Prioritization of antimicrobial targets by CRISPR-based oligo recombineering
Prioritization of antimicrobial targets by CRISPR-based oligo recombineering
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DOI：
10.1101/2021.02.04.429737
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发表时间：
2021-02
期刊：
影响因子：
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通讯作者：
H. Benns;M. Storch;J. Falco;F. Fisher;E. Alves;C. Wincott;J. Baum;G. Baldwin;E. Weerapana;E. Tate;M. Child
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文献类型：
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作者：
H. Benns;M. Storch;J. Falco;F. Fisher;E. Alves;C. Wincott;J. Baum;G. Baldwin;E. Weerapana;E. Tate;M. Child
亲核氨基酸在共价药物开发中是重要的，但作为抗菌靶点尚未得到充分利用。近年来，一些化学蛋白质组学技术已经被开发出来，通过它们对亲电探针的内在反应性来挖掘化学可接近的残基。然而，这些方法不能辨别哪些反应位点有助于蛋白质功能，因此应该优先用于药物发现。为了解决这个问题，我们开发了一个基于crispr的Oligo recombering （CORe）平台，根据它们对蛋白质功能的贡献系统地优先考虑活性氨基酸。我们的方法直接将蛋白质序列和功能与生物适应度相结合。在此，我们分析了真核病原体刚地弓形虫约700个蛋白上约1000个半胱氨酸的反应性，并利用CORe对功能位点进行了排序。我们竞争性地比较了74个半胱氨酸上370个密码子开关的适应度效应，并确定了多种蛋白质的功能位点。在我们的概念验证中，CORe的执行速度比标准遗传工作流程快800倍。修饰核糖体的活性半胱氨酸被发现对寄生虫的生长至关重要，随后基于靶标的筛选验证了顶复合体翻译机制作为共价配体发育的靶标。CORe是系统不可知的，支持在广泛的生物体和疾病中对反应位点的权宜之计识别、功能优先排序和合理靶向。
Nucleophilic amino acids are important in covalent drug development yet underutilized as antimicrobial targets. Over recent years, several chemoproteomic technologies have been developed to mine chemically-accessible residues via their intrinsic reactivity toward electrophilic probes. However, these approaches cannot discern which reactive sites contribute to protein function and should therefore be prioritized for drug discovery. To address this, we have developed a CRISPR-based Oligo Recombineering (CORe) platform to systematically prioritize reactive amino acids according to their contribution to protein function. Our approach directly couples protein sequence and function with biological fitness. Here, we profile the reactivity of >1,000 cysteines on ~700 proteins in the eukaryotic pathogen Toxoplasma gondii and prioritize functional sites using CORe. We competitively compared the fitness effect of 370 codon switches at 74 cysteines and identify functional sites in a diverse range of proteins. In our proof of concept, CORe performed >800 times faster than a standard genetic workflow. Reactive cysteines decorating the ribosome were found to be critical for parasite growth, with subsequent target-based screening validating the apicomplexan translation machinery as a target for covalent ligand development. CORe is system-agnostic, and supports expedient identification, functional prioritization, and rational targeting of reactive sites in a wide range of organisms and diseases.