Macrocyclic Rearrangement Ion Fragmentation of Glutathione Conjugates of Cyclobutane-Containing Covalent BTK Inhibitors.

IF 3.1 2区 化学 Q2 BIOCHEMICAL RESEARCH METHODS
Cathy A Muste, Chungang Gu, H George Vandeveer, Simone Sciabola, Martin K Himmelbauer
{"title":"Macrocyclic Rearrangement Ion Fragmentation of Glutathione Conjugates of Cyclobutane-Containing Covalent BTK Inhibitors.","authors":"Cathy A Muste, Chungang Gu, H George Vandeveer, Simone Sciabola, Martin K Himmelbauer","doi":"10.1021/jasms.4c00275","DOIUrl":null,"url":null,"abstract":"<p><p>Covalent BTK-inhibitor drugs often contain reactive acrylamide warheads designed to irreversibly bind to their protein targets at free thiol cysteines in the kinase active site. This reactivity also makes covalent inhibitors susceptible to conjugation to endogenous tripeptide glutathione (GSH), leading to clearance. During lead optimization efforts for the drug discovery of covalent BTK inhibitor BIIB129, some expected GSH adducts resulted in an unexpected and highly abundant rearrangement fragment ion in LC-MS/MS. By examining more than 30 inhibitors, the rearrangements were found to be dependent on the presence of a cycloalkane linker that connects the warhead to the kinase hinge binder motif of drug molecules. The proposed mechanism includes the formation of a 16-membered macrocyclic intermediate between the γ-glutamic acid residue (Glu) of GSH and a methyl-cyclobutyl cation, resulting in a rearrangement fragment originating from two distant parts of the adduct molecule separated by the warhead conjugated with the cysteine residue in between. Rich sets of chemical analogues available during the lead optimization enabled confirmation of the macrocyclic rearrangement. Proposed macrocyclic rearrangement was verified using GSH derivatives: N-acetylation of the γ-Glu blocked the rearrangement, and esterification of the γ-Glu side chain resulted in an expected shift in the mass of rearranged fragment ion. Proposed rearranged ion structures were supported by MS<sup>3</sup> and MS<sup>4</sup> fragmentations. Comparisons of the ion fragmentation of GSH conjugates between <i>cis</i> and <i>trans</i> matched pairs suggest a concerted mechanism for the cyclobutane linker and a stepwise mechanism for the methylcyclobutane linker, respectively.</p>","PeriodicalId":672,"journal":{"name":"Journal of the American Society for Mass Spectrometry","volume":" ","pages":""},"PeriodicalIF":3.1000,"publicationDate":"2025-03-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Journal of the American Society for Mass Spectrometry","FirstCategoryId":"92","ListUrlMain":"https://doi.org/10.1021/jasms.4c00275","RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"BIOCHEMICAL RESEARCH METHODS","Score":null,"Total":0}
引用次数: 0

Abstract

Covalent BTK-inhibitor drugs often contain reactive acrylamide warheads designed to irreversibly bind to their protein targets at free thiol cysteines in the kinase active site. This reactivity also makes covalent inhibitors susceptible to conjugation to endogenous tripeptide glutathione (GSH), leading to clearance. During lead optimization efforts for the drug discovery of covalent BTK inhibitor BIIB129, some expected GSH adducts resulted in an unexpected and highly abundant rearrangement fragment ion in LC-MS/MS. By examining more than 30 inhibitors, the rearrangements were found to be dependent on the presence of a cycloalkane linker that connects the warhead to the kinase hinge binder motif of drug molecules. The proposed mechanism includes the formation of a 16-membered macrocyclic intermediate between the γ-glutamic acid residue (Glu) of GSH and a methyl-cyclobutyl cation, resulting in a rearrangement fragment originating from two distant parts of the adduct molecule separated by the warhead conjugated with the cysteine residue in between. Rich sets of chemical analogues available during the lead optimization enabled confirmation of the macrocyclic rearrangement. Proposed macrocyclic rearrangement was verified using GSH derivatives: N-acetylation of the γ-Glu blocked the rearrangement, and esterification of the γ-Glu side chain resulted in an expected shift in the mass of rearranged fragment ion. Proposed rearranged ion structures were supported by MS3 and MS4 fragmentations. Comparisons of the ion fragmentation of GSH conjugates between cis and trans matched pairs suggest a concerted mechanism for the cyclobutane linker and a stepwise mechanism for the methylcyclobutane linker, respectively.

含环丁烷共价BTK抑制剂谷胱甘肽偶联物的大环重排离子断裂。
共价btk抑制剂药物通常含有活性丙烯酰胺弹头,旨在与激酶活性部位的游离巯基半胱氨酸蛋白不可逆地结合。这种反应性也使共价抑制剂容易与内源性三肽谷胱甘肽(GSH)结合,从而导致清除。在共价BTK抑制剂BIIB129药物发现的先导优化工作中,一些预期的GSH加合物在LC-MS/MS中导致了意想不到的高度丰富的重排片段离子。通过检查30多种抑制剂,发现重排依赖于将弹头连接到药物分子的激酶铰链结合基序的环烷烃连接体的存在。所提出的机制包括在谷胱甘肽的γ-谷氨酸残基(Glu)和甲基环丁基阳离子之间形成一个16元的大环中间体,导致重排片段起源于加合物分子的两个遥远部分,由中间与半胱氨酸残基共轭的战斗部分开。在先导优化过程中,丰富的化学类似物使大环重排的确认成为可能。用谷胱甘肽衍生物验证了提出的大环重排:γ-Glu的n -乙酰化阻止了重排,γ-Glu侧链的酯化导致重排片段离子的质量发生了预期的变化。提出的重排离子结构得到了MS3和MS4片段的支持。通过比较顺式和反式配对的谷胱甘肽偶联物的离子断裂,分别得出了环丁烷连接剂的一致机制和甲基环丁烷连接剂的逐步机制。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
求助全文
约1分钟内获得全文 求助全文
来源期刊
CiteScore
5.50
自引率
9.40%
发文量
257
审稿时长
1 months
期刊介绍: The Journal of the American Society for Mass Spectrometry presents research papers covering all aspects of mass spectrometry, incorporating coverage of fields of scientific inquiry in which mass spectrometry can play a role. Comprehensive in scope, the journal publishes papers on both fundamentals and applications of mass spectrometry. Fundamental subjects include instrumentation principles, design, and demonstration, structures and chemical properties of gas-phase ions, studies of thermodynamic properties, ion spectroscopy, chemical kinetics, mechanisms of ionization, theories of ion fragmentation, cluster ions, and potential energy surfaces. In addition to full papers, the journal offers Communications, Application Notes, and Accounts and Perspectives
×
引用
GB/T 7714-2015
复制
MLA
复制
APA
复制
导出至
BibTeX EndNote RefMan NoteFirst NoteExpress
×
提示
您的信息不完整,为了账户安全,请先补充。
现在去补充
×
提示
您因"违规操作"
具体请查看互助需知
我知道了
×
提示
确定
请完成安全验证×
copy
已复制链接
快去分享给好友吧!
我知道了
右上角分享
点击右上角分享
0
联系我们:info@booksci.cn Book学术提供免费学术资源搜索服务,方便国内外学者检索中英文文献。致力于提供最便捷和优质的服务体验。 Copyright © 2023 布克学术 All rights reserved.
京ICP备2023020795号-1
ghs 京公网安备 11010802042870号
Book学术文献互助
Book学术文献互助群
群 号:481959085
Book学术官方微信