奥皮诺内酯通过抑制OSBP破坏白血病对胆固醇转运的依赖性-动脉网

Orpinolide disrupts a leukemic dependency on cholesterol transport by inhibiting OSBP

Nature 等信源发布 2024-06-21 18:33

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AbstractMetabolic alterations in cancer precipitate in associated dependencies that can be therapeutically exploited. To meet this goal, natural product-inspired small molecules can provide a resource of invaluable chemotypes. Here, we identify orpinolide, a synthetic withanolide analog with pronounced antileukemic properties, via orthogonal chemical screening.

。为了实现这一目标，天然产物启发的小分子可以提供宝贵的化学型资源。在这里，我们通过正交化学筛选鉴定了orpinolide，一种具有明显抗白血病特性的合成withanolide类似物。

Through multiomics profiling and genome-scale CRISPR–Cas9 screens, we identify that orpinolide disrupts Golgi homeostasis via a mechanism that requires active phosphatidylinositol 4-phosphate signaling at the endoplasmic reticulum–Golgi membrane interface. Thermal proteome profiling and genetic validation studies reveal the oxysterol-binding protein OSBP as the direct and phenotypically relevant target of orpinolide.

通过多组学分析和基因组规模的CRISPR-Cas9筛选，我们发现orpinolide通过在内质网-高尔基体膜界面需要活性磷脂酰肌醇4-磷酸信号传导的机制破坏高尔基体稳态。热蛋白质组分析和遗传验证研究表明，氧固醇结合蛋白OSBP是orpinolide的直接和表型相关靶标。

Collectively, these data reaffirm sterol transport as a therapeutically actionable dependency in leukemia and motivate ensuing translational investigation via the probe-like compound orpinolide..

总的来说，这些数据重申固醇转运是白血病治疗上可行的依赖性，并通过类似探针的化合物orpinolide激发了随后的翻译研究。。

MainRewiring of metabolic networks contributes to the development, progression and resistance acquisition of cancers, including leukemia, and is a general hallmark of cancer1,2. Individuals with acute myeloid leukemia (AML) or acute lymphoblastic leukemia (ALL) display several metabolic changes, including alterations in lipid and cholesterol metabolism to enable rapid proliferation, thus establishing motivation to exploit associated metabolic dependencies3,4.

代谢网络的重新布线有助于癌症（包括白血病）的发展，进展和耐药性的获得，并且是癌症的一般标志1,2。患有急性髓细胞白血病（AML）或急性淋巴细胞白血病（ALL）的个体表现出几种代谢变化，包括脂质和胆固醇代谢的改变以实现快速增殖，从而建立了利用相关代谢依赖性的动机3,4。

A suite of genome-scale genetic perturbation screens, for instance, CRISPR–Cas9 knockout screens, provide a rich catalog of cell-autonomous essential cancer cell functions, including dependencies on lipid or sterol metabolic pathways. However, redundancy and genetic buffering inherent to biological systems often require modulation of more than one protein or modulation that goes beyond functional inhibition.

一套基因组规模的遗传扰动筛选，例如CRISPR-Cas9基因敲除筛选，提供了丰富的细胞自主基本癌细胞功能目录，包括对脂质或甾醇代谢途径的依赖性。然而，生物系统固有的冗余和遗传缓冲通常需要调节一种以上的蛋白质或超出功能抑制的调节。

These characteristics are often outside the reach of genetic perturbations but are attainable via small molecules.Natural products (NPs) are an invaluable source of bioactive molecules with remarkable therapeutic potential. Approximately one-third of all US Food and Drug Administration (FDA)-approved drugs in the last three decades originated from NPs or their derivatives5.

这些特征通常不在遗传扰动的范围内，但可以通过小分子实现。天然产物（NPs）是具有显着治疗潜力的生物活性分子的宝贵来源。。

The intricate biological relevance of NPs is defined by the rich and diverse chemical space embedded in their structures6. Yet, this structural complexity often encumbers the target identification and optimization of NP-derived bioactive compounds. To overcome these limitations, new strategies for identification and synthesis of simplified NP scaffolds are required.The expansion of tangible chemical space can on one hand be achieved through biology-oriented synthesis (BIOS)7.

NPs复杂的生物学相关性由其结构中嵌入的丰富多样的化学空间来定义6。然而，这种结构复杂性通常阻碍了NP衍生的生物活性化合物的目标鉴定和优化。为了克服这些限制，需要用于鉴定和合成简化NP支架的新策略。有形化学空间的扩展一方面可以通过面向生物学的合成（BIOS）7来实现。

BIOS simplifies NPs into core scaffolds with retained biological a.

BIOS将NPs简化为具有保留生物a的核心支架。

(1)

Similarities of phenotypic profiles (or biosimilarity) were calculated from the correlation distances between two profiles (biosimilarity = 1 – correlation distance; https://docs.scipy.org/doc/scipy/reference/generated/scipy.spatial.distance.correlation.html), and the compounds with the most similar profiles were determined from a set of 3,000 reference compounds that was also measured in the assay.

表型谱（或生物相似性）的相似性由两个谱之间的相关距离（生物相似性）=1-相关距离；https://docs.scipy.org/doc/scipy/reference/generated/scipy.spatial.distance.correlation.html)，并且从一组3000种参考化合物中确定了具有最相似特征的化合物，这些化合物也在测定中进行了测量。

UMAP analysis was performed as previously reported70.Western blottingCollected cell pellets were washed with PBS and lysed in RIPA buffer (50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 1× Halt protease inhibitor cocktail and 25 U ml–1 benzonase) by incubating on ice for at least 15 min.

如先前报道的那样进行UMAP分析70。蛋白质印迹收集的细胞沉淀用PBS洗涤，并在RIPA缓冲液（50mM Tris-HCl（pH 8.0），150mM NaCl，1%Triton X-100、0.5%脱氧胆酸钠，0.1%SDS，1x Halt蛋白酶抑制剂混合物和25umL-1苯甲酸酶）中裂解，方法是在冰上孵育至少15分钟。

The lysates were cleared through centrifugation (15 min, 20,000g, 4 °C), and the total protein concentration was determined by BCA protein assay (Pierce BCA Protein Assay kit, Thermo Scientific, 23225) following the manufacturer’s protocol. All lysates were supplemented with 4× Bolt LDS sample buffer (Invitrogen) and denatured for 5 min at 95 °C before loading onto polyacrylamide gels (20 μg of total protein).

。所有裂解物均补充有4x Bolt LDS样品缓冲液（Invitrogen），并在95℃变性5分钟，然后加载到聚丙烯酰胺凝胶（总蛋白20μg）上。

Proteins were separated on 4–12% SDS–PAGE gels (Invitrogen) and transferred to nitrocellulose membranes. The transfer efficiency was tested through staining with Ponceau-S. The membranes were blocked with 5% milk in Tris-buffered saline with Tween20 (TBST) (30 min, room temperature). Primary antibodies were incubated overnight at 4 °C in 1% milk in TBST, while the respective secondary antibodies were incubated in TBST for 1 h at room temperature.

蛋白质在4-12%SDS-PAGE凝胶（Invitrogen）上分离，并转移到硝酸纤维素膜上。通过用Ponceau-S染色测试转移效率。用含有吐温20（TBST）的Tris缓冲盐水中的5%牛奶（30分钟，室温）封闭膜。将一抗在4℃下在TBST中的1%牛奶中孵育过夜，而将各自的二抗在TBST中在室温下孵育1小时。

Blots were developed with chemiluminescence films. The following primary antibodies were used: OSBP (1:2,000; Bethyl, A304-553A), GAPDH (1:5,000; Santa Cruz Biotechnology, sc-365062),.

用化学发光膜显影印迹。使用了以下一抗：OSBP（1:2000；Bethyl，A304-553A），GAPDH（1:5000；Santa Cruz Biotechnology，sc-365062），。

Data availability

数据可用性

The MS proteomics data (Figs. 2a,b and 4a, Extended Data Figs. 4 and 6 and Supplementary Tables 2 and 5) have been deposited to the ProteomeXchange Consortium via the PRIDE83 partner repository with the dataset identifiers PXD040694 and PXD040694 (expression proteomics) as well as PXD040692 and PXD040692 (TPP).

MS蛋白质组学数据（图2a，b和4a，扩展数据图4和6以及补充表2和5）已通过PRIDE83合作伙伴存储库保存到ProteomeXchange Consortium，数据集标识符为PXD040694和PXD040694（表达蛋白质组学）以及PXD040692和PXD040692（TPP）。

Raw and analyzed RNA-sequencing and genome-wide CRISPR–Cas9 screening datasets (Figs. 2c,d and 3, Extended Data Fig. 5 and Supplementary Tables 3 and 4) are available in NCBI’s Gene Expression Omnibus under accession number GSE226849. Additionally, publicly available data from the following databases were used in this study: DepMap (22Q4 and 23Q2), The Human Protein Atlas project (v.22.0), UniProtKB (13.01.2023) and BioGRID (v.4.4.212).

原始和分析的RNA测序和全基因组CRISPR-Cas9筛选数据集（图2c，d和3，扩展数据图5和补充表3和4）可在NCBI的Gene Expression Omnibus中获得，登录号为GSE226849。此外，本研究使用了来自以下数据库的公开数据：DepMap（22Q4和23Q2），人类蛋白质图谱项目（v.22.0），UniProtKB（13.01.2023）和BioGRID（v.4.4.212）。

The data supporting all of the findings in this study are available within the paper, its supplementary files and the mentioned databases. Source data are provided with this paper..

支持本研究所有发现的数据可在论文，其补充文件和上述数据库中找到。本文提供了源数据。。

Code availability

代码可用性

The data analysis code is available at https://github.com/GWinterLab/W7.

数据分析代码可在https://github.com/GWinterLab/W7.

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Download referencesAcknowledgementsWe are grateful to all the members of the laboratory of G.E.W., in particular N. Scholes, A. Hanzl and V. Brennsteiner, for helpful discussions and editorial contributions. We thank A. Koren, S. Kubicek and the CeMM Molecular Discovery Platform for their assistance with the profiling of the withanolide-inspired compound collection and the CeMM Biomedical Sequencing Facility for next-generation sequencing sample processing, sequencing and data curation.

下载参考文献致谢我们感谢G.E.W.实验室的所有成员，特别是N.Scholes，A.Hanzl和V.Brennsteiner，感谢他们的有益讨论和编辑贡献。我们感谢A.Koren，S.Kubicek和CeMM分子发现平台在分析withanolide启发的化合物收集和CeMM生物医学测序设施以进行下一代测序样品处理，测序和数据管理方面提供的帮助。

We thank the Morph-Im platform and staff at UNamur for their microscopy support. We moreover thank J. Zuber at the Research Institute of Molecular Pathology for sharing iCas9 cell lines and plasmids. CeMM and the laboratories of G.E.W. and G.S.-F. are supported by the Austrian Academy of Sciences. The laboratory of G.E.W.

我们感谢Morph Im平台和联乌尔特派团的工作人员对显微镜的支持。此外，我们感谢分子病理学研究所的J.Zuber分享iCas9细胞系和质粒。CeMM以及G.E.W.和G.S.-F.的实验室得到了奥地利科学院的支持。G.E.W.实验室。

is further supported by funding from the European Research Council under the European Union’s Horizon 2020 research and innovation program (grant agreement 851478). The laboratory of L.L. was supported by funding from the Novo Nordisk Foundation (NNF21OC0067188) and the Independent Research Fund Denmark (9041-00248B).

由欧洲研究理事会根据欧盟地平线2020研究与创新计划（赠款协议851478）提供的资金进一步支持。L.L.实验室得到了诺和诺德基金会（NNF21OC0067188）和丹麦独立研究基金（9041-00248B）的资助。

A.F. is an FRS-FNRS Chercheur Qualifiée. This research was funded in whole or in part by the Austrian Science Fund (FW; P32125, P31690 and P7909). For open access purposes, the author has applied a CC BY public copyright license to any author-accepted manuscript version arising from this submission.Author informationAuthor notesCristina Mayor-RuizPresent address: IRB Barcelona—Institute for Research in Biomedicine, The Barcelona Institute of Science and Technology, Barcelona, SpainAuthors and AffiliationsCeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, AustriaMarko Cigler, Hana Imrichova, Fabian Fromm.

A、 F.是FRS-FNRS Chercheur Qualifieée。这项研究全部或部分由奥地利科学基金（FW；P32125，P31690和P7909）资助。出于开放获取的目的，作者已将CC BY public copyright许可证应用于本次提交产生的任何作者接受的稿件版本。作者信息作者注释Scristina Mayor Ruiz目前的地址：IRB巴塞罗那生物医学研究所，巴塞罗那科学技术研究所，巴塞罗那，SpainAuthors及其附属机构奥地利科学院分子医学研究中心，维也纳，澳大利亚马尔科·齐格勒，哈娜·伊姆里乔娃，法比安·弗洛姆。

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PubMed Google ScholarContributionsM.C., H.W. and G.E.W. conceptualized this study. M.C. designed and conducted the experiments with help from C.K. and C.M.-R. H.I. analyzed and visualized RNA-sequencing and CRISPR screening data. M.C. and A.R. performed the TPP experiment, while F.F.

PubMed谷歌学术贡献。C、，H.W.和G.E.W.将这项研究概念化。M、 C.在C.K.和C.M.-R.H.I.的帮助下设计并进行了实验。分析并可视化了RNA测序和CRISPR筛选数据。M、 C.和A.R.进行了TPP实验，而F.F。

analyzed and visualized the TPP data. F.F. performed the protein interaction analysis. L.C. performed immunofluorescence microscopy and anterograde trafficking assays under the supervision of A.F. L.D. developed and performed the recombinant binding and sterol transfer assays under the supervision of L.L.

分析并可视化TPP数据。F、 F.进行蛋白质相互作用分析。五十、 C.在A.F.L.D.的监督下进行免疫荧光显微镜检查和顺行运输测定。在L.L.的监督下开发并进行重组结合和甾醇转移测定。

S.S. performed the cell painting assay. J.T.H. supervised the proteomics experiments. G.S.-F. supervised the TPP analyses. M.C. generated the figures with input from all authors. M.C. and G.E.W. wrote the manuscript with input from all authors.Corresponding authorsCorrespondence to.

S、 S.进行了细胞绘画测定。J、 T.H.监督了蛋白质组学实验。G、 S.-F.监督了TPP分析。M、 C.根据所有作者的输入生成数字。M、 C.和G.E.W.在所有作者的意见下撰写了手稿。通讯作者通讯。

Herbert Waldmann or Georg E. Winter.Ethics declarations

。道德宣言

Competing interests

相互竞争的利益

G.E.W. and G.S.-F. are scientific founders and shareholders of Proxygen and Solgate. G.E.W. is on the Scientific Advisory Board of Nexo Therapeutics. The laboratories of G.E.W. and G.S.-F. received research funding from Pfizer. C.M.-R. is part of the Scientific Advisory Board of Nostrum Biodiscovery.

G、 E.W.和G.S.-F.是Proxygen和Solgate的科学创始人和股东。G、 E.W.是Nexo Therapeutics科学顾问委员会成员。G.E.W.和G.S.-F.的实验室获得了辉瑞公司的研究资助。C、 M.-R.是Nostrum Biodiscovery科学顾问委员会的成员。

The C.M.-R. lab receives research funding from Aelin Therapeutics and Almirall. The remaining authors declare no competing interests..

C.M.-R.实验室获得了Aelin Therapeutics和Almirall的研究资助。其余作者声明没有利益冲突。。

Peer review

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Nature Chemical Biology thanks Minetaro Arita, Markus Schirle and the other, anonymous, reviewers for their contribution to the peer review of this work.

《自然化学生物学》感谢Minetaro Arita，Markus Schirle和其他匿名审稿人对这项工作的同行评审做出的贡献。

Additional informationPublisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Extended dataExtended Data Fig. 1 Withanolide-inspired compound collection.Structural overview of the 52-membered compound library based on the type A withanolide scaffold.

Additional informationPublisher的注释Springer Nature在已发布的地图和机构隶属关系中的管辖权主张方面保持中立。扩展数据扩展数据图1与内酯启发的化合物集合。基于A型withanolide支架的52元化合物文库的结构概述。

The cytotoxic vinylogous uretanes W5-W11, their putative hydrolysis product W23 and the ring-fragmented product W52 are highlighted as well. SMILES representations of the compound collection are available in the Supplementary Table 1.Extended Data Fig. 2 Phenotypic profiling of the withanolide-inspired compound collection in leukemia cells.(a-g) Representative dose response curves for seven cytotoxic vinylogous urethans (W5-W11) as well as their (h) putative hydrolysis product (W23) and (i) the ring-fragmented product (W52).

细胞毒性乙烯基脲W5-W11，其推定的水解产物W23和环片段化产物W52也被突出显示。化合物集合的SMILES表示可在补充表1中获得。扩展数据图2白血病细胞中withanolide启发的化合物集合的表型分析。。

Mean ± s.e.m.; n = 3 independent treatments.Source dataExtended Data Fig. 3 Cell viability profiling of W7 in additional non-malignant and non-leukemic cell lines.(a) Overview of the luminescence-based cell viability screening results for W7 across a panel of 30 different cell lines. The results are represented as area under the curve (A.U.C.), calculated from 10-concentration-point dose-response curves.

平均值±标准误。；n=3个独立治疗。来源数据扩展数据图3 W7在其他非恶性和非白血病细胞系中的细胞活力分析。（a）在一组30种不同的细胞系中，基于发光的W7细胞活力筛选结果概述。结果表示为曲线下面积（A.U.C.），由10个浓度点剂量反应曲线计算得出。

The cells were exposed to the drug for 72 hours, with the exception of PBMCs, which had a 48-hour treatment period. (b-i) Dose-resolved, normalized viability of non-malignant (b), blood cancer (c), bone cancer (d), cervical/prostate adenocarcinoma (e), colorectal adenocarcinoma (f), pancreatic adenocarcinoma (g), lung cancer (h) and other cancer cells (i) after 72 h of W7 treatment.

将细胞暴露于药物72小时，但PBMC除外，其具有48小时的治疗期。。

Mean ± s.e.m.; n = 3 independent treatments.Source dataExtended Data Fig. 4 W7 induces global destabilization of Golgi-related proteins.(a) Subce.

平均值±标准误。；n=3个独立治疗。源数据扩展数据图4 W7诱导高尔基体相关蛋白的整体不稳定。（a）分条款。

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Reprints and permissionsAbout this articleCite this articleCigler, M., Imrichova, H., Frommelt, F. et al. Orpinolide disrupts a leukemic dependency on cholesterol transport by inhibiting OSBP.

转载和许可本文引用本文Cigler，M.，Imrichova，H.，Frommelt，F。等人。Orpinolide通过抑制OSBP破坏白血病对胆固醇转运的依赖性。

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