Screening Method for the Discovery of Potential Cancer Chemoprevention Agents Based on Mass Spectrometric Detection of Alkylated Keap1

详细信息    查看全文

• 作者：Guowen Liu ; Aimee L. Eggler ; Birgit M. Dietz ; Andrew D. Mesecar ; Judy L. Bolton ; John M. Pezzuto ; and Richard B. van Breemen
• 刊名：Analytical Chemistry
• 出版年：2005
• 出版时间：October 1, 2005
• 年：2005
• 卷：77
• 期：19
• 页码：6407 - 6414
• 全文大小：167K
• 年卷期：v.77,no.19(October 1, 2005)
• ISSN：1520-6882

文摘

Natural products are important sources of drugs such ascancer chemopreventive agents, but most assays for thediscovery of compounds in natural product extracts arelow throughput and provide little information about leadcompounds in these complex mixtures. The induction ofenzymes such as quinone reductase, glucuronyl transferases, glutathione S-transferases, and sulfotransferasescan protect cells against the toxic and neoplastic effectsof carcinogens. An increase in the concentration of Nrf2in the nucleus of a cell upregulates the antioxidantresponse element and induces the expression of thesechemopreventive enzymes. Based on the hypothesis thatubiquitination and proteosome-mediated degradation ofNrf2 in the cytoplasm decreases upon the covalentmodification of 1 or more of the 27 cysteine sulfhydrylgroups on Keap1 (a protein that sequesters Nrf2 in thecytoplasm) and results in higher Nrf2 levels both in thecytoplasm and in the nucleus, a high-throughput massspectrometry-based screening assay was designed todetect alkylation of sulfhydryl groups of human Keap1.As an initial high-throughput screening step, matrix-assisted laser desorption time-of-flight mass spectrometrywas used to determine whether incubation of Keap1 witha botanical sample produced adducts of Keap1. Testextracts found to form adducts with Keap1 were thenincubated with the alternative biological nucleophile glutathione and characterized using LC-UV-MS-MS. Aftervalidation of the assay using two model alkylating agents,fractions of an extract of hops (Humulus lupulus L.) fromthe brewing industry were screened, and several compounds were detected as potential chemopreventive agents.Two of these electrophilic hops constituents were identified as xanthohumol and xanthohumol D. In a subsequentcell-based assay, xanthohumol and xanthohumol D wereconfirmed to be potent inducers of quinone reductase,and reaction with Keap1 was also confirmed. Therefore,this new mass spectrometric screening assay was demonstrated to facilitate the discovery of chemopreventionagents in complex natural product mixtures.