An Activity-Based Oxaziridine Platform for Identifying and Developing Covalent Ligands for Functional Allosteric Methionine Sites: Redox-Dependent Inhibition of Cyclin-Dependent Kinase 4.
An Activity-Based Oxaziridine Platform for Identifying and Developing Covalent Ligands for Functional Allosteric Methionine Sites: Redox-Dependent Inhibition of Cyclin-Dependent Kinase 4.
复制标题
DOI：
10.1021/jacs.2c04039
复制
发表时间：
2022-12-21
影响因子：
15
通讯作者：
Chang, Christopher J.
中科院分区：
文献类型：
--
作者：
Gonzalez-Valero, Angel;Reeves, Audrey G.;Page, Annika C. S.;Moon, Patrick J.;Miller, Edward;Coulonval, Katia;Crossley, Steven W. M.;Xie, Xiao;He, Dan;Musacchio, Patricia Z.;Christian, Alec H.;McKenna, Jeffrey M.;Lewis, Richard A.;Fang, Eric;Dovala, Dustin;Lu, Yipin;McGregor, Lynn M.;Schirle, Markus;Tallarico, John A.;Roger, Pierre P.;Toste, F. Dean;Chang, Christopher J.
基于活性的蛋白质图谱(ABPP)是一种用于识别和表征用于治疗开发的功能蛋白质位点和化合物的通用策略。然而，绝大多数用于共价药物发现的ABPP方法针对的是半胱氨酸或赖氨酸等高度亲核的氨基酸。在这里，我们报告了一个使用氧化还原激活的化学标签(Reaction)的蛋氨酸导向的ABPP平台，它利用仿生氧化连接策略来选择性地修饰蛋氨酸。应用对癌蛋白细胞周期蛋白依赖性激酶4(CDK4)的反应作为具有代表性的高价值药物靶点，发现了三个新的可配基的蛋氨酸位点。然后我们合成了一个以蛋氨酸为靶标的共价配体文库，该文库含有一系列不同的杂环、杂原子和富含立体化学的取代基。ABPP筛选发现1oxF11是CDK4在变构M169位点的共价修饰物。该化合物以剂量依赖的方式抑制纯化蛋白和乳腺癌细胞中的激酶活性。对1oxF11的进一步研究发现，显著的阳离子-π和氢键相互作用稳定了该片段在M169位点的结合。定量质谱学研究证实了乳腺癌细胞裂解物中CDK4的1oxF11连接。进一步的生化分析揭示了M169氧化和T172磷酸化之间的串扰，其中M169氧化阻止CDK4上激活的T172位点的磷酸化，并阻止细胞周期进展。通过识别CDK4上变构蛋氨酸氧化还原调节的新机制并为其治疗干预开发一种独特的方式，这项工作展示了一个可推广的平台，为参与更广泛的化学蛋白质组学和蛋白质配体发现工作提供了一个起点，以寻找和靶向以前不可药物的蛋氨酸位点。
Activity-based protein profiling (ABPP) is a versatile strategy for identifying and characterizing functional protein sites and compounds for therapeutic development. Yet, the vast majority of ABPP methods for covalent drug discovery target highly nucleophilic amino acids such as cysteine or lysine. Here, we report a methionine-directed ABPP platform using Redox-Activated Chemical Tagging (ReACT), which leverages a biomimetic oxidative ligation strategy for selective methionine modification. Application of ReACT to oncoprotein cyclin-dependent kinase 4 (CDK4) as a representative high-value drug target identified three new ligandable methionine sites. We then synthesized a methionine-targeting covalent ligand library bearing a diverse array of heterocyclic, heteroatom, and stereochemically-rich substituents. ABPP screening of this focused library identified 1oxF11 as a covalent modifier of CDK4 at an allosteric M169 site. This compound inhibited kinase activity in a dose-dependent manner on purified protein and in breast cancer cells. Further investigation of 1oxF11 found prominent cation-π and H-bonding interactions stabilizing the binding of this fragment at the M169 site. Quantitative mass-spectrometry studies validated 1oxF11 ligation of CDK4 in breast cancer cell lysates. Further biochemical analyses revealed crosstalk between M169 oxidation and T172 phosphorylation, where M169 oxidation prevented phosphorylation of the activating T172 site on CDK4 and blocked cell cycle progression. By identifying a new mechanism for allosteric methionine redox regulation on CDK4 and developing a unique modality for its therapeutic intervention, this work showcases a generalizable platform that provides a starting point for engaging in broader chemoproteomics and protein ligand discovery efforts to find and target previously undruggable methionine sites.
登录
查看更多内容
影响因子：
15
作者：
Christian, Alec H.;Jia, Shang;Toste, F. Dean
通讯作者：
Toste, F. Dean
DOI：
10.1073/pnas.94.18.9932
发表时间：
1997-09-02
影响因子：
11.1
作者：
Ciorba, MA;Heinemann, SH;Hoshi, T
通讯作者：
Hoshi, T
影响因子：
5.3
作者：
Bockstaele, Laurence;Kooken, Hugues;Coulonval, Katia
通讯作者：
Coulonval, Katia
影响因子：
2.9
作者：
Choi, Eun Jeong
通讯作者：
Choi, Eun Jeong
影响因子：
2.9
作者：
deGruyter JN;Malins LR;Baran PS
通讯作者：
Baran PS