图确定这些化合物是否通过Cullin E3泛素连接酶依赖机制发挥其抗增殖效应。为此，我们在UBE2M敲除的hyponeddylation细胞系中与dCeMM1进行了对照筛选，dCeMM1是由Mayor-Ruiz等人先前鉴定的一种阳性对照分子粘合剂降解剂，以CRL4 DCAF15 -依赖方式诱导RBM39的降解（图1C和1D）。
Figure 1Discovery of a covalent molecular glue degrader with anti-proliferative activities in HAP1 leukemia cancer cells 图1 在HAP1白血病癌细胞中发现具有抗增殖活性的共价分子粘合剂降解剂
(A) HAP1 cell viability screen of cysteine-reactive covalent ligands. DMSO vehicle or cysteine-reactive covalent ligands (50 μM) were treated in HAP1 cells for 24 h and cell viability was assessed by Hoescht staining. Compounds highlighted in red and labeled are those that impaired HAP1 cell viability by greater than 90% compared with vehicle-treated controls. This screen was performed with n = 1 biological replicate/group.
(B) The 11 hit compounds from (A) were rescreened with n = 3 biologically independent replicates/group in HAP1 cells under the same conditions. Individual replicate values shown. Among these hits, EN222, EN226, and EN450 showed reproducible impaired HAP1 cell viability of greater than 90% compared with DMSO vehicle-treated controls.
(C) Knockdown of UBE2M. HAP1 cells were transiently transfected with siControl or siUBE2M oligonucleotides and knockdown of UBE2M was assessed by western blotting compared with GAPDH loading control.
(D) HAP1 cell viability in siControl and siUBE2M cells treated with DMSO vehicle, the positive control molecular glue degrader dCEMM1, EN222, EN266, or EN450 (50 μM) for 24 h, assessed by Hoescht staining.
(E) Structure of EN450 with the reactive acrylamide handle highlighted in red.
(F) Attenuation of HAP1 cell viability impairments by NEDDylation inhibitor MLN4924. HAP1 cells were pre-treated with DMSO vehicle or MLN4924 (1 μM) for 1 h prior to treatment of cells with DMSO vehicle, dCEMM1, or EN450 (50 μM) for 24 h, and cell viability was assessed by Hoechst staining.
(G) Attenuation of HAP1 cell viability impairments by proteasome inhibitor bortezomib. HAP1 cells were pre-treated with DMSO vehicle or bortezomib (1 μM) for 1 h prior to treatment of cells with DMSO vehicle or EN450 (50 μM) for 24 h, and cell viability was assessed by Hoechst staining. Data in (D), (F), and (G) are average ±SEM of n = 3–6 biologically independent replicates/group with individual replicate values also shown. Statistical significance is expressed as ∗p < 0.05 compared with siControl or DMSO control groups in (D), (F), and (G), #p < 0.05 compared with corresponding siControl or DMSO pre-treated treatment groups, and calculated with Student’s unpaired two-tailed t tests. Related to Table S1.
Based on these data, we conjectured that EN450 was a molecular glue inducing the proximity of a target protein with a component of the ubiquitin-proteasome system (UPS) to form a ternary complex leading to the ubiquitination and proteasome-mediated degradation of the target protein and subsequent anti-proliferative effects in HAP1 cells. To identify proteome-wide covalent targets of EN450, we performed cysteine chemoproteomic profiling using isotopic tandem orthogonal proteolysis-activity-based protein profiling (isoTOP-ABPP).
Through competitive profiling of EN450 covalent protein targeting in HAP1 cells against cysteine-reactive alkyne-functionalized iodoacetamide probe labeling, we identified 81 targets that showed significant engagement—control/EN450-treated ratio of >1.3 with adjusted p values <0.05—among 4,501 cysteines quantified (Figure 2A and Table S2). Given that we and others have previously shown that the UPS is highly ligandable with covalent ligands, that even partial covalent occupancy of a UPS effector protein can lead to significant degradation of the target protein due to the catalytic nature of degraders, and that EN450 is a small-molecule fragment that is not likely to engage in high-affinity interactions, we conjectured that the covalent interaction of EN450 is likely occurring with a component of the ubiquitin-proteasome system, rather than the target protein.
Among these targets, only one protein was involved in the UPS and the Cullin E3 ligase machinery—C111 of ubiquitin-conjugating enzyme E2D (UBE2D). C111 is an allosteric cysteine in UBE2D that is distal from the cysteine (C85) that undergoes ubiquitin conjugation during ubiquitin transfer. Because of the high degree of sequence identity between UBE2D isoforms, we could not distinguish whether EN450 targeted C111 on UBE2D1, UBE2D2, UBE2D3, or UBE2D4. Nonetheless, UBE2D is one of the key E2 ligases that are coupled with the Cullin E3 ligase complex, which is consistent with attenuation of EN450 anti-proliferative effects with a NEDDylation inhibitor of Cullin E3 ligases.
(A) Cysteine chemoproteomic profiling of EN450 targets in HAP1 cells using isoTOP-ABPP. HAP1 cells were treated with DMSO vehicle or EN450 (50 μM) for 3 h, after which resulting cell lysates were labeled with an alkyne-functionalized iodoacetamide cysteine-reactive probe (200 μM) for 1 h, and an isotopically light (for DMSO) or heavy (for EN450) biotin-azide handle bearing a TEV protease recognition peptide was appended by CuAAC. Control and treated proteomes were combined in a 1:1 ratio, taken through the isoTOP-ABPP procedure, and light/heavy probe-modified peptides were analyzed by LC-MS/MS and quantified. Shown in blue are probe-modified peptides with light/heavy ratios >1.3 with adjusted p < 0.05. Shown in red is C111 of UBE2D. Data shown are average ratios from n = 4 biologically independent replicates/group.
(B) Structure of alkyne-functionalized probe derivative of EN450—EK-1-8 with cysteine-reactive acrylamide warhead highlighted in red.
(C) Attenuation of HAP1 cell viability impairments by NEDDylation inhibitor MLN4924. HAP1 cells were pre-treated with DMSO vehicle or MLN4924 (1 μM) for 1 h prior to treatment of cells with DMSO vehicle, EK-1-8 (100 μM) for 24 h, and cell viability was assessed by Hoechst staining.
(D) EK-1-8 labeling of recombinant pure human UBE2D1 C85S mutant protein. UBE2D1 C85S mutant protein was labeled with DMSO vehicle or EK-1-8 for 30 min, after which an azide-functionalized rhodamine handle was appended onto probe-labeled protein by CuAAC, after which proteins were resolved by SDS-PAGE and visualized by in-gel fluorescence. Protein loading was assessed by silver staining. Gels are representative of n = 3 biologically independent replicates/group.
(E) UBE2D1 pulldown from HAP1 cells with EK-1-8 probe. HAP1 cells were pre-treated with DMSO vehicle or EN450 (50 μM) for 1 h prior to treatment of cells with DMSO vehicle or EK-1-8 (10 μM) for 3 h. Probe-labeled proteins from resulting cell lysates were subsequently appended to an azide-functionalized biotin handle by CuAAC, avidin-enriched, eluted and separated by SDS/PAGE and blotted for UBE2D1 and unrelated protein GAPDH. Shown on the blot are also input UBE2D1 and GAPDH protein levels among the three groups shown. Blots are representative of n = 3 biologically independent replicates/group.
(F) Confirmation of UBE2D1 knockdown. HAP1 cells were transiently transfected with siControl or siUBE2D1 oligonucleotides and UBE2D1 and loading control GAPDH expression were assessed after 72 h by western blotting. Blot is representative of n = 3 biologically independent replicates/group.
(G) Percent HAP1 cell proliferation in siControl and siUBE2D1 cells treated with EN450 for 24 h compared with DMSO vehicle-treated controls. Data shown in (C) and (G) are average ±SEM of n = 6–30 biologically independent replicates/group with individual replicate values also shown. Statistical significance in (C) is expressed as ∗p < 0.05 compared with DMSO-pre-treated control groups and #p < 0.05 compared with DMSO-pre-treated EK-1-8-treated groups and in (G) is expressed as ∗p < 0.05 compared with the corresponding treatment group in siControl cells. Related to Table S2, Figures S1, and S2.
To further characterize interactions of EN450 with UBE2D, we synthesized an alkyne-functionalized probe derivative of EN450—EK-1-8 (Figure 2B). EK-1-8 still impaired HAP1 cell viability in a NEDDylation-dependent manner (Figure 2C). EK-1-8 directly and covalently labeled recombinant UBE2D1 C85S mutant protein in a dose-responsive manner (Figure 2D). This labeling was attenuated, albeit not completely, upon mutation of both C85 and C111 on UBE2D1 to serines (Figure S1A). We interpreted the residual labeling evident in the double mutant as another allosteric cysteine on UBE2D1 that may be additionally reactive with EN450, potentially in the absence of its main reactive C111. We further demonstrated that EK-1-8 engaged UBE2D1, but not an unrelated protein such as GAPDH, in HAP1 cells and that this engagement was outcompeted in part by EN450, as assessed by enrichment of UBE2D1 in HAP1 cells by EK-1-8 treatment, appendage of biotin through azide-alkyne cycloaddition (CuAAC), avidin-enrichment, elution, and blotting (Figure 2E). We also synthesized the non-reactive analog of EN450, EK-1-37, as well as its alkyne-functionalized derivative, EK-1-38, to ascertain the contribution of the covalent acrylamide warhead to the observed effects (Figure S1B). EK-1-37 did not impair HAP1 cell viability and EK-1-38 expectedly did not show any covalent modification of UBE2D1 C85S, compared with EK-1-8 (Figures S1C and S1D). 为了进一步表征EN450与UBE2D的相互作用，我们合成了EN450的炔基功能化探针衍生物EK-1-8（图2B）。EK-1-8仍然以NEDDylation依赖的方式影响HAP1细胞的存活能力（图2C）。EK-1-8以剂量响应的方式直接和共价地标记了重组的UBE2D1 C85S突变蛋白（图2D）。尽管在UBE2D1的C85和C111都突变为丝氨酸时，这种标记有所减弱，但并非完全消失（图S1A）。我们将双突变体中残留的标记解释为UBE2D1上另一个可能与EN450发生反应的变构半胱氨酸，可能在其主要反应位点C111缺失的情况下发生反应。我们进一步证明了EK-1-8与UBE2D1发生了相互作用，但与HAP1细胞中的GAPDH等无关蛋白不发生相互作用，并且通过叠氮-炔基环加成（CuAAC）连接生物素，通过亲和素富集、洗脱和印迹分析（图2E）部分地被EN450所取代。 我们还合成了EN450的非反应性类似物EK-1-37，以及其炔基功能化衍生物EK-1-38，以确定共价丙烯酰胺战斧对观察到的效应的贡献（图S1B）。EK-1-37不影响HAP1细胞的存活能力，EK-1-38预期地与EK-1-8相比没有显示对UBE2D1 C85S的任何共价修饰（图S1C和S1D）。
To understand the contribution of the individual UBE2D isoforms to EN450 effects, we assessed dose-responsive effects of EN450 on HAP1 cell proliferation upon knockdown of UBE2D1, UBE2D2, UBE2D3, and UBE2D4 and knockdown of all four enzymes (Figures 2F, 2G, and S2). Knockdown of UBE2D1 conferred the greatest degree of resistance to EN450 anti-proliferative effects, followed by knockdown of UBE2D4 and knockdown of all four enzymes. Intriguingly, knockdown of UBE2D2 and UBE2D3 led to hypersensitization to EN450, suggesting divergent roles of each individual E2 ligase isoform in relation to interactions with EN450 despite their high degree of sequence identity (Figures 2F, 2G, and S1). Nonetheless, our data indicated that UBE2D1 and UBE2D4 are likely the UPS components covalently engaged by EN450 to, presumably along with the recruitment of its associated Cullin E3 ligase complex, ubiquitinate and degrade a yet unknown target protein to exert its anti-proliferative effects. 为了理解个体UBE2D同工酶对EN450效应的贡献，我们评估了在敲除UBE2D1、UBE2D2、UBE2D3和UBE2D4以及敲除所有四种酶的情况下，EN450对HAP1细胞增殖的剂量响应效应（图2F、2G和S2）。敲除UBE2D1对EN450的抗增殖效应产生了最大程度的抵抗，其次是敲除UBE2D4和敲除所有四种酶。有趣的是，敲除UBE2D2和UBE2D3导致对EN450的过敏反应，表明每个单独的E2连接酶同工酶在与EN450的相互作用中可能具有不同的作用，尽管它们具有高度相似的序列（图2F、2G和S1）。尽管如此，我们的数据表明，UBE2D1和UBE2D4很可能是EN450共价结合的UPS组分，可能与其相关的Cullin E3连接酶复合物一起，泛素化和降解一个尚未知的靶蛋白，以发挥其抗增殖效应。
To identify the degraded target protein, we next performed tandem mass tagging (TMT)-based quantitative proteomic profiling to map protein level changes from EN450 treatment in HAP1 cells. This profiling effort revealed only one protein—the NFKB1 p105 subunit—that was significantly reduced in levels by >4-fold upon EN450 treatment compared with vehicle-treated controls (Figure 3A and Table S3). Loss of both the precursor p105 protein and further processed p50 product of NFKB1 was confirmed by western blotting (Figures 3B–3D). The non-reactive EK-1-37 control did not lower NFKB1 levels (Figure S3A). We further confirmed that the reduction in NFKB1 p105 and p50 levels by EN450 treatment was not observed when HAP1 cells were pre-treated with the NEDDylation inhibitor (Figures 3E and 3F). MLN4924 treatment itself reduced NFKB1 levels potentially due to its inherent toxicity in leukemia cancer cells,
but EN450 did not further lower NFKB1 levels (Figures 3E and 3F). 为了识别降解的靶蛋白，我们接下来进行串联质谱标记（TMT）基础的定量蛋白质组学分析，以绘制HAP1细胞中EN450处理引起的蛋白水平变化。这项分析揭示了只有一个蛋白质——NFKB1 p105亚基，在EN450处理后与车辆处理对照相比水平显著降低了超过4倍（图3A和表S3）。通过免疫印迹验证了NFKB1的前体p105蛋白和进一步加工的p50产物的丧失（图3B-3D）。非反应性的EK-1-37对照未降低NFKB1水平（图S3A）。我们进一步确认，当HAP1细胞预先用NEDDylation抑制剂处理时，EN450处理不会导致NFKB1 p105和p50水平的降低（图3E和3F）。MLN4924处理本身降低了NFKB1水平，可能是由于其在白血病癌细胞中的固有毒性，但EN450并未进一步降低NFKB1水平（图3E和3F）。
Figure 3Identification of the protein degraded by EN450 图3：鉴定被EN450降解的蛋白质
(A) TMT-based quantitative proteomic profiling of EN450 in HAP1 cells. HAP1 cells were treated with DMSO vehicle or EN450 (50 μM) for 24 h. Shown in red is the only protein reduced in levels by >4-fold with p value <0.05—NFKB1. Data shown are from n = 3 biologically independent replicates/group.
(B) Western blotting analysis of p105 and p50 isoforms of NFKB1 and loading control vinculin levels in HAP1 cells treated with DMSO vehicle or EN450 (50 μM) for 24 h.
(C and D) Quantification of experiment described in (B).
(E) NEDDylation inhibitor attenuates NFKB1 loss by EN450 treatment in HAP1 cells. HAP1 cells were pre-treated with DMSO vehicle or MLN4924 (1 μM) for 1 h prior to treatment of cells with DMSO vehicle or EN450 (50 μM) for 24 h. NFKB1 and loading control vinculin levels were assessed by western blotting.
(F) Quantification of experiment described in (E).
(G) Ternary complex formation between UBE2D1, EN450, and NFKB1. Recombinant pure human GST-NFKB1, and UBE2D1 proteins were incubated with DMSO vehicle or EN450 (50 μM) for 1 h, after which GST-NFKB1 was enriched and the pulldown eluate was blotted for NFKB1 and UBE2D1.
(H) Quantification of pulldown experiment described in (G).
(I and J) Ubiquitination assay with UBE2D1, ATP, FLAG-Ubiquitin, and NFKB1 pure proteins with the exclusion or addition of the CUL4A/RBX1/NEDD8 Cullin complex. This incubation was treated with DMSO vehicle or EN450 (50 μM) for 1 h. NFKB1 ubiquitination was detected by western blotting visualizing FLAG-Ub in (I) and NFKB1 in (J). Blots shown in (B), (E), and (G) are representative of n = 3 biologically independent replicates/group. Data shown in (C), (D), (F), and (H) are average ±SEM of n = 3–8 biologically independent replicates/group with individual replicate values also shown. Statistical significance in (C), (D), (F), and (H) is expressed as ∗p < 0.05 compared with DMSO vehicle-treated controls and NS denotes not significant. Related to Table S3 and Figure S3.
Next, we sought to demonstrate EN450-mediated ternary complex formation between UBE2D and NFKB1. First, we demonstrated that EN450 promoted ternary complex formation between UBE2D1 and NFKB1, through pulldown studies with recombinant GST-NFKB1 and UBE2D1 protein (Figures 3G and 3H). We then showed that both p105 and p50 NFKB1 isoforms, but not an unrelated GAPDH control, were pulled down by EK-1-8 from HAP1 cells in addition to our previously described results showing pulldown of UBE2D1 (Figures 2E and S3B). Third, reconstitution of UBE2D1-mediated ubiquitination activity showed that EN450 enhanced ubiquitination of NFKB1 only in the presence of the full CUL4A/RBX1/NEDD8 complex (Figures 3I and 3J). We also demonstrated that UBE2D1 knockdown alone has no influence on NFKB1 levels (Figure S3C). Overall, these results were consistent with EN450 forming a productive ternary complex between UBE2D and NFKB1 in cells that resulted in NFKB1 ubiquitination and degradation. 接下来，我们试图证明EN450介导的UBE2D和NFKB1之间的三元复合物形成。首先，我们通过重组GST-NFKB1和UBE2D1蛋白的拉下研究表明EN450促进了UBE2D1和NFKB1之间的三元复合物形成（图3G和3H）。然后，我们展示了除了我们先前描述的UBE2D1的拉下结果外，EK-1-8还从HAP1细胞中拉下了p105和p50 NFKB1亚型，而与无关的GAPDH对照无关（图2E和S3B）。第三，UBE2D1介导的泛素化活性的重构显示，EN450仅在完整的CUL4A/RBX1/NEDD8复合物存在时增强了NFKB1的泛素化（图3I和3J）。我们还证明，仅仅UBE2D1的沉默对NFKB1水平没有影响（图S3C）。总的来说，这些结果与EN450在细胞中形成了一个有益的UBE2D和NFKB1之间的三元复合物，导致了NFKB1的泛素化和降解是一致的。
EN450 is clearly not a selective compound, as demonstrated by ∼80 potential cysteine targets identified by chemoproteomic profiling. Nonetheless, we sought to link the degradation of NFKB1, a known oncogenic transcription factor,
to the mechanism underlying EN450 anti-proliferative effects. Stable overexpression of NFKB1 in HAP1 cells led to significant attenuation of EN450-mediated anti-proliferative effects in a dose-dependent manner (Figures 4A and 4B ). We also transiently transfected NFKB1 in HEK293T cells, where we also demonstrated NEDDylation-dependent cell viability impairments and reduction in NFKB1 levels with EN450 treatment (Figures 4D and 4E). We observed an even more robust rescue of EN450-mediated anti-proliferative effects (Figure 4F). While we believe that EN450 likely has additional polypharmacological mechanisms underlying its anti-proliferative effects, our data suggest that NFKB1 is at least partially involved in the anti-cancer effects of EN450. EN450显然不是一种选择性化合物，这是通过化学蛋白质组学分析鉴定出的约80个潜在半胱氨酸靶点所证明的。尽管如此，我们试图将已知的致癌转录因子NFKB1的降解与EN450抗增殖效应的机制联系起来。在HAP1细胞中稳定过表达NFKB1显著减弱了EN450介导的抗增殖效应，且呈剂量依赖性（图4A和4B）。我们还在HEK293T细胞中瞬时转染NFKB1，结果显示NEDDylation依赖的细胞存活受损以及EN450处理后NFKB1水平降低（图4D和4E）。我们观察到EN450介导的抗增殖效应得到了更强有力的恢复（图4F）。虽然我们认为EN450可能具有其他多药机制来支持其抗增殖效应，但我们的数据表明NFKB1至少部分参与了EN450的抗癌效应。
Figure 4Understanding the role of NFKB1 in EN450-mediated effects 图4：理解NFKB1在EN450介导效应中的作用
(A) Stable lentiviral overexpression of FLAG-NFKB1 in HAP1 cells assessed by western blotting for NFKB1, FLAG, and loading control GAPDH.
(B) HAP1 eGFP control or FLAG-NFKB1 overexpressing cell viability from cells treated with DMSO vehicle or EN450 for 24 h.
(C) Attenuation of HEK293T cell viability impairments by NEDDylation inhibitor MLN4924. HEK293T cells were pre-treated with DMSO vehicle or MLN4924 (1 μM) for 1 h prior to treatment of cells with DMSO vehicle or EN450 (50 μM) for 24 h, and cell viability was assessed by Hoechst staining.
(D) NFKB1 and loading control vinculin levels in HEK293T cells pre-treated with DMSO vehicle or MLN4924 (1 μM) 1 h prior to treatment of cells with DMSO vehicle or EN450 (50 μM) for 24 h, assessed by western blotting.
(E) Transient transfection and overexpression of FLAG-NFKB1 in HEK293T cells assessed by western blotting of NFKB1, FLAG, and loading control GAPDH.
(F) HEK293T eGFP control or FLAG-NFKB1 overexpressing cell viability from cells treated with DMSO vehicle or EN450 (50 μM) for 24 h. Blots shown in (A), (D), and (E) are representative of n = 3 biologically independent replicates/group. Data shown in (C) and (F) are average ±SEM of n = 3–8 biologically independent replicates/group with individual replicate values also shown. Statistical significance in (C) and (F) is expressed as ∗p < 0.05 compared with DMSO vehicle-treated controls or eGFP-expressing control cells and as #p < 0.05 compared with DMSO pre-treated EN450-treated groups in (C) or EN450-treated eGFP-expressing cells in (F).
In this study, we have used covalent chemoproteomic approaches to discover a covalent molecular glue degrader that uniquely induces the proximity of an E2 conjugating enzyme UBE2D1 to the oncogenic nuclear transcription factor NFKB1 to ubiquitinate and degrade NFKB1 in a proteasome- and NEDDylation-dependent manner. Our study also reveals that allosteric sites within E2 ubiquitin-conjugating enzymes, rather than E3 ligases or E3 ligase substrate receptor or adapter proteins, can be directly targeted by small molecules to engage in molecular glue interactions with neo-substrate proteins. Our study is reminiscent of previously reported studies by Słabicki et al. demonstrating that the CDK8 inhibitor CR8 acts as a molecular glue degrader of cyclin K through engaging a core adapter protein of the CUL4 E3 ligase machinery DDB1, thereby bypassing the requirement for a substrate receptor for ubiquitination and degradation.
Both our and the Słabicki et al. study demonstrate that core proteins within the ubiquitin-proteasome machinery, such as E2 ligases and DDB1, can be exploited for targeted protein degradation application. This indicates that small-molecule recruiters can be developed against these core components of the UPS for use in heterobifunctional degraders. While this paper was under revision, we also demonstrated that both EN450 and more potent UBE2D covalent recruiters could be used in PROTACs to degrade neo-substrate proteins.
Taken more broadly, our study also demonstrates the utility of coupling covalent ligand screening with chemoproteomic and quantitative proteomic platforms to rapidly discover molecular glue degraders and their ternary complex components. Furthermore, we may identify even more molecular glue degraders through the development of bespoke compound collections, including covalent enantiopair libraries
and by coupling these specialized libraries with not only chemoproteomic platforms but also target-based cellular screens or size-exclusion chromatography.
Our study reveals a covalent molecular glue degrader that engages the E2 ubiquitin-conjugating enzyme UBE2D to engage in molecular glue interactions with NFKB1 leading to the degradation of NFKB1 in cancer cells. Our study nonetheless still has several limitations and open questions. While the NEDDylation-dependency indicates that UBE2D still needs to be coupled to the Cullin E3 ligase complex to mark NFKB1 for degradation and confer anti-proliferative effects based on the attenuation of these effects by a NEDDylation inhibitor, we do not yet know whether recruitment of the E2 bypasses the necessity for a substrate adapter protein to induce the ubiquitination and degradation of NFKB1. We also acknowledge that the NEDDylated CUL4/RBX1 used to reconstitute the ternary complex here is not the ideal RING E3 ligase to address the role of C111 on UBE2D1 on the activated structure since NEDD8 binds UBE2D. As such, the ubiquitination activity that we observe may not exclusively rely on the RING causing the folded back UBE2D1-ubiquitin arrangement, as described in previous literature.
We also note that due to limitations on garnering sufficient pure protein to perform all necessary control experiments for the reconstitution assays, we were not able to test negative control compounds in these assays, and as such cannot exclude an effect of the small molecule beyond what we hypothesized. The mechanism for NFKB1 degradation may also operate through initial mono-ubiquitination of NFKB1 through the EN450-mediated UBE2D/NFKB1 molecular glue interaction that subsequently sensitizes NFKB1 to polyubiquitination and degradation through a combination of or specific E3 ligases. Of future interest is the investigation of whether NFKB1 already interacts weakly with this allosteric site on UBE2D, and whether EN450 thus just strengthens an already existing interaction or whether NFKB1 truly represents a neo-substrate that engages UBE2D only upon EN450 binding. While we demonstrate here that EN450 directly engages with UBE2D1 alone without NFKB1, it is unclear at this point whether EN450 has any inherent reversible affinity for NFKB1 or whether UBE2D1 binding to EN450 is necessary to recruit NFKB1. We acknowledge that EN450 is a primary screening hit that is not selective or potent enough and likely has polypharmacological action in cancer cells. Future improved UBE2D-based NFKB1 degraders can potentially be used to identify particularly sensitive cancer cells that may respond therapeutically to NFKB1 degradation beyond leukemias. 我们的研究揭示了一种共价分子粘合剂降解剂，它与E2泛素连接酶UBE2D相互作用，与NFKB1发生分子粘合作用，导致癌细胞中NFKB1的降解。尽管我们的研究仍然存在一些限制和未解之谜。虽然NEDDylation依赖性表明UBE2D仍然需要与Cullin E3连接酶复合物结合，将NFKB1标记为降解物，并通过NEDDylation抑制剂减弱这些效应来提供抗增殖效应，但我们尚不清楚E2的招募是否绕过了需要底物适配蛋白来诱导NFKB1的泛素化和降解。我们也承认，在这里重建三元复合物所使用的NEDDylated CUL4/RBX1并不是理想的RING E3连接酶，无法解决C111对UBE2D1在激活结构上的作用，因为NEDD8结合UBE2D。因此，我们观察到的泛素化活性可能不仅仅依赖于RING引起的折回的UBE2D1-泛素排列，正如先前文献中所描述的那样。 由于在获取足够纯净蛋白进行所有必要的对照实验以进行重组实验的限制，我们无法在这些实验中测试阴性对照化合物，因此无法排除小分子产生超出我们假设的影响。 NFKB1降解的机制也可能通过NFKB1的初级单泛素化，通过EN450介导的UBE2D/NFKB1分子粘合相互作用，随后使NFKB1对多泛素化和降解产生敏感性，通过一种或特定的E3连接酶的组合。未来感兴趣的是调查NFKB1是否已经与UBE2D的这个变构位点弱交互，并且EN450是否仅加强已经存在的相互作用，或者NFKB1是否真正代表一个新底物，只有在EN450结合时才与UBE2D发生相互作用。虽然我们在这里证明EN450直接与UBE2D1结合而不需要NFKB1，但目前尚不清楚EN450是否具有与NFKB1的任何固有可逆亲和力，或者UBE2D1结合EN450是否需要招募NFKB1。 我们承认EN450是一个初步筛选命中物，不够选择性或有效，并且可能在癌细胞中具有多药作用。未来改进的基于UBE2D的NFKB1降解剂有可能用于识别特别敏感的癌细胞，这些细胞可能对NFKB1降解产生治疗反应，超出白血病范围。
Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Daniel K. Nomura (dnomura@berkeley.edu). 进一步的信息和资源以及试剂的请求应直接发送至并将由主要联系人 Daniel K. Nomura（dnomura@berkeley.edu）履行。
Materials availability 材料可用性
Compounds and protocols generated in this study will be available upon request. Any raw data or images generated in this proposal will be provided upon request. 本研究生成的化合物和方案将根据要求提供。本提案生成的任何原始数据或图像将根据要求提供。
Experimental model and subject details 实验模型和受试者细节
Cell culture 细胞培养
HAP1 cells were purchased from Horizon Discovery and were cultured in IMDM containing 10% (v/v) fetal bovine serum (FBS) and maintained at 37°C with 5% CO2. HAP1 cells are male in origin. HEK293T cells were obtained from the American Type Culture Continued. HEK293 cells were originally derived from a female fetus but the sex is unclear. HEK293T cells were cultured in DMEM containing 10% (v/v) fetal bovine serum (FBS) and maintained at 37°C with 5% CO2. Unless otherwise specified, all cell culture materials were purchased from Gibco. It is not known whether HEK293T cells are from male or female origin. HAP1 细胞是从 Horizon Discovery 购买的，并在含有 10%（体积/体积）胎牛血清（FBS）的 IMDM 中培养，并在 37°C 下以 5% CO 2 维持。HAP1 细胞源自男性。HEK293T 细胞来自美国类型培养继续。HEK293 细胞最初来源于女性胎儿，但性别不明。HEK293T 细胞在含有 10%（体积/体积）胎牛血清（FBS）的 DMEM 中培养，并在 37°C 下以 5% CO 2 维持。除非另有说明，所有细胞培养材料均从 Gibco 购买。不清楚 HEK293T 细胞是男性还是女性来源。
Method details 方法细节
Materials 材料
Cysteine-reactive covalent ligand libraries were either previously synthesized and described or for the compounds starting with “EN” were purchased from Enamine, including EN450. Other chemicals synthesized for this study are described in Supporting Information. 半胱氨酸反应性共价配体库要么是先前合成和描述的，要么是从 Enamine 购买的，包括 EN450 开头的化合物。本研究中合成的其他化学品在支持信息中有描述。
Preparation of cell lysates 细胞裂解的准备
Cells were washed twice with cold PBS, scraped, and pelleted by centrifugation (1,400 g, 4 min, 4°C). Pellets were resuspended in PBS containing protease inhibitor cocktail (ThermoFisher A32955), sonicated, clarified by centrifugation (21,000 g, 10 min, 4°C), and lysate was transferred to another low-adhesion microcentrifuge tubes. Proteome concentrations were determined using BCA assay and lysate was diluted to appropriate working concentrations. 细胞用冷PBS洗涤两次，刮取，经离心（1,400 g，4 分钟，4°C）沉淀。沉淀物在含有蛋白酶抑制剂混合物（ThermoFisher A32955）的PBS中重悬，经声波处理，离心澄清（21,000 g，10 分钟，4°C），裂解物转移到另一个低粘附微离心管中。用BCA测定蛋白组浓度，裂解物稀释至适当的工作浓度。
Cell viability
Cell viability assays were performed using Hoechst 33342 dye (Invitrogen H3570) according to the manufacturer’s protocol. For survival assays, cells were seeded into 96-well plates (20,000 per well) in a volume of 100 μL and allowed to adhere overnight, cells were treated with an additional 50 μL of media containing DMSO vehicle or compound (150 μM) in 0.1% DMSO for 24 h. For rescue experiments, cell media was changed to media (100 μL per well) containing Bortezomib (1 μM) (Calbiochem 5043140001) or MLN4924 (1 μM) (Calbiochem 5054770001) for 1 h prior to compound treatment. For dose response experiments, dilutions of compound were prepared in DMSO prior to dilution in media. After incubation, the media was aspirated from each well, and 100 μL of staining solution containing 10% formalin and Hoechst 33,342 dye (Invitrogen H3570) was added to each well and incubated for 25 min in the dark at room temperature. Staining solution was then removed, and wells were washed with 3x with PBS before fluorescent imaging. 细胞存活率测定采用Hoechst 33342染料（Invitrogen H3570）按照制造商的方案进行。对于存活率测定，细胞被分装到96孔板中（每孔20,000个细胞）中，体积为100μL，并允许其在夜间附着，细胞接受额外50μL含有DMSO载体或化合物（150μM）的培养基，含0.1% DMSO，处理时间为24小时。对于救助实验，细胞培养基在化合物处理前1小时更换为含有Bortezomib（1μM）（Calbiochem 5043140001）或MLN4924（1μM）（Calbiochem 5054770001）的培养基。对于剂量响应实验，化合物的稀释在DMSO中进行，然后在培养基中稀释。孵育后，每孔的培养基被抽取，加入含有10%甲醛和Hoechst 33,342染料（Invitrogen H3570）的100μL染色溶液，暗处室温下孵育25分钟。然后去除染色溶液，用PBS洗涤孔板3次，然后进行荧光成像。
Production of recombinant UBE2D1 重组 UBE2D1 的生产
A UBE2D1(1–147) expression plasmid was synthesized by Twist Biosciences with an N-terminal 8xHistidine tag and HRV 3C cleavage site. The C85S and C111S mutations were produced using a Q5 Site Directed Mutagenesis Kit (New England Biolabs E0554S) and standard cloning techniques. HI-Control BL21(DE3) E. coli cells (Lucigen 60,435-1) were transformed with expression plasmid and a single colony was used to start an overnight culture in LB media, followed by inoculation of a 1 L culture in Terrific Broth supplemented with 50 mM sodium phosphate monobasic pH 7.0 and 50 μg/mL kanamycin. This culture grew at 37°C until the OD600 reached approximately 1.2, at which point the temperature was reduced to 19°C along with immediate addition of 0.5 mM (final) IPTG. The cells were allowed to grow overnight prior to harvesting via centrifugation. The cell pellet was resuspended in IMAC_A Buffer (50 mM Tris pH 8.0, 400 mM NaCl, 1 mM TCEP, 20 mM imidazole) and lysed with three passages through a cell homogenizer at 18,000 psi. Cell lysate was then clarified with centrifugation at 45,000 x g for 30 min. The clarified lysate was flowed through a 5 mL HisTrap Excel column (Cytiva) pre-equilibrated with IMAC_A Buffer. After loading, the resin was washed with IMAC_A Buffer until the UV absorbance reached baseline, after which the protein was eluted with a linear gradient of IMAC_B Buffer (50 mM Tris pH 8.0, 400 mM NaCl, 1 mM TCEP, 500 mM imidazole). The eluate was treated with HRV 3C protease until cleavage of the histidine tag was complete as determined by ESI-LC/MS. Cleavage proceeded while the protein dialyzed into IMAC_A Buffer, enabling reverse-IMAC purification which was conducted in batch using 3 mL of Ni-NTA resin pre-equilibrated with IMAC_A Buffer. The flow-through was collected, concentrated, and subjected to size exclusion chromatography using a Superdex 75 16/600 column (Cytiva) pre-equilibrated with SEC Buffer (25 mM HEPES pH 7.5, 150 mM NaCl, 1 mM TCEP). Fractions within the included volume were analyzed by SDS-PAGE and those containing pure protein were pooled and concentrated. This protocol was used to produce wild-type, C85S, and C85S/C111S variants of UBE2D1 with an approximate yield of ∼90 mg/L. 使用 Twist Biosciences 合成了一个带有 N 端 8xHistidine 标记和 HRV 3C 切割位点的 UBE2D1(1-147)表达质粒。使用 Q5 Site Directed Mutagenesis Kit（New England Biolabs E0554S）和标准克隆技术产生了 C85S 和 C111S 突变体。将 HI-Control BL21(DE3) E. coli 细胞（Lucigen 60,435-1）转化为表达质粒，使用单个菌落开始在 LB 培养基中进行过夜培养，然后接种到含有 50 mM 磷酸氢二钠 pH 7.0 和 50 μg/mL 卡那霉素的 Terrific Broth 培养基中的 1 L 培养中。该培养在 37°C 下生长，直到 OD600 达到约 1.2，此时将温度降至 19°C，并立即加入 0.5 mM（最终浓度）IPTG。细胞在收获之前允许过夜生长，然后通过离心收获。将细胞沉淀在 IMAC_A 缓冲液（50 mM Tris pH 8.0，400 mM NaCl，1 mM TCEP，20 mM 咪唑）中重悬，并在 18,000 psi 的细胞均质器中进行三次通行后裂解。然后通过以 45,000 x g 离心 30 分钟澄清细胞裂解液。 经过澄清的裂解物通过预先用 IMAC_A 缓冲液平衡的 5 mL HisTrap Excel 柱（Cytiva）进行流动。加载后，用 IMAC_A 缓冲液洗涤树脂直到紫外吸收达到基线，然后用 IMAC_B 缓冲液（50 mM Tris pH 8.0，400 mM NaCl，1 mM TCEP，500 mM 咪唑唑）的线性梯度洗脱蛋白质。洗脱物经 HRV 3C 蛋白酶处理，直到通过 ESI-LC/MS 确定组氨酸标签的裂解完全。在蛋白质透析到 IMAC_A 缓冲液时，裂解继续进行，从而实现了使用预先用 IMAC_A 缓冲液平衡的 3 mL Ni-NTA 树脂进行批量进行反向 IMAC 纯化。收集流过液，浓缩，并使用预先用 SEC 缓冲液（25 mM HEPES pH 7.5，150 mM NaCl，1 mM TCEP）平衡的 Superdex 75 16/600 柱（Cytiva）进行凝胶排阻色谱。在包含体积内的分数通过 SDS-PAGE 分析，含有纯蛋白质的分数被混合并浓缩。该方案用于生产 UBE2D1 的野生型，C85S 和 C85S/C111S 变体，产量约为∼90 mg/L。
Labeling of recombinant UBE2D1 with EK-1-8 and EK-1-38 probes 使用 EK-1-8 和 EK-1-38 探针标记重组 UBE2D1
For in vitro labeling of UBE2D1 C85S, recombinant pure human protein (0.1 μg per sample) was treated with either DMSO vehicle, EK-1-8, or EK-1-38 at 37°C for 30 min in 25 μL PBS. Each sample was incubated with 1 μL of 30 μM rhodamine-azide (in DMSO) (Click Chemistry Tools AZ109-5), 1 μL of 50 mM TCEP (in water) (Strem 15–7400), 3 μL of TBTA ligand (0.9 mg/mL in 1:4 DMSO/t-BuOH) (Sigma 678,937), and 1 μL of 50 mM Copper (II) Sulfate (Sigma 451,657) for 1 h at room temperature. Samples were then diluted with 10 μL of 4x reducing Laemmli SDS sample loading buffer (Alfa Aesar J60015), boiled at 95°C for 5 min, and separated by SDS/PAGE. Probe-labeled proteins were analyzed by in-gel rhodamine fluorescence using a ChemiDoc MP (Bio-Rad). Protein loading was assessed by silver staining. 对于 UBE2D1 C85S 的体外标记，每个样品中的重组纯人类蛋白（每个样品 0.1 微克）在 25 微升 PBS 中分别与 DMSO 载体、EK-1-8 或 EK-1-38 在 37°C 处理 30 分钟。每个样品与 1 微升 30 μM 罗丹明-叠氮基（在 DMSO 中）（Click Chemistry Tools AZ109-5）、1 微升 50 mM TCEP（在水中）（Strem 15-7400）、3 微升 TBTA 配体（0.9 mg/mL 在 1:4 DMSO/t-BuOH 中）（Sigma 678,937）和 1 微升 50 mM 硫酸铜（II）（Sigma 451,657）在室温下孵育 1 小时。然后用 10 微升 4x 还原 Laemmli SDS 样品加载缓冲液（Alfa Aesar J60015）稀释，95°C 煮沸 5 分钟，并通过 SDS/PAGE 分离。探针标记的蛋白质通过使用 ChemiDoc MP（Bio-Rad）进行凝胶内罗丹明荧光分析。蛋白负载通过银染色进行评估。
Pulldown of UBE2D1 from HAP1 cells with EK-1-8 probe
HAP1 WT cells were pre-treated at 70% confluency with DMSO or 50 μM EN450 in situ for 1 h followed by treatment with DMSO or 10 μM EK-1-8 in situ for 3 h. Cells were harvested, lysed via sonication, and the resulting lysate normalized to 6 mg/mL per sample. Following normalization, 30 μL of each lysate sample was removed for Western blot analysis of input, and 500 μL of each lysate sample was incubated for 2 h at room temperature with 10 μL of 5 mM biotin picolyl azide (in DMSO) (Sigma Aldrich 900,912), 10 μL of 50 mM TCEP (in water), 30 μL of TBTA ligand (0.9 mg/mL in 1:4 DMSO/t-BuOH), and 10 μL of 50 mM Copper (II) Sulfate. Proteins were precipitated, washed 3 x with cold MeOH, resolubilized in 200 μL of 1.2% SDS/PBS (w/v), heated for 5 min at 98C, and centrifuged to remove any insoluble components. Each resolubilized sample was then transferred to 1.5 mL eppendorf low-adhesion tubes containing 1 mL PBS with 30 μL high-capacity streptavidin resin (ThermoFisher 20,357) to give a final SDS concentration of 0.2%. Samples were incubated with the streptavidin beads at 4°C overnight on a rotator. The following day the samples were warmed to room temperature and washed with 0.2% SDS and further washed 3 x with 500 μL PBS and 3 x with 500 μL water to remove non-probe-labeled proteins. For western blot analysis, the washed beads were resuspended in 30 μL PBS, transferred to 1.5 mL eppendorf low-adhesion tubes, combined with 10 μL Laemmli Sample Buffer (4 x), heated to 95°C for 5 min, and analyzed by Western blotting to look for enriched UBE2D1 versus non-enriched control Vinculin. HAP1 WT细胞在70%的密度下预先用DMSO或50μM EN450原位处理1小时，然后用DMSO或10μM EK-1-8原位处理3小时。细胞被收集，通过声波裂解，裂解液被标准化为每个样本6毫克/毫升。标准化后，每个裂解液样本中取出30μL用于Western印迹分析输入，每个裂解液样本中取出500μL，与5mM生物素吡啶基叠氮（在DMSO中）（Sigma Aldrich 900,912）的10μL，50mM TCEP（在水中）的10μL，TBTA配体（0.9mg/mL在1:4 DMSO/t-BuOH中）的30μL和50mM硫酸铜（II）的10μL在室温下孵育2小时。蛋白质被沉淀，用冷MeOH洗涤3次，重新溶解在200μL的1.2% SDS/PBS（w/v）中，98°C加热5分钟，离心去除任何不溶性成分。然后将每个重新溶解的样品转移到含有30μL高容量链霉亲和素树脂（ThermoFisher 20,357）的1.5mL Eppendorf低粘附管中，使最终SDS浓度为0.2%。样品在4°C的转动器上过夜与链霉亲和素珠一起孵育。第二天，样品升温至室温并用0.2% SDS 和进一步用 500 μL PBS 洗涤 3 次，然后用 500 μL 水洗涤 3 次，以去除未标记的蛋白质探针。对于免疫印迹分析，洗涤后的珠子在 30 μL PBS 中重悬，转移到 1.5 mL Eppendorf 低粘附管中，与 10 μL Laemmli 采样缓冲液（4 倍）结合，加热至 95°C 5 分钟，并通过免疫印迹分析寻找富集的 UBE2D1 与非富集的对照 Vinculin。
Western blotting 免疫印迹分析
Antibodies to NFKB p105/p52 (Abcam ab323670), SFT (Abcam ab176561), UBE2D2 (Origene TA806600), UBE2D3 (Abcam ab176568), UBE2D4 (Invitrogen MA5-27358), UBE2M/UBC12 (Abcam ab109507), DYKDDDDK Tag (D6W5B) (Cell Signaling Technology Cat#14793S), Vinculin (Bio-Rad Cat#MCA465S), GAPDH (Proteintech Cat#60004-I-Ig) were obtained commercially and prepared at dilutions recommended by the manufacturers. Proteins were resolved by SDS/PAGE and transferred to nitrocellulose membranes using the Trans-Blot Turbo transfer system (Bio-Rad). Membranes were blocked with 5% BSA in Tris-buffered saline containing Tween 20 (TBS-T) solution for 30 min at RT, washed in TBS-T, and probed with primary antibody diluted 5% BSA in TBS-T overnight at 4°C. After 3 washes with TBS-T, the membranes were incubated in the dark with IR680-or IR800-conjugated secondary antibodies obtained commercially from LI-COR at 1:10,000 dilution in 5% BSA in TBS-T at RT for 1 h. After 3 additional washes with TBST, blots were visualized using an Odyssey Li-Cor fluorescent scanner. The membranes were stripped using Re-Blot Plus Strong Antibody Stripping Solution (EMD Millipore) when additional primary antibody incubations were performed. 抗体 NFKB p105/p52（Abcam ab323670）、SFT（Abcam ab176561）、UBE2D2（Origene TA806600）、UBE2D3（Abcam ab176568）、UBE2D4（Invitrogen MA5-27358）、UBE2M/UBC12（Abcam ab109507）、DYKDDDDK 标签（D6W5B）（Cell Signaling Technology Cat#14793S）、Vinculin（Bio-Rad Cat#MCA465S）、GAPDH（Proteintech Cat#60004-I-Ig）是商业获得的，并按制造商推荐的稀释度准备。蛋白质通过SDS/PAGE分离，并使用Trans-Blot Turbo转移系统（Bio-Rad）转移到硝酸纤维素膜上。膜用含Tween 20的Tris缓冲盐水（TBS-T）溶液中的5% BSA在室温下阻断30分钟，用TBS-T洗涤，然后用5% BSA在TBS-T中稀释的初级抗体在4°C过夜探测。用TBS-T洗涤3次后，膜在黑暗中与从LI-COR商业获得的IR680或IR800结合的二级抗体在5% BSA中以1:10,000稀释度在室温下孵育1小时。用TBST再洗涤3次后，使用Odyssey Li-Cor荧光扫描仪可视化印迹。 使用 Re-Blot Plus 强抗体去除溶液（EMD Millipore）去除膜，当需要进行额外的一次性抗体孵育时。
Cells were treated for 3 h with either DMSO vehicle or EN450 (50 μM) before cell lysate preparation as described above. Proteomes were subsequently labeled with IA-alkyne labeling (200 μM) (Chess Gmbh 3187) for 1 h at room temperature. CuAAC was used by sequential addition of tris(2-carboxyethyl)phosphine (1 mM), tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (34 μM), copper(II) sulfate (1 mM) and biotin-linker-azide—the linker functionalized with a tobacco etch virus (TEV) protease recognition sequence as well as an isotopically light or heavy valine for treatment of control or treated proteome, respectively. After CuAAC, proteomes were precipitated by centrifugation at 6,500g, washed in ice-cold methanol, combined in a 1:1 control:treated ratio, washed again, then denatured and resolubilized by heating in 1.2% SDS–PBS to 95 °C for 5 min. Insoluble components were precipitated by centrifugation at 6,500g and soluble proteome was diluted in 5 mL 0.2% SDS–PBS. Labeled proteins were bound to streptavidin-agarose beads (170 μL resuspended beads per sample, ThermoFisher 20,349) while rotating overnight at 4 °C. Bead-linked proteins were enriched by washing three times each in PBS and water, then resuspended in 6 M urea/PBS, reduced in TCEP (1 mM), alkylated with iodoacetamide (18 mM, ACROS AC122270050), before being washed and resuspended in 2 M urea/PBS and trypsinized overnight with 0.5 μg/μL sequencing grade trypsin (Promega V5111). Tryptic peptides were eluted off. Beads were washed three times each in PBS and water, washed in TEV buffer solution (water, TEV buffer, 100 μM dithiothreitol (Invitrogen 15508013)) and resuspended in buffer with Ac-TEV protease (Invitrogen 12575-015) and incubated overnight. Peptides were diluted in water, acidified with formic acid (1.2 M, Fisher A117-50), and prepared for LC-MS/MS analysis. IsoTOP-ABPP研究已按先前报道的方法进行。
IsoTOP-ABPP mass spectrometry analysis IsoTOP-ABPP 质谱分析
Peptides from all chemoproteomic experiments were pressure-loaded onto a 250 μm inner diameter fused silica capillary tubing packed with 4 cm of Aqua C18 reverse-phase resin (Phenomenex, 04A-4299), which was previously equilibrated on an Agilent 600 series high-performance liquid chromatograph using the gradient from 100% buffer A to 100% buffer B over 10 min, followed by a 5 min wash with 100% buffer B and a 5 min wash with 100% buffer A. The samples were then attached using a Micro-Tee PEEK 360 μm fitting (Thermo Fisher Scientific p-888) to a 13 cm laser pulled column packed with 10 cm Aqua C18 reverse-phase resin and 3 cm of strong-cation exchange resin for isoTOP-ABPP studies. Samples were analyzed using an Q Exactive Plus mass spectrometer (ThermoFisher Scientific) using a five-step Multidimensional Protein Identification Technology (MudPIT) program, using 0, 25, 50, 80 and 100% salt bumps of 500 mM aqueous ammonium acetate and using a gradient of 5–55% buffer B in buffer A (buffer A: 95:5 water:acetonitrile, 0.1% formic acid; buffer B 80:20 acetonitrile:water, 0.1% formic acid). Data were collected in data-dependent acquisition mode with dynamic exclusion enabled (60 s). One full mass spectrometry (MS1) scan (400–1,800 mass-to-charge ratio (m/z)) was followed by 15 MS2 scans of the nth most abundant ions. Heated capillary temperature was set to 200 °C and the nanospray voltage was set to 2.75 kV.
Data were extracted in the form of MS1 and MS2 files using Raw Extractor v.1.9.9.2 (Scripps Research Institute) and searched against the Uniprot human database using ProLuCID search methodology in IP2 v.3-v.5 (Integrated Proteomics Applications, Inc.).
Cysteine residues were searched with a static modification for carboxyaminomethylation (+57.02146) and up to two differential modifications for methionine oxidation and either the light or heavy TEV tags (+464.28596 or +470.29977, respectively). Peptides were required to be fully tryptic peptides and to contain the TEV modification. ProLUCID data were filtered through DTASelect to achieve a peptide false-positive rate below 5%. Only those probe-modified peptides that were evident across two out of three biological replicates were interpreted for their isotopic light to heavy ratios. For those probe-modified peptides that showed ratios greater than two, we only interpreted those targets that were present across all three biological replicates, were statistically significant and showed good quality MS1 peak shapes across all biological replicates. Light versus heavy isotopic probe-modified peptide ratios are calculated by taking the mean of the ratios of each replicate paired light versus heavy precursor abundance for all peptide-spectral matches associated with a peptide. The paired abundances were also used to calculate a paired sample t-test p value to estimate constancy in paired abundances and significance in change between treatment and control. p values were corrected using the Benjamini–Hochberg method. 数据以MS1和MS2文件的形式使用Raw Extractor v.1.9.9.2（斯克里普斯研究所）提取，并使用IP2 v.3-v.5（综合蛋白质组学应用公司）中的ProLuCID搜索方法针对Uniprot人类数据库进行搜索。
半胱氨酸残基使用静态修饰进行卡氧氨基甲基化（+57.02146），甲硫氨酸氧化和轻型或重型TEV标签（+464.28596或+470.29977）的差异修饰最多可有两种。肽段需要是完全的胰蛋白酶肽段，并且包含TEV修饰。 ProLUCID数据通过DTASelect进行过滤，以实现肽段假阳性率低于5％。仅解释那些在三个生物重复中至少有两个显示的探针修饰肽段的同位素轻重比的数据。对于显示比率大于两的探针修饰肽段，我们仅解释那些在所有三个生物重复中都存在、在统计上显著且在所有生物重复中都显示良好质量MS1峰形的目标。 轻重同位素探针修饰的肽比率是通过计算与肽相关的所有肽-光谱匹配的每个复制品配对的轻重前体丰度比率的平均值来计算的。这些配对的丰度也被用来计算配对样本 t 检验 p 值，以估计配对丰度的稳定性和治疗与对照之间变化的显著性。p 值使用 Benjamini–Hochberg 方法进行校正。
HAP1 WT cells were treated with either DMSO vehicle or EN450 (50 μM) for 24 h and lysate was prepared as described above. Briefly, 25–100 μg protein from each sample was reduced, alkylated and tryptically digested overnight. Individual samples were then labeled with isobaric tags using commercially available TMTsixplex (ThermoFisher 90,061) kits, in accordance with the manufacturer’s protocols. Tagged samples (20 μg per sample) were combined, dried with SpeedVac, resuspended with 300 μL 0.1% TFA in H2O, and fractionated using high pH reversed-phase peptide fractionation kits (ThermoFisher 84,868) according to manufacturer’s protocol. Fractions were dried with SpeedVac, resuspended with 50 μL 0.1% FA in H2O, and analyzed by LC-MS/MS as described below. HAP1 WT细胞分别用DMSO载体或EN450（50μM）处理24小时，制备上述所述的裂解物。简而言之，每个样品中的25-100μg蛋白质经还原、烷基化和胰蛋白酶消化过夜。然后，使用商业可用的TMTsixplex（ThermoFisher 90061）套件，按照制造商的方案对各个样品进行同位素标记。标记的样品（每个样品20μg）被结合，用SpeedVac干燥，用300μL 0.1% TFA在H2O中重悬，然后根据制造商的方案使用高pH反相肽分离套件（ThermoFisher 84868）进行分级。分级后用SpeedVac干燥，用50μL 0.1% FA在H2O中重悬，然后按照以下所述的方法进行LC-MS/MS分析。
Quantitative TMT-based proteomic analysis was performed as previously described using a Thermo Eclipse with FAIMS LC-MS/MS.
Trypsin cleavage specificity (cleavage at K, R except if followed by P) allowed for up to 2 missed cleavages. Carbamidomethylation of cysteine was set as a fixed modification, methionine oxidation, and TMT-modification of N-termini and lysine residues were set as variable modifications. Reporter ion ratio calculations were performed using summed abundances with most confident centroid selected from 20 ppm window. Only peptide-to-spectrum matches that are unique assignments to a given identified protein within the total dataset are considered for protein quantitation. High confidence protein identifications were reported with a <1% false discovery rate (FDR) cut-off. Differential abundance significance was estimated using ANOVA with Benjamini-Hochberg correction to determine p values. 使用先前描述的方法，使用 Thermo Eclipse 和 FAIMS LC-MS/MS 进行定量 TMT 基础蛋白质组学分析。
RNAi was performed using siRNA purchased from Dharmacon. HAP1 cells were seeded at 100,000 cells per 6 cm plate and allowed to adhere overnight. Cells were transfected with 25 nM of either non-targeting (Dharmacon D-001810-10), anti-UBE2M (Dharmacon L-004348-00), or anti-UBE2D1 siRNA (Dharmacon L-0093870-00) using 7.5 μL of transfection reagent DharmaFECT 1 (Dharmacon T-2001-02) per well. For quadruple knockdown studies, 12.5 nM of anti-UBE2D1, -UBE2D2 (Dharmacon L-010383-00), -UBE2D3 (Dharmacon L-008478-00), and -UBE2D4 (Dharmacon L-009435-00) siRNA with 15 μL of DharmaFECT1 was used. Transfection reagent was added to OPTIMEM (ThermoFisher 31985070) media and allowed to incubate for 5 min at room temperature. Meanwhile siRNA was added to an equal amount of OPTIMEM. Solutions of transfection reagent and siRNA in OPTIMEM were then combined and allowed to incubate for 30 min at room temperature. These combined solutions were diluted with complete DMEM to provide 2 mL per well, and the media exchanged. Cells were incubated with transfection reagents for 48h, at which point the media was replaced with media containing DMSO or 50 μM EN450 and incubated for another 24 h. Cells were then harvested, and protein abundance analyzed by Western blotting. RNAi是使用从Dharmacon购买的siRNA进行的。HAP1细胞以每个6cm培养皿100,000个细胞的密度接种，并允许其在过夜后附着。细胞使用25 nM的非靶向（Dharmacon D-001810-10）、抗UBE2M（Dharmacon L-004348-00）或抗UBE2D1 siRNA（Dharmacon L-0093870-00）进行转染，每孔使用7.5 μL的转染试剂DharmaFECT 1（Dharmacon T-2001-02）。对于四重基因敲除研究，使用12.5 nM的抗UBE2D1、UBE2D2（Dharmacon L-010383-00）、UBE2D3（Dharmacon L-008478-00）和UBE2D4（Dharmacon L-009435-00）siRNA，每孔使用15 μL的DharmaFECT1。转染试剂加入到OPTIMEM（ThermoFisher 31985070）培养基中，并在室温下孵育5分钟。同时，siRNA加入到相同量的OPTIMEM中。将OPTIMEM中的转染试剂和siRNA溶液混合，并在室温下孵育30分钟。将这些混合溶液与完整的DMEM稀释，每孔提供2 mL，并更换培养基。细胞与转染试剂共孵育48小时，然后将培养基更换为含有DMSO或50 μM EN450的培养基，并再孵育24小时。 然后收集细胞，通过 Western blotting 分析蛋白质丰度。
Lentiviral overexpression of NFKB p105 NFKB p105 的慢病毒过表达
In separate 15 mL conicals, 1 μg of expression clone cDNA (Origene RC208384L3) or control cDNA (Origene PS100093) was mixed with packaging plasmids MD2G (1 μg, Addgene 12,259) and PSPAX2 (1 μg, Addgene 12,260) in 600 μL per plate OPTIMEM and Lipofectamine 2000 transfection reagent (Invitrogen 11668027) was incubated with an equal volume of OPTIMEM (1:30 v/v) for 5 min prior to tubes being combined and incubated for 40 min at room temperature. The DNA-Lipofectamine mix was diluted with 8 mL of DMEM and added to HEK293T cells at 40% confluency in 10 cm plates. The next day, media was replaced with 6 mL fresh DMEM for 24 h. 在单独的 15 mL 圆锥瓶中，将 1 μg 表达克隆 cDNA（Origene RC208384L3）或对照 cDNA（Origene PS100093）与包装质粒 MD2G（1 μg，Addgene 12259）和 PSPAX2（1 μg，Addgene 12260）混合在每个培养皿的 600 μL OPTIMEM 和 Lipofectamine 2000 转染试剂（Invitrogen 11668027）与相等体积的 OPTIMEM（1:30 v/v）孵育 5 分钟，然后将管子组合并在室温下孵育 40 分钟。 DNA-Lipofectamine 混合物用 8 mL DMEM 稀释并加入 40%密度的 HEK293T 细胞在 10 cm 培养皿中。第二天，将培养基更换为 6 mL 新鲜 DMEM，培养 24 小时。
For each control or expression clone, media was removed from HEK293T cells, filtered through a 0.45 micron syringe filter, mixed with 10 μL polybrene transfection reagent, and added to HAP1 WT cells at 50% confluency. HEK293T media was replaced with 6 mL fresh DMEM for 24 h and the infection process was repeated. 24 h after the second infection, the Hap1 WT infection media was removed and cells were seeded for proliferation experiments and Western blot analysis.
Transient overexpression of NFKB p105 in HEK293T cells
Prior to transfection, HEK293T cells were seeded into a 96-well plate (35,000 cells/well in 100 μL) or 6-well plate. Flag-tagged NFKB plasmid (Origene RC208384) or GFP control plasmid (Ward et al., 2019) was diluted into Opti-MEM medium (0.2 μg DNA into 25 μL Opti-MEM per each well). Lipofectamine 2000 (Invitrogen, 11668019) was diluted into Opti-MEM I medium (0.5 μL Lipofectamine 2000 into 25 μL Opti-MEM I per each well). DNA and diluted Lipofectamine 2000 were combined in a 1:1 ratio then incubated at room temperature for 30 min. The DNA-Lipofectamine 2000 mixture was diluted with 50 μL of FBS-complete DMEM and then all 100 μL was added to each well. 24 h post-transfection, media was carefully aspirated from each well, and 100 μL of fresh media containing either DMSO or compound was added, then assayed for proliferation. Briefly, for 6-well plate experiments, cells were seeded at 400,000 cells/well in 2 mL media, 4 μg of DNA and 10 μL of Lipofectamine 2000 per well were each diluted into 250 μL of Opti-MEM, and final transfection volume was 2 mL per well. Cells were analyzed via Western blot analysis after 48 h. 转染前，HEK293T 细胞被接种到 96 孔板（每孔 35,000 个细胞/100μL）或 6 孔板中。Flag 标记的 NFKB 质粒（Origene RC208384）或 GFP 对照质粒（Ward 等人，2019 年）被稀释到 Opti-MEM 培养基中（每孔 0.2μg DNA 稀释到 25μL Opti-MEM 中）。Lipofectamine 2000（Invitrogen, 11668019）被稀释到 Opti-MEM I 培养基中（每孔 0.5μL Lipofectamine 2000 稀释到 25μL Opti-MEM I 中）。DNA 和稀释后的 Lipofectamine 2000 以 1:1 的比例混合后在室温下孵育 30 分钟。DNA-Lipofectamine 2000 混合物用 50μL 完整 FBS DMEM 稀释，然后全部 100μL 加入每个孔中。转染后 24 小时，小心地从每个孔中吸出培养基，加入含 DMSO 或化合物的新鲜培养基，然后进行增殖分析。简而言之，对于 6 孔板实验，每孔接种 400,000 个细胞，每孔 4μg DNA 和 10μL Lipofectamine 2000 被稀释到 250μL Opti-MEM 中，最终转染体积为每孔 2mL。细胞在 48 小时后通过 Western blot 分析进行分析。
In vitro pulldown of UBE2D1 with GST-tagged NFKB1 用带有 GST 标记的 NFKB1 进行 UBE2D1 的体外拉下实验
Glutathione Sepharose 4B beads (1 μL of packed beads per sample, Cytiva 17075605) were washed 3 x with wash buffer (30 mM Tris (pH 7.5), 100 nM NaCl, 5 mM MgCl2, 2 mM DTT, 0.1 mg/mL BSA, 10% glycerol, 0.01% Triton X-) with bead collection at 2000 x g between each wash, then resuspended in blocking buffer (30 mM 30 mM Tris (pH 7.5), 100 nM NaCl, 5 mM MgCl2, 2 mM DTT, 100 mg/mL BSA, 10% glycerol, 0.01% Triton X-) with gentle agitation at room temperature for 1 h, then washed twice more. GST-NFKB p105 (1 pmol per μL of beads, Novus H00004790-P01) in 100 μL was incubated with the beads for 2 h at 4°C with gentle agitation, then beads were washed three times. Beads were resuspended in wash buffer (45 μL per sample) containing 50 nM UBE2D1 (Boston Biochem Inc., E2-616-100) then aliquoted into PCR tubes. Input control was prepared via immediate addition of 15 μL Laemmli’s buffer. Beads were treated with 5 μL of vehicle (10% DMSO, 0.25% CHAPS) or EN450 (50 μM) for a 50 μL final volume and incubated with gentle agitation at 4°C overnight. Beads were washed 4x with wash buffer (50 μL), resuspended in 20 μL of 1x Laemmli’s in PBS, boiled at 95°C for 5 min, pelleted at 1000 x g for 5 min, then analyzed via Western blotting. 谷胱甘肽琼脂糖4B珠（每个样品1μL包装珠，Cytiva 17075605）用洗涤缓冲液（30mM Tris（pH 7.5），100nM NaCl，5mM MgCl2，2mM DTT，0.1mg/mL BSA，10%甘油，0.01% Triton X-）洗涤3次，每次洗涤后以2000 x g收集珠子，然后在室温下轻轻摇动悬浮在阻断缓冲液中（30mM Tris（pH 7.5），100nM NaCl，5mM MgCl2，2mM DTT，100mg/mL BSA，10%甘油，0.01% Triton X-）中1小时，然后再洗涤两次。 GST-NFKB p105（每μL珠子1pmol，Novus H00004790-P01）在100μL中与珠子一起在4°C下轻轻摇动孵育2小时，然后珠子被洗涤三次。将珠子悬浮在含有50nM UBE2D1（Boston Biochem Inc.，E2-616-100）的洗涤缓冲液中（每个样品45μL），然后分装到PCR管中。通过立即加入15μL Laemmli缓冲液制备输入对照。珠子用5μL载体（10% DMSO，0.25% CHAPS）或EN450（50μM）处理，最终体积为50μL，并在4°C下轻轻摇动孵育过夜。 珠子用洗涤缓冲液（50 μL）洗涤 4 次，再用 20 μL 1x Laemmli's in PBS 悬浮，95°C 煮沸 5 分钟，以 1000 x g 离心 5 分钟，然后通过免疫印迹分析。
In vitro ubiquitination assay 体外泛素化实验
UBE2D1 (5.2 μL, 25 μM, Boston Biochem. Inc., E2-616-100) was incubated with 0.5 μL of DMSO vehicle or EN450 (10 μM final concentration) for 30 min at 37°C. Subsequently, UBE1 (0.95 μL, 1 μM, Boston Biochem. Inc., E−305-025) was added followed by Cul4A/RBX/NEDD8 (0.7 μL, 5 μM, Boston Biochem. Inc., E3-441-025), NFΚB p105 (19.1 μL, 0.91 μM, Novus Biologicals, H00004790-Q01), FLAG-ubiquitin (0.5 μL, 10 mg/mL, Boston Biochem. Inc., U12001M), MgCl2 (0.5 μL, 500 mM), DTT (0.5 μL, 500 mM) and ATP (0.5 μL, 100 mM) to achieve a final volume of 25.5 μL. The mixture was incubated at 37°C for 4 h with agitation. Then, 10 μL of Laemmli SDS sample loading buffer was added to quench the reaction and proteins were analyzed by Western Blot. All dilutions were made using 50 mM TBS (pH 7.5). UBE2D1（5.2 μL，25 μM，Boston Biochem. Inc.，E2-616-100）与 0.5 μL DMSO 溶剂或 EN450（最终浓度为 10 μM）在 37°C 孵育 30 分钟。随后，加入 UBE1（0.95 μL，1 μM，Boston Biochem. Inc.，E-305-025），然后加入 Cul4A/RBX/NEDD8（0.7 μL，5 μM，Boston Biochem. Inc.，E3-441-025），NFΚB p105（19.1 μL，0.91 μM，Novus Biologicals，H00004790-Q01），FLAG-泛素（0.5 μL，10 mg/mL，Boston Biochem. Inc.，U12001M），MgCl2（0.5 μL，500 mM），DTT（0.5 μL，500 mM）和 ATP（0.5 μL，100 mM）以达到最终体积 25.5 μL。将混合物在 37°C 下搅拌孵育 4 小时。然后加入 10 μL Laemmli SDS 样品加载缓冲液以停止反应，并通过免疫印迹分析蛋白质。所有稀释均使用 50 mM TBS（pH 7.5）进行。
Synthetic methods and characterization for EN450 and EK-1-8 EN450 和 EK-1-8 的合成方法和表征
Preparation of N-(2-chloro-5-(N,N-dimethylsulfamoyl)phenyl)acrylamide 制备 N-(2-氯-5-(N,N-二甲基磺酰)苯基)丙烯酰胺
To a solution of dimethylamine (0.198 mL, 2.35 mmol) in DCM (5 mL) was added triethylamine (0.326 mL, 2.35 mmol) dropwise. The solution was stirred at 0C for 45 min. Then, 3-nitro-4-chlorobenzenesulfonyl chloride (500 mg, 1.95 mmol) in 2:1 DCM/THF (5 mL) was added dropwise, and the solution was warmed to room temperature for 5 min when the reaction was diluted with DCM, washed with water, brine, dried over NaSO4, filtered, and the filtrate concentrated in vacuo. The residue was purified by flash chromatography on silica gel (0–30% EtOAc/Hexanes) to give the desired product 4-chloro-N,N-dimethyl-3-nitrobenzenesulfonamide (464 mg, 90%) as a light yellow solid. LCMS. 1H NMR (400 MHz, CDCl3) δ 8.27 (t, J = 2.2 Hz, 1H), 7.93 (dd, J = 8.4, 2.1 Hz, 1H), 7.78 (d, J = 8.4 Hz, 1H), 2.81 (d, J = 1.7 Hz, 6H). LCMS Rt 0.066 min; m/z 264.8 [M + H]. 将二甲胺（0.198 毫升，2.35 毫摩尔）溶解在 DCM（5 毫升）中，滴加三乙胺（0.326 毫升，2.35 毫摩尔）。在 0 摄氏度搅拌 45 分钟。然后，将 3-硝基-4-氯苯磺酰氯（500 毫克，1.95 毫摩尔）溶解在 2:1 DCM/THF（5 毫升）中滴加，反应混合物升温至室温 5 分钟，反应混合物用 DCM 稀释，用水、食盐水洗涤，经过 NaSO4 干燥，过滤，滤液在减压下浓缩。残渣经硅胶快速色谱纯化（0-30% EtOAc/Hexanes）得到所需产物 4-氯-N,N-二甲基-3-硝基苯磺酰胺（464 毫克，90%）为淡黄色固体。LCMS。H NMR（400 兆赫，CDCl）δ 8.27（t，J = 2.2 赫兹，1H），7.93（dd，J = 8.4，2.1 赫兹，1H），7.78（d，J = 8.4 赫兹，1H），2.81（d，J = 1.7 赫兹，6H）。LCMS Rt 0.066 分钟；m/z 264.8 [M + H]。
To a solution of ammonium chloride (346 mg, 6.46 mmol) in 1:1 EtOH/H2O (10 mL) was added iron powder (362 mg, 6.46 mmol). The solution was stirred at 60°C for 30 min. Then, 4-chloro-N,N-dimethyl-3-nitrobenzenesulfonamide (286 mg, 1.08 mmol) was added. The solution was stirred at 80C for 1 h, then diluted with DCM, washed with water, brine, dried over NaSO4, filtered, and the filtrate concentrated to give 3-amino-4-chloro-N,N-dimethylbenzenesulfonamide (183 mg, 78%) as a white powder. LCMS Rt 0.085 min; m/z 259.1 [M + H]. 将氯化铵（346毫克，6.46毫摩尔）溶解在1:1的乙醇/水（10毫升）溶液中，加入铁粉（362毫克，6.46毫摩尔）。将溶液在60°C搅拌30分钟。然后加入4-氯-N,N-二甲基-3-硝基苯磺酰胺（286毫克，1.08毫摩尔）。将溶液在80°C搅拌1小时，然后用二氯甲烷稀释，用水、食盐水洗涤，经过硫酸钠干燥，过滤，浓缩产物得到3-氨基-4-氯-N,N-二甲基苯磺酰胺（183毫克，78%）为白色粉末。LCMS Rt 0.085分钟；m/z 259.1 [M + H]。
To a solution of 3-amino-4-chloro-N,N-dimethylbenzenesulfonamide (100 mg, 0.37 mmol) in DCM (15 mL) was added triethylamine (80 μL, 0.57 mmol) dropwise. The solution was stirred at 0C for 5 min. Acryloyl chloride (67 μL, 0.83 mmol) was added and the solution was warmed to room temperature. After 2 h, the mixture was diluted with water, extracted with DCM, washed with brine, dried over NaSO4, filtered, and the filtrate concentrated in vacuo. The residue was purified by flash chromatography on silica gel (20–50% EtOAc/Hexanes) to give N-(2-chloro-5-(N,N-dimethylsulfamoyl)phenyl)acrylamide (mg, %) as a white powder. 1H NMR (500 MHz, CDCl3) δ 8.91 (d, J = 2.1 Hz, 1H), 7.85 (s, 1H), 7.58 (d, J = 8.4 Hz, 1H), 7.52 (dd, J = 8.4, 2.1 Hz, 1H), 6.52 (dd, J = 16.8, 1.0 Hz, 1H), 6.35 (dd, J = 16.8, 10.2 Hz, 1H), 5.91 (dd, J = 10.2, 1.0 Hz, 1H), 2.80 (s, 6H). 将 3-氨基-4-氯-N，N-二甲基苯磺酰胺（100 毫克，0.37 毫摩尔）溶解于 DCM（15 毫升）中，滴加三乙胺（80 微升，0.57 毫摩尔）。将溶液在 0 摄氏度搅拌 5 分钟。加入丙烯酰氯（67 微升，0.83 毫摩尔），并将溶液加热至室温。2 小时后，将混合物稀释至水，用 DCM 萃取，用卤水洗涤，过硫酸钠干燥，过滤，将滤液浓缩至干燥。残渣经硅胶快速色谱纯化（20-50%乙酸乙酯/己烷）得到 N-(2-氯-5-(N,N-二甲基磺酰基)苯基)丙烯酰胺（毫克，%）为白色粉末。H NMR（500 兆赫，CDCl）δ8.91（d，J = 2.1 赫兹，1H），7.85（s，1H），7.58（d，J = 8.4 赫兹，1H），7.52（dd，J = 8.4，2.1 赫兹，1H），6.52（dd，J = 16.8，1.0 赫兹，1H），6.35（dd，J = 16.8，10.2 赫兹，1H），5.91（dd，J = 10.2，1.0 赫兹，1H），2.80（s，6H）。
To a solution of 3-amino-4-chloro-N,N-dimethylbenzenesulfonamide (60 mg, 0.26 mmol) in DCM (5 mL) was added triethylamine (54 μL, 0.38 mmol) dropwise. The solution was stirred at 0C for 5 min. Propionoyl chloride (25 μL, 0.28 mmol) was added and the solution was warmed to room temperature. After 10 min, the mixture was diluted with water, extracted with DCM, washed with brine, dried over NaSO4, filtered, and the filtrate concentrated in vacuo. The residue was purified by flash chromatography on silica gel (30–60% EtOAc/Hexanes) to give N-(2-chloro-5-(N,N-dimethylsulfamoyl)phenyl)acrylamide (48 mg, 65%) as a white powder. 将 3-氨基-4-氯-N,N-二甲基苯磺酰胺（60 毫克，0.26 毫摩尔）溶解于 DCM（5 毫升）中，滴加三乙胺（54 微升，0.38 毫摩尔）。将溶液在 0 摄氏度搅拌 5 分钟。加入丙酰氯（25 微升，0.28 毫摩尔），并将溶液加热至室温。10 分钟后，将混合物稀释水，用 DCM 萃取，用盐水洗涤，经过 NaSO4 干燥，过滤，将滤液在真空中浓缩。残渣经硅胶快速色谱纯化（30-60% 乙酸乙酯/己烷），得到 N-(2-氯-5-(N,N-二甲基磺酰基)苯基)丙烯酰胺（48 毫克，65%）为白色粉末。
To a solution of 3-amino-4-chloro-N-methyl-N-(prop-2-yn-1-yl)benzenesulfonamide (50 mg, 0.19 mmol) in DCM (2 mL) was added triethylamine (78 μL, 0.56 mmol) dropwise. The solution was stirred at 0C for 5 min. Propionyl chloride (50 μL, 0.56 mmol) was added and the solution was warmed to room temperature. After 10 min, the mixture was diluted with water, extracted with DCM, washed with brine, dried over NaSO4, filtered, and the filtrate concentrated in vacuo. The residue was purified by flash chromatography on silica gel (30–60% EtOAc/Hexanes) to give N-(2-chloro-5-(N-methyl-N-(prop-2-yn-1-yl)sulfamoyl)phenyl)propionamide (35 mg, 58%) as a white powder. 将 3-氨基-4-氯-N-甲基-N-(丙-2-炔-1-基)苯磺酰胺（50 毫克，0.19 毫摩尔）溶解于 DCM（2 毫升）中，滴加三乙胺（78 微升，0.56 毫摩尔）。将溶液在 0 摄氏度搅拌 5 分钟。加入丙酰氯（50 微升，0.56 毫摩尔），并将溶液加热至室温。10 分钟后，将混合物稀释水，用 DCM 萃取，用盐水洗涤，经过 NaSO4 干燥，过滤，将滤液浓缩至干燥。残渣经硅胶快速色谱纯化（30-60%乙酸乙酯/己烷）得到 N-(2-氯-5-(N-甲基-N-(丙-2-炔-1-基)磺酰基)苯基)丙酰胺（35 毫克，58%）为白色粉末。
HRMS calcd for C13H15ClN2O3S(M + H)+ 315.05702, found 315.05641.
Quantification and statistical analysis 定量和统计分析
Statistical details can be found in the figure legends as well as Supplemental Tables. Statistical analyses for TMT-based quantitative proteomics were performed as follows. High confidence protein identifications were reported with a <1% false discovery rate (FDR) cut-off. Differential abundance significance was estimated using ANOVA with Benjamini-Hochberg correction to determine p values. IsoTOP-ABPP data analysis was performed as follows. Light versus heavy isotopic probe-modified peptide ratios are calculated by taking the mean of the ratios of each replicate paired light versus heavy precursor abundance for all peptide-spectral matches associated with a peptide. The paired abundances were also used to calculate a paired sample t-test p value to estimate constancy in paired abundances and significance in change between treatment and control. p values were corrected using the Benjamini–Hochberg method. All other statistical analyses were performed by Student’s two-tailed t-tests. 统计细节可以在图例和补充表中找到。基于 TMT 的定量蛋白质组学的统计分析如下进行。高置信度的蛋白质鉴定报告使用<1%的假阳性发现率（FDR）截断。差异丰度显著性是使用 ANOVA 与 Benjamini-Hochberg 校正来确定 p 值进行估计。IsoTOP-ABPP 数据分析如下进行。轻重同位素探针修饰的肽比率是通过计算每个重复配对的轻重前体丰度的比率的平均值来计算的，所有与一个肽相关的肽-光谱匹配。这些配对的丰度还用于计算配对样本 t 检验 p 值，以估计配对丰度的恒定性和治疗与对照之间的变化的显著性。p 值使用 Benjamini-Hochberg 方法进行校正。所有其他统计分析均通过学生的双尾 t 检验进行。
Data and code availability 数据和代码可用性
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All proteomic raw data files are available via ProteomeXchange with Project accession PXD039924. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the database identifier PXD039924. 所有蛋白质组学原始数据文件均可通过 ProteomeXchange 获得，项目访问号为 PXD039924。质谱蛋白质组学数据已通过 PRIDE 合作伙伴库存储到 ProteomeXchange 联盟中，数据库标识符为 PXD039924。
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Data processing and statistical analysis algorithms for chemoproteomics from our lab can be found on our lab’s Github site: https://github.com/NomuraRG, and we can make any further code from this study available upon request. 我们实验室的化学蛋白质组学数据处理和统计分析算法可在我们实验室的 Github 网站上找到：https://github.com/NomuraRG，我们可以根据请求提供本研究的任何进一步代码。
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Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request. 本文报告的数据重新分析所需的任何额外信息可根据请求从主要联系人处获得。
Acknowledgments 致谢
We thank the members of the Nomura Research Group and Novartis Institutes for BioMedical Research for critical reading of the manuscript. This work was supported by Novartis Institutes for BioMedical Research and the Novartis-Berkeley Center for Proteomics and Chemistry Technologies (NB-CPACT) for all listed authors. This work was also supported by the Nomura Research Group and the Mark Foundation for Cancer Research ASPIRE Award for D.K.N., E.A.K. This work was also supported by grants from the National Science Foundation Molecular Foundations for Biotechnology Award (2127788) (for D.K.N.) and the National Science Foundation Graduate Research Fellowship (for E.A.K.). We also thank Drs. Hasan Celik, Alicia Lund, and UC Berkeley’s NMR facility in the College of Chemistry (CoC-NMR) for spectroscopic assistance. Instruments in the CoC-NMR are supported in part by NIH S10OD024998. 我们感谢野村研究小组和诺华生物医学研究所的成员对手稿的重要阅读。本工作得到了诺华生物医学研究所和诺华-伯克利蛋白质组学和化学技术中心（NB-CPACT）对所有列出作者的支持。此工作还得到了野村研究小组和马克癌症研究基金会颁发给 D.K.N.、E.A.K. 的 ASPIRE 奖的支持。此工作还得到了国家科学基金会分子生物技术奖（2127788）（D.K.N.）和国家科学基金会研究生研究奖学金（E.A.K.）的资助。我们还感谢 Hasan Celik 博士、Alicia Lund 博士和加州大学伯克利分校化学学院的核磁共振设施（CoC-NMR）提供的光谱学帮助。CoC-NMR 中的仪器部分得到了 NIH S10OD024998 的支持。
Author contributions 作者贡献
E.A.K. and D.K.N. conceived of the project idea, designed experiments, performed experiments, analyzed and interpreted the data, and wrote the paper. E.A.K., Y.C., N.S.H., D.D., and D.K.N. performed experiments, analyzed and interpreted data, and provided intellectual contributions. E.A.K., D.D., J.A.T., J.M.K., and M.S. provided intellectual contributions to the project and overall design of the project. E.A.K.和 D.K.N.构思了项目的想法，设计了实验，执行了实验，分析和解释了数据，并撰写了论文。E.A.K.，Y.C.，N.S.H.，D.D.和 D.K.N.执行了实验，分析和解释了数据，并提供了智力贡献。E.A.K.，D.D.，J.A.T.，J.M.K.和 M.S.为项目和项目整体设计提供了智力贡献。
Declaration of interests 利益声明
J.A.T., J.M.K., and D.D. are employees of Novartis Institutes for BioMedical Research. This study was funded by the Novartis Institutes for BioMedical Research and the Novartis-Berkeley Translational Chemical Biology Institute. D.K.N. is a co-founder, shareholder, and on the scientific advisory boards for Frontier Medicines and Vicinitas Therapeutics; a member of the board of directors for Vicinitas Therapeutics; on the scientific advisory boards of The Mark Foundation for Cancer Research, Photys Therapeutics, Apertor Pharmaceuticals, Ecto Therapeutics, Oerth Bio, and Chordia Therapeutics; and on the investment advisory board of Droia Ventures. J.A.T.，J.M.K.和 D.D.是 Novartis 生物医学研究所的雇员。本研究由 Novartis 生物医学研究所和 Novartis-Berkeley 转化化学生物学研究所资助。D.K.N.是 Frontier Medicines 和 Vicinitas Therapeutics 的联合创始人，股东，并担任科学顾问委员会成员；Vicinitas Therapeutics 董事会成员；The Mark Foundation for Cancer Research，Photys Therapeutics，Apertor Pharmaceuticals，Ecto Therapeutics，Oerth Bio 和 Chordia Therapeutics 科学顾问委员会成员；以及 Droia Ventures 投资顾问委员会成员。
Table S2. Cysteine chemoproteomic profiling of EN450 targets in HAP1 cells using isoTOP-ABPP, related to Figure 2 表格 S2. 使用 isoTOP-ABPP 在 HAP1 细胞中对 EN450 靶点进行半胱氨酸化学蛋白质组学分析，与图 2 相关
HAP1 cells were treated with DMSO vehicle or EN450 (50 μM) for 3 h, after which resulting cell lysates were labeled with an alkyne-functionalized iodoacetamide cysteine-reactive probe (200 μM) for 1 h, and an isotopically light (for DMSO) or heavy (for EN450) biotin-azide handle bearing a TEV protease recognition peptide was appended by CuAAC. Control and treated proteomes were combined in a 1:1 ratio, taken through the isoTOP-ABPP procedure and light/heavy probe-modified peptides were analyzed by LC-MS/MS and quantified HAP1 细胞分别用 DMSO 载体或 EN450（50 μM）处理 3 小时，随后将得到的细胞裂解物用烷基化碘乙酰胺半胱氨酸反应探针（200 μM）标记 1 小时，然后通过 CuAAC 附加一个同位素轻（对于 DMSO）或重（对于 EN450）生物素-偶氮手柄，带有 TEV 蛋白酶识别肽。对照组和处理组的蛋白质组以 1:1 比例混合，经过 isoTOP-ABPP 程序处理，轻/重探针修饰的肽段通过 LC-MS/MS 分析并定量
Table S3. TMT-based quantitative proteomic profiling of EN450 in HAP1 cells, related to Figure 3 表S3. TMT基于定量蛋白质组学分析EN450在HAP1细胞中的相关性，参见图3。
HAP1 cells were treated with DMSO vehicle or EN450 (50 μM) for 24 h. Data shown are from n = 3 biologically independent replicates/group HAP1细胞分别用DMSO载体或EN450（50μM）处理24小时。所示数据来自n = 3个生物独立重复/组。
References 参考
Spradlin J.N. 斯普拉德林 J.N.
Zhang E. 张 E.
Nomura D.K. 野村 D.K.
Reimagining druggability using chemoproteomic platforms. 重新构想药物可达性，利用化学蛋白质组学平台。
Proteolysis targeting chimeras (PROTACs) come of age: entering the third decade of targeted protein degradation. 蛋白质降解靶向嵌合物（PROTACs）已经进入第三个有针对性蛋白质降解的十年。
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