Department of Chemistry, University of Illinois, Urbana, IL, United States.
Department of Chemistry, University of Illinois, Urbana, IL, United States; Carl R. Woese Institute for Genomic Biology, University of Illinois, Urbana, IL, United States; Department of Microbiology, University of Illinois, Urbana, IL, United States.
Methods Enzymol. 2022;665:177-208. doi: 10.1016/bs.mie.2021.11.018. Epub 2021 Dec 21.
Natural products have traditionally been a fruitful source of chemical matter that has been developed into novel therapeutics. Actinomycetes and several other bacterial taxa are especially gifted in biosynthesizing natural products. However, many decades of intense bioactivity-based screening led to a large rediscovery problem, rendering industrial natural product discovery pipelines uneconomical. Numerous methods for circumventing the rediscovery problem have been developed, among them various chemistry-focused strategies, including reactivity-based screening. Emerging from the field of chemical proteomics, reactivity-based screening relies on a reactive probe that chemoselectively modifies a functional group of interest in the context of a complex biological sample. Reactivity-based probes for several distinct functional groups have been deployed to discover new polyketide and peptidic natural products. This chapter describes the protocols to conduct a reactivity-based screening campaign, including bacteria cultivation and screening of cellular extracts with phenylglyoxal-, tetrazine-, thiol-, and aminooxy-functionalized probes, which respectively target primary uriedo, electron-rich olefins, Michael acceptors, and reactive carbonyls. In addition, a recent case study is presented that employs reactivity-based screening as a component of a forward genetics screen to identify a previously unknown peptidyl arginine deiminase. We anticipate that these methods will be useful for those interested in discovering natural products that evade detection by traditional, bioassay-guided methods and others who wish to rapidly connect metabolic chemotype with genotype.
天然产物一直是化学物质的丰富来源，这些物质已被开发成新的治疗方法。放线菌和其他几种细菌类群特别擅长生物合成天然产物。然而，数十年来基于生物活性的高强度筛选导致了大量的重新发现问题，使得工业天然产物发现管道变得不经济。已经开发出了许多规避重新发现问题的方法，其中包括各种以化学为重点的策略，包括基于反应性的筛选。基于反应性的筛选源于化学蛋白质组学领域，它依赖于一种反应性探针，该探针在复杂生物样品的背景下化学选择性地修饰感兴趣的功能基团。已经部署了几种不同功能基团的基于反应性的探针来发现新的聚酮和肽天然产物。本章描述了进行基于反应性的筛选活动的方案，包括细菌培养和使用苯乙二醛、四嗪、巯基和氨氧基功能化探针对细胞提取物进行筛选，这些探针分别针对初级尿嘧啶、富电子烯烃、迈克尔受体和反应性羰基。此外，还提出了一个最近的案例研究，该研究将基于反应性的筛选作为正向遗传学筛选的一部分，以鉴定一种以前未知的肽基精氨酸脱亚氨酶。我们预计这些方法将对那些有兴趣发现逃避传统生物测定指导方法检测的天然产物的人以及那些希望快速将代谢化学型与基因型联系起来的人有用。
